This PDF file contains the front matter associated with SPIE Proceedings Volume 11070, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

To understand the origin of life on Earth, and to evaluate the potential for life on exoplanets, we must understand the pathways that lead from chemistry to biology. Recent experiments suggest that a chemically rich environment that provides the building blocks of membranes, nucleic acids and peptides, along with sources of chemical energy, could result in the emergence of replicating, evolving cells. Diverse photochemical processes are thought to have been important for the origin of life, from photochemical steps in prebiotic synthetic pathways, to the generation of useful forms of chemical energy, to the selection of the canonical nucleotides based on their photostability. I will discuss the many ways in which UV light may have influenced the origin of life.

We will discuss product target profiles that photosensitizers for PDT should met to satisfy the requirements of clinical PDT. Next, we will review the spectroscopy and photochemistry of the main families of photosensitizers with an impact in clinical PDT. We will critically examine the factors that made such photosensitizers appealing for PDT and their shortcomings. Finally, an overview of opportunities ahead will be presented. Topics addressed – The phototherapeutic window – Electronic spectroscopy of porphyrin derivatives, phthalocyanines and BODIPYs – Photochemistry and ROS – Factors contributing to the performance of PDT sensitizers

This Conference Presentation, "Intraoperative photodynamic therapy of glioblastoma: the Japanese experience," was recorded at the International Photodynamic Association World Congress 2019 in Boston, Massachusetts, United States.

Contamination of food takes place in all stages of production process, packaging, storage, transportation, sales and cooking. Therefore large amounts of clean water are used throughout the food production process such as cleaning, sanitizing, peeling, cooling or cooking. Besides physical or chemical contamination a major part represents the biological contamination by different microbial species. In recent years Enterohaemorrhagic Escherichia coli (EHEC) caused a serious outbreak of food borne illness worldwide, where patients suffered from severe diarrhoea and in some cases from haemolytic-uremic syndrome. The source of infection for humans was contaminated food (e.g. bean sprout) with EHEC. Furthermore pigs, colonised with methicillin resistant Staphylococcus aureus (MRSA), has become an emerging risk to be a source of infection for humans. Therefore one important task will be preventing contamination of food with pathogens and spreading from people, pets and pests. One new technology for successful eradication of bacteria is the photodynamic process. This talk summarizes the potential of photodynamic decontamination of foodstuff and decolonization of porcine skin as a novel approach for improving hygiene standards as well as to safe clean water in the future. Summary In general photodynamic decontamination of living surfaces like plant food or porcine skin is possible. However successful eradication of pathogens (killing efficacy of > 99.9%) depends on the constitution and geometry of the living object.

Food safety challenges are present in modern life in many ways. The whole supply chain from harvesting, processing, storage and reaching consumers are full of contaminations possibilities. The problem causes a significant food los and waste. It is estimated today that about 30 % of the produced food never gets to the people. Besides that fact, we have today many examples of fresh food contamination like meet, chicken and fish, putting in risk the consumers. To control, in part, the situation, the abusive use of toxic substances, antibiotics and excess of acid water has been used causing a server for health and for environment. In this presentation we shall discuss the challenges ahead in this area and how photo reaction and photodynamic reactions are able to help to minimize the problems. Examples of project for fresh vegetables decontamination as well as for meat will be described in this presentation. The discussion about modification of food tast, composition or other characteristics will be presented as well as a study of the dynamic involving microorganism inactivation. Work supported by FAPESP, CNPq and CAPES – all Brazilian agencies.

Despite tremendous progress in biomedical science, the number of reported food-borne diseases continues to rise. Health experts estimate that every year food-borne illnesses in USA cost 86 billion dollars. Obviously, existing antibacterial technologies for microbial control of foods are not enough effective. Photosensitization is a treatment involving the interaction of the two non-toxic factors, photosensitizer and light, which in the presence of oxygen results in the destruction of the target cell without leakage of harmful by-products in the environment. According to our results, chlorophyllin (food additive E140) exhibits perfect photosensitizing properties. After excitation with light (405 nm) it inactivates food pathogens, their spores and biofilms, yeasts/microfungi. ROS-induced oxidative stress and following membrane damage was the main reason of photosensitization- based inactivation of microorganisms. Afterwards we applied photosensitization for microbial control of fresh produce. Obtained results indicate that this treatment significantly (2-3 log CFU/g) reduces microbial load on fruits (strawberries, apricots, plums), vegetables (cauliflower, cucumber, lettuce, basil) and sprouts without thermal effects on food matrix. Moreover, this treatment extended the shelf-life of treated produce by 2-4 days what is economically very important. No reduction of nutritional value (antioxidant activity, chlorophyll content) or organoleptic properties (color, texture, taste) of treated produce has been observed. In order to decontaminate fresh produce from Gram (+) and Gram (-) pathogens in uniform way, chlorophyllin was conjugated with chitosan. Obtained data reveal that such photoactivated conjugate is very effective against all pathogens and can be applied for coating of fresh produce. Therefore, a photosensitization phenomenon might open a new avenue for the development of non-thermal, effective and ecologically friendly antimicrobial technology for preservation of fresh produce.

Streptococcus mutans is recognized as the major causative agent of dental caries, especially in fissures and on smooth surfaces. Thus, the purpose of this study was to evaluate the susceptibility of S. mutans (ATCC 25175) biofilm to photodynamic therapy after sensitization with chlorin e-6 (PS) at 0.6 and 2.5μM by exposure to red light at 660nm (BioTable®) under 36.1mW, 15J/cm2 and 5 min of pre-irradiation time. Biofilm was induced in BHI broth supplemented with 1% sucrose for 7 days in a 96 well-plate. Serial dilutions were seeded onto brain heart infusion agar to determine viability in colony-forming units per milliliter (CFU/mL). Additionally, the metabolism of the biofilm by XTT and confocal laser scanning microscopy (CLSM) was performed using BacLight LIVE/DEAD system. Different groups were analyzed: L-D- (negative control), L-D+ (drug), L+D- (light), L+D+ (PDT) and clhorexidine at 0.2% (positive control). Results were analyzed by two-way ANOVA and Tukey’s test (p<0.05). Biofilms were taken for observation at confocal microscopy, and qualitative analyzes were obtained as to the distribution of non-viable viable/cells using LIVE/DEAD® Baclight™ before and after treatments. For CFU/mL and metabolism, the data were submitted to analysis of variance (ANOVA) and Tukey's test at 5% significance. It was observed that there was statistically significant difference in all PDT and chlorhexidine Groups compared to negative control and light Group (p<0.05). No dark cytotoxicity was observed (p>0.05). PDT using chlorin e-6 as photosensitizer can be an adjunct and effective method to control Streptococcus mutans biofilm responsible to dental caries.

Different processing technologies have been applied by the food industry to control food spoilage and disease causing microorganisms. Due to changing consumer expectations for fresh and safe food, there is a need for alternative processing technologies that are clean and green with no harmful residual effects. Light acting on some natural plant compounds produces reactive oxygen species which are lethal to some pathogens and this phenomenon of photosensitization would be an attractive alternative treatment to chemical preservatives for the decontamination of food and extending shelf life of fresh produce. Curcumin is a natural polyphenolic compound from the spice turmeric (Curcuma longa L.) and has been identified as an efficient photosensitizer for inactivation of a broad range of food related microorganisms. Curcumin-based photosensitization has shown promise in extending the shelf life of fresh date fruit, oysters, salads and other minimally processed foods. It has been very effective against pathogenic bacteria such as Escherichia coli O157:H7, Listeria monocytogenes and Vibiro parahaemolyticus. It has significantly reduced the Aspergillus flavus conidia, hyphae and carcinogenic fungal toxin aflatoxin B1 in maize kernels. The promising results obtained from the application of photoactivated curcumin in food systems suggest that this treatment could be an efficacious and cost-effective method to inactivate a broad range of food borne pathogens.

This study evaluated Curcuma longa, Citrus lemon, Hamamelis virginiana and Hypericum perforatum available on the market as photosensitizers (FS) in Antimicrobial Photodynamic Therapy (aPDT). Each FS were analyzed in a spectrophotometer between 350 and 750 nm, to determine the ideal light source. Once the absorption bands were determined, 3 light sources were selected to determine the concentration of use, the compounds were verified at different concentrations on bovine dentin samples to evaluate the risk of staining. Once the concentration was determined, FS were evaluated for cytotoxicity on fibroblast culture. Each compound was then irradiated with each light source and evaluated for indirect reactive oxygen species (ROS) production. The bacterial reduction was tested in culture of E. faecalis in planktonic form and in biofilm using energy of 10 J and Energy Density of 26 J/cm2. The tested compounds had light absorption in three bands of the visible spectrum: violet (405 nm), blue (470 nm) and red (660 nm). At 1:6 concentration, none of the compounds promoted tooth staining, as none of the compounds showed significant toxicity in cells or bacterial suspension. In addition, when the compounds were irradiated at each wavelength, significant ROS production was observed. When the aPDT was performed on the planktonic and biofilm bacteria, significant microbial reduction was observed for both cases, reaching up to 5Logs of reduction. In conclusion, extracts of Curcuma longa, Citrus lemon, Hamamelis virginiana and Hypericum perforatum, showed potential for use in aPDT as photosensitizing agents.

Aminolevulinic acid (ALA) is a prodrug that is converted in the heme biosynthesis pathway to protoporphyrin IX (PpIX) for photodynamic therapy (PDT) and fluorescence-guided tumor detection and resection. Although ALA-based therapy has been clinically used for the treatment of various types of tumors, clinical outcomes of ALA applications are not satisfactory due to issues such as low tumor PpIX production, high PpIX fluorescence heterogeneity, and low tumor to normal fluorescence contrast. We argue that a personalized ALA-based therapy would overcome these limitations and result in enhanced therapeutic outcomes. Since PpIX is endogenously produced in the complex heme biosynthesis pathway composed of 4 cytoplasmic and 4 mitochondrial enzymes and subject to cell membrane transporters, ALA-based therapy needs to be tailored to tumor phenotypic and genotypic characteristics that affect tumor PpIX production and accumulation. We found that genetic alterations in heme biosynthesis enzymes in tumor cells could cause significant changes in ALA-PpIX production. In these tumors, a low dose of ALA was able to achieve better PpIX fluorescence contrast between tumor and normal cells than a high dose of ALA that is commonly used. For tumors with elevated transporter activity, combination of ALA and a clinical transporter inhibitor is necessary for increasing ALA-PpIX fluorescence and reducing PpIX fluorescence heterogeneity. Overall we hope to demonstrate that a personalized ALA protocol optimized to fit tumor phenotype and genotype offers better treatment outcomes than applying ALA based on a one-size-fits-all approach.

Photodynamic therapy (PDT) is one of cancer treatments using a specific photosensitizer and a particular type of light. There have been many trials to improve the effectiveness of the therapy. Immunotoxin (IT), on the other hand, has been developed as a class of highly potent biopharmaceuticals designed as a molecular targeted therapy for the treatment of malignant tumor. However, the internalization of IT into cytoplasm remains as the big issue. In order to overcome this issue, NG-PDT is developed which target the cancer specific membrane protein by IT in combination of PDT. The authors challenges to develop more effective next generation-PDT. We present here an example of NG-PDT using a saporin-conjugated antibody against the tumor specific membrane protein, Robo1, and a photosensitizer which generates reactive oxygen by light to destruct endocytic membranes. Though the treatment with IT only showed little effect on cancer cells examined, the addition of light illumination augmented the cytotoxic effect several tens of times. By longer exposure, we observed a significant effect even on a cancer cell line which has ten times less Robo1 expression. Thus the results suggested that the NG-PDT widen the therapeutic measure both in the specific drug delivery and in the drug target.

Background: Patients with centrally located early lung cancer (CLELC) are often heavy smokers with a considerably high risk of multiple lung cancer (MPLC) lesions; treatment strategies for such patients must preserve the cardiopulmonary function. Methods: Between June 2013 and June 2018, 10 patients with CLELC underwent photodynamic therapy (PDT) using NPe6, second-generation photosensitizer at Nippon Medical School Hospital. Among these patients, we retrospectively analyzed MPLC which were treated by surgery plus PDT or PDT alone, and examined the effectiveness of PDT and we propose a treatment strategy for patients with MPLC. Results: Eight patients underwent surgery (five lobectomies and three partial resections) for primary lung cancer and then underwent NPe6-PDT for the treatment of secondary primary CLELC, two patients were performed PDT alone. In 6 of these 10 patients, CLELC was found as metachronous lesions and in 4 patients as synchronous lesions using sputum cytology and a bronchoscopical examination using autofluorescence bronchoscopy. All 10 lesions were CLELC and identified by autofluorescence bronchoscopy. Among the 10 patients with MPLC including peripheral-type lung cancers which were resected by surgery, all 10 CLELC lesions exhibited a complete response after PDT. Conclusions: For lung cancer patients with a long-term history of smoking, careful follow-up examinations after surgical resection are needed considering the incidence of metachronous primary lung cancers. PDT can play an important role for the treatment strategy for MPLC.

Plantar warts, infected by human papillary virus, occur in the heel, metatarsal head or metatarsal. Presently, the methods of treating plantar warts include cryotherapy, local injection of pingyangmycin, etc. However, these treatments have many problems such as severe pain, poor transdermal absorption and failure to reduce recurrence rate. In clinical practice, we found that for some refractory warts, surgical curettage combined with photodynamic therapy can be an ideal effect.

As salvage surgery after failure of chemoradiotherapy (CRT) for esophageal cancer shows high morbidity and mortality, curative and less invasive salvage treatment has been needed. Photodynamic therapy (PDT) is a candidate for local failure after CRT. Taraporfin sodium (Leserphyrin) is the new generation photosensitizer which was made in Japan, and has an advantage of low skin toxicity because of rapid clearance. We conducted multi-institutional trial to evaluate the safety and efficacy of PDT using taraporfin sodium for patients with histologically proven local failure limited within the muscularis propria after 50Gy or more CRT for esophageal cancer. The PDT procedure commenced with intravenous administration of a 40mg/m2 dose of talaporfin sodium followed by diode laser irradiation at a 664nm wavelength. We set the primary endpoint as local complete response (L-CR) per patients. In this study, 26 patients were enrolled and all were treated with PDT. Twenty three patients were assessed L-CR after PDT; the L-CR rate per patient was 88.5 % (95% CI: 69.8%－97.6%). No skin phototoxicity was observed only with two weeks’ sun shade, and no grade 3 or worse non-hematological toxicities related to PDT were observed. At the timing of all enrolled cases were followed 3 years or longer, the median local progression free and overall survival time were 3.1 and 4.2 years, respectively. PDT using new generation photosensitizer and a diode laser is a safe and potentially curative salvage treatment for local failure after CRT for patients with esophageal cancer.

Imaging technologies such as Ultrasound, OCT, MRI and CT are useful for diagnostics and tomographic assessment of therapy response. Fluorescence imaging with its high sensitivity is a promising approach and is extensively used for lesion localization, surgical guidance and monitoring response to therapies. While conjugating fluorophores to antibodies improves specificity, further conjugation of drugs provides a dual function, where fluorescence monitoring may simultaneously reveal drug pharmacokinetics. Fluorescence imaging, however, is limited by low penetration of light, which can be partially overcome by photoacoustic imaging (PAI). In this study, by conjugating a fluorophore and photoacoustic molecule to an antibody (Dual Function Antibody Conjugate (DFAC)), we evaluate whether PAI can significantly improve deep-tissue imaging. To provide a systematic comparison of these imaging modalities we developed a DFAC, comprising of Cetuximab (anti-EGFR antibody) conjugated with a fluorophore (AF647) and a photoacoustic dye (IRDye800) in a 1:2:2 ratio. We hypothesize that, conjugating quantifiable probes to an antibody, would provide information about different depths within the confines of optical approaches. Such quantification is particularly important in photodynamic therapy, for determination of tissue concentration of photosensitizers and in chemotherapy for quantification of drug concentrations non-invasively. The relationship between the photoacoustic and fluorescence signals from the DFAC is demonstrated through spectroscopic techniques and their EGFR specificity along with deep tissue photoacoustic quantification is established using EGFR positive/negative cell lines and tissue mimicking phantoms. The DFAC, presented in this study, demonstrates a combination of two complimentary imaging modalities for non-invasive determination of pharmacokinetics and in vivo drug quantification.

Photodynamic Therapy (PDT) has recently gain attention as alternative treatment of Central Nervous System (CNS) cancer diseases, due to the demonstration of successfully elimination of gliomas in patients. The implementation of PDT for brain tumors, and especially glioblastoma (GBM), has already been approved in some countries. Due to their superb light absorption and photostability conjugated polymer nanoparticles (CPNs) are promising photosensitizers (PS) for use in PDT. Recently, we developed metallated porphyrin-doped CPNs for PDT and demonstrated that they were effective eliminating glioma cells trough ROS-mediated photoinduced damage. A problem of many therapies used to eradicate brain gliomas is the difficulty of arrival and preferential accumulation of the active drug into the tumor upon systemic administration due to the selective permeability of the blood-brain barrier (BBB). To solve this problem our approach employs mononuclear cells, which can cross BBB and infiltrate tumors, as stealth carriers for drug delivery into brain tumors. In this study loading of CPNs into monocytes/macrophages was demonstrated and the cellular functionality, chemotaxis and penetration of these loaded monocytes/macrophages into GBM spheroids (3D tumor models) was tested. CPNs loading was successfully achieved using human monocytes THP-1 and mouse bone marrow-derived monocytes (BMdM) without disturbing cell viability and differentiation potential towards macrophage state. CPNs-loaded monocytes were found to better infiltrate spheroids as compared to CPNs. Furthermore, PDT efficacy on GBM spheroids was improved when using our monocyte-mediated delivery strategy

Introduction: Fluorescence-guided surgery could potentially reduce local recurrence after pancreatic cancer resection. However, the ideal contrast agent for this purpose is not yet determined. The monoclonal antibody cetuximab targets the EGFR receptor, which is overexpressed in 64% of pancreatic cancers. We investigated the efficacy of near-infrared emitting silver sulphide Quantum Dot (QD)-cetuximab nanoconjugates for targeting EGFR-positive pancreatic cancer. Methods: 2-Mercaptopropionic acid-coated QDs were prepared from AgNO₃ and Na₂S. Pancreatic cancer cell lines PANC-1 and CFPAC-1 were confirmed EGFR-positive using a commercial AlexaFluor488-cetuximab probe. Nonconjugated QD and cetuximab-conjugated QD (QD-cetuximab) toxicity was assessed after 24 and 48 hours using MTT assay. Fluorescence microscopy was performed following a) formaldehyde-fixed immunofluorescence and b) live staining with QD-cetuximab for four hours at concentrations corresponding to 0, 10, 50, 100, 200, 400 and 600μg ml-1 of silver. Results: Untargeted QDs were non-toxic in both cell lines after 48 hours at all investigated concentrations, whereas QDcetuximab was toxic at 100µg ml-1 after 24 hours in PANC-1 and at 10µg ml-1 in CFPAC^-1. Fixed immunofluorescence demonstrated EGFR targeting by QD-cetuximab at concentrations of 50μg ml^-1 upwards in both cell lines. Live staining demonstrated similar efficacy of EGFR targeting up to 50μg ml^-1 , although a reduction of fluorescence at higher concentrations was observed when compared to fixed immunofluorescence. Conclusion: Silver sulphide QD-cetuximab nanoconjugates have the potential to target live EGFR-positive pancreatic cancer cells at doses of up to 50 μg ml^-1 . The reduction in QD fluorescence observed at higher concentrations is likely to be secondary to cetuximab toxicity.

While many have contributed to the field of ‘photodynamic therapy’, Thomas J. Dougherty played the central role. Without his efforts, it seems unlikely that PDT would have advanced much beyond being a medical curiosity. This summary discusses both his many contributions to the field and the wide range of discoveries made possible by his pioneering studies that brought PDT to the attention of the funding agencies.

Though my area of expertise (synthetic organic chemistry) is different from those of Tom Dougherty, we nonetheless managed to actively collaborate on several PDT projects through the intermediacy of Professor Ravi Pandey. This brief talk will concentrate on those projects that we published jointly, and on my memories of Tom, a great scientist, and an even greater friend.

This Conference Presentation, "Much more than an impactful cancer scientist: Tom Dougherty as a graduate student advisor, mentor, advocate, and friend," was recorded at the International Photodynamic Association World Congress 2019 in Boston, Massachusetts, United States.

The effect of PDT on the host immune response and the role the host immune response plays in PDT efficacy against cancer has been the subject of intensive research for the past 2 plus decades. During that time we have learned that 1) the patient’s immune health can affect the efficacy of PDT; 2) PDT can both enhance and suppression immunity; 3) treatment regimens can be devised to enhance anti-tumor immunity; and 4) PDT has the potential to be an effective immunotherapy. This presentation will focus on how these findings came about and the role Dr. Dougherty played in the discoveries.

Since 1978 the initial research on PDTand PDD using canine lung cancer model and then clinical research of early stage cancers of lung, esophagus, stomach and cervix was performed with hematoporphyrin derivative and argon dye laser. A lot of meetings on PRT which was called initially, mainly in USA, Late in 1980's IPA was founded during the International Meeting of Clinical Application of PRT in Tokyo. After the extended clinical trials of PDT on early stage cancers by the support of Government in 1980' Japanese Government approved PDT with Photofrin and argon dye laser or excimer dye laser for clinical use in 1992. A New photosensitizer of Laserphyrin ( Chrolin e6) wad developed after the preclinical investigation by Prof. David Kessel. This photosensitizer showed less skin photosensitibity than previous Photofrin.

This Conference Presentation, "Tom Dougherty and the FDA approval of PDT photosensitizers," was recorded at the International Photodynamic Association World Congress 2019 in Boston, Massachusetts, United States.

Standard chemoradiation often enriches drug-resistant tumor cell populations that can lead to recurrent and treatment-refractory disease. For instance, preclinical models of several cancers suggest that the cancer stem cell subpopulation becomes enriched and re-populates the tumor milieu following conventional therapies. Here, we show evidence that photodynamic therapy (PDT) is effective against several patient-derived glioblastoma stem cell cultures. Moreover, sub-lethal PDT results in re-sensitization of cancer cell phenotypes with induced drug-resistance to chemotherapy. This talk will also introduce some of our efforts to establish a new program aimed at developing personalized and targeted PDT to overcome drug-resistance.

Glioblastoma is a highly aggressive and common brain cancer in adults with a grave prognosis, and aggressive radio and chemotherapy provide only a 15 months median survival. We evaluated the tolerability, and efficacy of the Ruthenium-based photosensitizer TLD-1433 in the formulation with apo-Transferrin (Rutheriin®) in the RG2, rat glioblastoma model. The specific tumour uptake ratio, PDT threshold, of the RG2 rat glioblastoma models and normal brain in vivo were determined as well as the survival post-PDT and the extent of CD8+T cell infiltration post-PDT. Results were compared with those obtained by 5-ALA-induced PpIX mediated PDT in the same animal model. As both photosensitizers have different photophysical properties, the number of absorbed photons required to achieve an equal cell kill is compared during in-vitro and in vivo studies. A significantly lower absorbed energy was enough to achieve LD50 with Rutherrin® versus -PpIX mediated PDT. Rutherrin® provides higher selective uptake ratio (SUR>20) in RG2 tumours compared to normal brain, whereas the SUR for ALA-induced PpIX was 10.6 in the same tumour model. To evaluate the short-term tissue response in vivo enhanced T2-weighted MR images provided the spatial extent of edema, which is twice post PpIX-PDT versus Rutherrin®-PDT suggesting reduced non-specific damage typically associated with a secondary wave of neuronal damage. A significant survival increase was observed in Rutherrin® treated rats bearing RG2 versus PpIX-PDT for the selected treatment conditions, associated with an increased CD8+T cell infiltration in the tumours. Rutherrin®-PDT was well tolerated providing safe and effective treatment of RG2 -induced glioblastoma.

Background and Objective Glioblastoma (GBM) is a malignant brain tumor with a median overall survival of approximately 15 months with the current standard of care (SOC). Although it is a rare neoplastic disease with low prevalence (0.3/10,000 persons), it remains the most frequent primary malignant brain tumor in adults. Currently, there is still no therapeutic option to prevent GBM recurrence and total resection is rarely feasible because of tumor cells infiltrating the surrounding brain. Thus, adjuvant therapies to improve local control are highly expected. 5-ALA photodynamic therapies have been reported with promising results. We present here an ongoing clinical trial (INDYGO) to evaluate 5-ALA PDT delivered intraoperatively to treat newly diagnosed GBM. Materials and Methods Our group has introduced a specific light applicator to deliver PDT in the surgical cavity early after maximal resection. 5-ALA PDT is delivered in combination with the SOC recommended by the current guidelines and enabling to simultaneously investigate the potential synergistic effects. Results and Discussion Between May 2017 and June 2018 ten patients have been enrolled. Currently, therapy has been delivered without significant toxicity or adverse event and are fulfilling the primary endpoint of this feasibility study. Secondary endpoints still being under investigation are progression-free survival, overall survival and patients' quality of life. Conclusions Finally, after the feasibility and the absence of adverse effects, multicentric, parallel-group, randomized controlled trial (RCT) is planned to assess the efficacy of 5-ALA PDT for the treatment of newly diagnosed GBM.

Background: 5-aminolevulinic acid (5-ALA) induces red fluorescence in malignant brain tumors that has been used for intraoperative guidance for tumor removal. Positron emission tomography with 11C-Methionine (Met-PET) is a promising imaging modality to depict clear boundary of infiltrating glioma. We studied the correlation between preoperative Met-PET uptake and intraoperative 5-ALA fluorescence. Method: Patients who underwent preoperative Met-PET study and tumor removal using 5-ALA over a period of two years were analyzed. The regional uptake of Met-PET was expressed as the ratio of the maximum of standardized uptake value (SUV-max) to the contralateral normal brain. 5-ALA fluorescence from tumor sample was immediately measured during surgery using blue laser and spectrometer. Fluorescence intensity was categorized into four groups (None, Weak, Moderate, Strong) with ten-hold differences. Results: 16 cases with pathological diagnosis of astrocytic tumors were analyzed. Met-PET uptake was markedly high in “Strong” fluorescence group (3.81±0.77). Particularly, among 11 newly-diagnosed cases, the difference was statistically significant (p=0.017, Kruskal-Wallis test). "Strong" fluorescence group still showed significant difference when compared with other groups together, in all cases and newly-diagnosed cases (p=0.01 and p=0.004, respectively, Mann- Whitney U test). Discussion/Conclusion: There are limited number of reports regarding 5-ALA and Met-PET, stating that Met-PET and 5- ALA fluorescence should be considered separately. However, the fluorescence was grossly distinguished whether positive or negative. Our study utilized objective spectroscopic measurement of fluorescence intensity. The results suggested that strong fluorescence intensity induced by 5-ALA reflects high Met-PET uptake. This could indicate the importance of bright fluorescence by 5-ALA in glioma surgery.

This talk will introduce spatiotemporal synchronization of photodynamic therapy (PDT) combined with molecular targeted therapies to suppress multiple treatment escape pathways in parallel. We newly developed photoactivatable multi-inhibitor nanoliposomes (PMILs) for photodynamic tumor cell and microvessel damage in concert with photo-initiation of tumor-confined, multikinase inhibitor release. The PMIL is a biodegradable delivery system for precision medicine comprised of a nanoliposome carrying a photoactivable chromophore in its bilayer. A multikinase inhibitor-loaded PEG-PLGA nanoparticle is encapsulated within the liposome, which acts a barrier to nanoparticle erosion and drug release. Following intravenous PMIL administration, near infrared irradiation of tumors triggers PDT and initiates tumor-confined drug release from the nanoparticle. Preclinical data in mouse models of pancreatic cancer demonstrates that this concept facilitates suppression of the VEGF and MET signaling pathways—both critical to cancer progression, metastasis and treatment escape. Remarkably, a single PMIL treatment using low doses of a multikinase inhibitor (less than 1/1,000th of the standard cumulative dose) achieves sustained tumor reduction and suppresses metastatic escape, whereas combination therapy by co-administration of the individual agents compromises spatiotemporal synchronization and has significantly reduced efficacy. The PMIL concept is amenable to a number of molecular inhibitors and offers new prospects for precise and efficient use of potent but toxic multimolecular targeted therapies, reducing systemic drug exposure and associated toxicities.

The reactive oxygen species (ROS)-mediated mechanism is the major cause underlying the efficacy of photodynamic therapy (PDT). The PDT procedure is based on the cascade of synergistic effects between light, a photosensitizer (PS) and oxygen, which greatly favors the spatiotemporal control of the treatment. This procedure has also evoked several unresolved challenges at different levels including (i) the limited penetration depth of light, which restricts traditional PDT to superficial tumors; (ii) oxygen reliance does not allow PDT treatment of hypoxic tumors; (iii) light can complicate the phototherapeutic outcomes because of the concurrent heat generation; (iv) specific delivery of PSs to sub-cellular organelles for exerting effective toxicity remains an issue; and (v) side effects from undesirable white-light activation and self-catalyzation of traditional PSs. In this talk, the current status and the possible opportunities of nanomedicine for ROS generation for cancer therapy will be discussed in detail.

Over the past few decades, considerable attention has been given to improving the photoactivity and biocompatibility of hydrophobic photosensitizing drugs for light-activatable biomedical applications. It is increasingly clear that photosensitizing biomolecules, based on chemical conjugation or association of photosensitizers with biomolecules (e.g., lipids, polymers, antibodies, and Pluronic), strongly influence the performance of a given photosensitizer in biological environments. However, the numerous studies that have revealed PSBMs are not readily comparable as they cover a wide range of macromolecules, evaluated across a range of experimental conditions. Here, we prepared and characterized a series of well-defined PSBMs and pure drug crystal based on a clinically used photosensitizer—benzoporphyrin derivative (BPD). Our results illuminate the variable trafficking and end effects of clinically relevant PSBMs and BPD nanocrystals, providing valuable insights into methods of PSMB evaluation as well as strategies to select PSMBs based on subcellular targets and cytotoxic mechanisms. More importantly, these results demonstrate that biologically-informed combinations of PSBMs and carrier-free photosensitizers to target multiple subcellular organelles may lead to enhanced therapeutic effects in gliomas.

We examined the efficacy of Indocyanine Green (ICG) loaded plasmonic gold nanorods (AuNP) assisted combined photothermal and photodynamic therapy for laser induced choroidal neovascularization (CNV) in pigmented mice. Eight C57BL/6 wild type pigmented mice were divided into two treatment groups. Each mouse received multiple 532 nm laser induced CNV in both eyes. Control mice were untreated while treatment group received intravenous injection of ICG loaded gold nanorods on day 7 prior to single near-infrared (NIR 808 nm) laser treatment session. For assessing progression of CNV lesion volume, in-vivo ocular imaging was performed on each animal at day 1, 7, and 14 using optical coherence tomography volumetric imaging. Early and late phase fluorescence angiography was performed for CNV leakage analysis. Infrared reflectance imaging and were used to analyze in-vivo laser injury and healing assessment. ICG-AuNP assisted combined photothermal/photodynamic therapy resulted in significantly increased healing and decreased fluorescein leakage at the CNV site compared to control mice who did not receive NIR laser treatment. Enhanced permeability and retention of AuNP assists in localization of therapeutic agents to the CNV lesion, which allows for targeted therapy while minimizing laser induced damage to healthy tissues.

The present study is aimed at the development of drug-in-cyclodextrin-in-liposome (DCL) nanoconstruct by coupling two independent delivery systems: cyclodextrin/mTHPC inclusion complexes and liposomal vesicles to improve the transport of mTHPC to the target tissue and to strengthen its intra-tissue accumulation in the tumor. Liposomes offer an excellent opportunity to achieve selective drug, targeting what is expected to prevent local irritation and reduce drug toxicity. Сyclodextrins (CDs) have been utilized as independent carriers for improvement of pharmaceutical properties such as solubility, stability, and bioavailability of various drug molecules, including mTHPC. Therefore, we assumed that encapsulation of CD-complexed drug into liposomes might increase drug loading capacity, entrapment efficiency, may restrain the dissociation of drug-CD complexes and prolong its systemic circulation. DCL nanoparticles have been prepared with various compositions to optimize the structure aiming to alter more favorably the distribution of temoporfin in tumor tissue. To enhance the encapsulation efficiency, double loaded DCLs, which include mTHPC in lipid bilayer along with (CD-mTHPC) inclusion complexes in the inner aqueous lumen, were prepared. It was demonstrated that DCLs possessed higher serum stability compared with conventional mTHPC liposomes (Foslip). In fine, we showed that the presence of serum in the medium less affected cellular uptake of mTHPC delivered by double loaded MDCL compared with Foslip.

The potency of a photosensitizer is conveniently measured in vitro in terms of its EC50 (extracellular photosensitizer concentration needed to cause 50% cell kill in vitro upon illumination). The potency in vivo may likewise be defined in terms of the drug dose required to elicit an effect of a given intensity. The quest for increased potency drives medicinal chemistry to optimize molecular structures, and in PDT led to literally hundreds of new photosensitizers more potent than Photofrin® . Photosensitizers with EC50 in the sub-nanomolar range are at hand. Nevertheless, only very exceptionally more potent photosensitizers succeeded in clinical trials and reached commercialization. One of the reasons for the resilience of less potent photosensitizers is that photosensitizers in general have large “phototherapeutic” windows and the use of higher doses to compensate for lower potency is not necessarily associated with an exacerbation of adverse reactions. Although pursuing the development of more potent photosensitizer systems may seem to be futile when drug doses can be safely increased, it is shown that higher potency can be an advantage in applications where the dosage is limited, such as in topical applications. This work distinguishes the potency of a photosensitizer from its efficacy, i.e., the maximum effect that it can reach without toxicity in the dark. The molecular properties that contribute to the potency and to the efficacy of photosensitizers are discussed with special emphasis on porphyrins, chlorins and bacteriochlorins.

This paper, originally published on 7 August 2019, was withdrawn at the request of the authors on 12 August 2019.

Porphyrins and porphyrin-derivatives have been described as successful PSs for PDT, some of them being approved for clinical use. Trying to advance in the development of PDT PSs, other chromophoric scaffolds (phtalocyanine, fullerene, BODIPY, etc.) have been reported as potentially interesting to develop advanced systems able to overcome some of the drawbacks. In this context, BODIPY have revealed as valuable dyes for the development of new PSs due to their outstanding chemical and physical features such as high absorption coefficients in the Vis region, high resistance to photobleaching low dependence of the photophysical signatures with the environmental conditions (e.g., solvent polarity), as well as small size and high lipophilicity, facilitating biological-membrane penetration. On the other hand, the high chemical versatility of the BODIPY core, amenable by the rich well-known BODIPY chemistry, allows exhaustive and selective modification of the chromophoric BODIPY core (BODIPY functionalization), making possible the fine modulation of photophysical properties. This work focuses on the contributions of our research group on the application of novel BODIPYs as singlet oxygen photosensitizers from the two perspectives that are commonly considered as effective (1) Attaching heavy atoms (mainly iodine and bromine, but also transition metals) to the BODIPY and (2) orthogonal BODIPY dimers. Moreover, the accessible fine chemical modulation of BODIPY properties makes possible to balance 1O2 photogeneration and fluorescence, which is interesting to develop PDT + bioimaging theranostic agentsas well as to modulate other physical and chemical properties interesting for the searched photo-biological activity (e.g., water solubility and membrane permeability Finally, BODIPYs with functional groups of interest to be anchored to different nanocarriers are also described.

Ruthenium and osmium-based photosensitizers (PS) are compounds of interest for use in photodynamic therapy (PDT). These PS’s can be activated by light wavelengths in the range of 400-675 nm, which can be selected based on the tumor environment, treatment area, and available light sources. The objective of this study was to explore these PS’s for the treatment of several relatively aggressive cancer cell lines. A human adenocarcinoma cell line (A549) was treated with ruthenium-based compounds at concentrations of 1, 5, or 10 uM, followed by light treatment of 93 J/cm2 at either 532 nm or 630 nm. Similarly, osmium-based compounds were used to treat A549, murine melanoma (B16F10) and squamous cell carcinoma (SCCVII) cell lines at concentrations of 0.05, 1, or 3 uM, followed by light treatment of 93 J/cm2 at 630 nm. Cells survival was assessed 24 hours after PDT treatment using either alamarBlue or MTT cell viability assays. In-vitro MTT viability assays revealed that ruthenium-based compounds activated with 630 nm light showed high SCCVII cell toxicity at 5uM. AlamarBlue assays have shown that ruthenium based compounds activated by 532 nm light show high A549 cell toxicity at 1uM in-vitro. Osmium-based compounds showed optimal PDT-mediated cytotoxicity in SCCVII, A549, and B16F10 cell lines at a concentration of 1uM activated by 630 nm light, while exhibiting minimal dark toxicity. The Ruthenium and Osmium-based compounds are potentially potent PSs against lung, melanoma and squamous cell carcinoma cells, in-vitro.

Lung cancer has high incidence and mortality rates worldwide and so there is strong need for the development of novel therapies. Photodynamic therapy (PDT) is a photochemotherapeutic cancer treatment that utilizes a photosensitizer (PS) drug that, when activated by laser light at a specific wavelength, yields reactive oxygen species, which in turn induces cell death. However, due to the passive diffusion of PSs, normal surrounding cells are sometimes affected and their targeted concentrations in cancer cells tends to be minimal, thus limiting the effectiveness of this treatment. Therefore, a multicomponent drug targeting strategy is often applied to improve PS specific delivery and concentration in cancer cells, which in turn can improve the effectiveness of PDT. The intention of this study was to improve the PS drug delivery of Zn(II) Phthalocyanine tetrasulfonic acid (ZnPcS₄) in lung cancer cells, by enhancing its chemical structure. ZnPcS₄ was successfully conjugated to pegylated gold nanoparticles in order to maximize its solubility and stability, as well as bound to specific active tumour-associated antibody-antigens (Cetuximab: Anti-EGFR1 Ab) to aid specific targeted PS delivery. Within in vitro cultured lung cancer cells, this molecular drug delivery system noted improved and specific sub-cellular location of ZnPcS₄. Furthermore, after conducting in vitro PDT experiments, a significant amount of cell death and cytotoxicity was found. Overall, this nano immunotherapy drug conjugation combination of ZnPcS₄ with AuNP and Cetuximab, proved to enhance concentrated PS uptake in lung cancer cells and so improve PDT treatment outcomes for this form of cancer.

Re-excision due to inability to visualize positive margins following breast-conserving surgery is a significant clinical challenge. 5-aminolevulinic acid (5-ALA), a non-fluorescent prodrug, leads to intracellular accumulation of fluorescent porphyrins in tumor cells. This single-centre Phase II randomized controlled trial evaluated the clinical safety, feasibility and diagnostic performance of a new handheld fluorescence imaging device (PRODIGI) combined with 5-ALA hydrochloride (HCl) for intraoperative visualization of invasive breast carcinomas. Fifty-four patients were enrolled in the study and randomized to receive no 5-ALA HCl or orally administered 5-ALA HCl (15 mg/kg or 30 mg/kg BW). Forty-five patients (n = 15/group) were included in the analysis. Fluorescence imaging of the excised surgical specimen was performed and biopsies were collected from within and outside the clinically-demarcated tumor border for blinded histopathological analysis. In the absence of 5-ALA HCl, tissue autofluorescence imaging lacked tumor-specific fluorescent contrast. Administration of 5-ALA HCl resulted in tumors that fluoresced bright red with improved visualization of tumor contrasted against normal tissue autofluorescence. In the 15 mg/kg 5-ALA dose group the positive predictive value (PPV) for detecting tumor inside and outside the grossly demarcated tumor border was 100.0% and 55.6%, respectively and 100.0% and 50.0% respectively in the 30 mg/kg dose group. No drug or device-related adverse events were observed and technical feasibility and clinical integration of this intraoperative tumor visualization approach were confirmed. This is the first known clinical report of visualization of 5-ALA HCl-induced fluorescence in invasive lobular and ductal breast carcinoma using a real-time handheld intraoperative fluorescence imaging device.

THIS IS FOR THE SESSION "CAPABILITIES OF 5-ALA" The accumulation of the fluorescent photosensitizer protoporphyrin IX (PpIX) after systemic administration of 5-ALA proved high tumor selectivity and led to intra-operative fluorescence guidance during resection of malignant glioma. 5-ALA therefore also promises to be useful for PDT of malignant glioma. Preclinical investigations resulted in the establishment of an appropriate treatment planning strategy and treatment parameters. This can be outlined as follows: interstitial placement of cylindrical diffuser fibers (1 to 4 cm diffuser length) by stereotaxy after careful planning on CT/MRI scans, such that fibers are approximately 1 cm apart and as close as 3-4 mm from the contrast uptaking tumor border. Aim of the irradiation parameters (200 mW/cm diffuser length for 1 hour) is to reach >95% photobleaching of PpIX in at least one optical penetration depth (approx. 3 mm) from the fibers. The therapeutic penetration depth can be expected to reach significantly deeper and kill glioma cells within parts of the infiltration zone, which are no more highlighted by Gd-contrast uptake. So far, inoperable de novo and recurrent malignant glioma with sizes up to 3 cm in diameter proved amenable in clinical investigations. Intriguing longterm progression free and overall survivals led to the design of prospective clinical trials, which are expected to start recruitment in 2019. Further research should focus on individualizing treatment parameters, further improve PpIX accumulation, including the sensitization of cancer stem cells and explore the role of immune stimulation by PDT, including the possible synergistic effect of immune checkpoint inhibitors.

SBI Pharmaceuticals Co., Ltd. (SBI) is a pharmaceutical authorization holder of the orally available 5-aminolevulinic acid (5-ALA) in Japan for visualization of glioma and bladder cancer. SBI provides 5-ALA as a research material and makes mutual collaborations with academic institutions. We examined four published studies of 5-ALA-FD (5-ALA fluorescence guided detection) conducted in Japan. 5-ALA was orally administrated at 20mg/kg body weight or 1g per patient at 2-4 hours before surgery or diagnosis. Bladder cancer: The presence of CIS (carcinoma in situ) affects recurrence of non-muscle-invasive bladder cancer. Additional CISs were detected and diagnosed only by 5-ALA-FD in 9 out of 60 patients and they were classified as high risk group. Peritoneal metastasis of gastric cancer: The addition of 5-ALA-FD to conventional laparoscopy enabled us to identify usually invisible peritoneal metastases in 5 of 24 patients with advanced gastric cancer. Pancreatic cancer: Peritoneal nodules suspected to be peritoneal metastases were observed under white light in 9 of 34 patients. The nodules in 4 of 9 patients showed fluorescence by 5-ALA-FD and were histopathologically diagnosed as metastases. Lung cancer: Fluorescence was confirmed in 14 of 14 PL1-PL3 patients and 10 of 24 PL0 patients. PL0 patients showed red fluorescence were preoperatively diagnosed as stage PL1. Accurate intraoperative diagnosis for visceral pleural invasion in lung cancer was performed by 5-ALA-FD. Four studies indicate that 5-ALA-FD is useful for distinction of tumors such as small lesions and contributes to selection of an appropriate therapy without a non-curative resection.

Background and Objective Chronic and infected leg ulcers (LUs) are painful, debilitating, resistant to antibiotics, immensely reducing patient's quality of life. The purpose of the new clinical research project was to demonstrate the the efficacy of photodynamic therapy (PDT) as an alternative to antibiotic therapy for the treatment of infected chronic LUs. Patients and Methods PDT using irradiation at fluency 80 J / cm2 and 20% 5-aminolevulinic acid topically applied for 4 hrs, was performed during 10 days of hospitalization in 10 patients both sexes aged 40-85, with chronic leg ulcers, who had not responded well to conventional treatment. This was a randomized, placebo-controlled trial (local Octenidine dihydrochloride). The numerical scale of pain was used. Treatments were carried out at 3-week intervals. Result During 8 months follow up, complete remission (CR) was obtained in 4 patients (40%), partial response (>50% reduction in ulcer diameter), in 3 patients (30%), no response in 1 patients (10%). In two patients (20%) was observed deterioration of the local condition, with swelling, erythema and inflammation. Conclusion PDT allowed healing of chronic leg ulcers through the antimicrobial effect, modulation of inflammatory process and improving the oxygenation of ulcer tissue with a reduction in healing time and improving scarring results with the reduction of pathological bacterial culture. Further studies on the larger group of patients are needed to confirm the benefit role of PDT in the treatment of LUs.

A total of 62 female patients who underwent ALA-PDT at the Gynecology Department of Henan Province People's Hospital in 2018 were retrospectively assessed. Lesions included 24 case of LSIL, 13 case of HSIL, 12 case of VAIN, 2 case of VIN and 11 case of VL. Three-six sessions of ALA-PDT were carried out at intervals of ten days. The treatment efficacy was evaluated by colposcopy, TCT and HPV dectection. After one month follow-up, The effective rate of LSI, HSIL, VAIN, VIN and VL were 70.8%, 84.6%, 83.3%, 100% and 100%. After three month follow-up, The effective rate of LSIL, HSIL, VAIN, VIN and VL were 83.3%, 92.3%, 83.3%, 100% and 100%. After six month follow-up, The effective rate of LSIL, HSIL, VAIN, VIN and VL were 87.5%, 92.3%, 91.7%, 100% and 100%. Our experience show that ALA-PDT is an effective treatment modality for female genital precancerous lesions. Keywords: 5-aminolevulinic acid photodynamic therapy, Low-grade cervical squamous intraepithelial lesions,

For patients failing standard Bacillus Calmette-Guerin based immunotherapy for Non-Muscle Invasive Bladder Cancer (NMIBC), PDT may delay or prevent cystectomy. A Phase IB clinical trial evaluated the feasibility and safety of TLD1433, a novel Ruthenium coordination-complex as photosensitizer (PS) for PDT. The clinical trial combined PS instillation for one hour and the use of strongly attenuated green (525nm) light to reduce PDT caused damage to the bladder wall. The low and high PS doses were defined as 0.35mg and 0.7mg TLD1433 per cm^2 bladder surface (N=3 each) and 90+/-9J/cm^2 as target radiant exposure on the bladder wall. The PS concentration in the urine and blood at 24hrs post instillation was below 1ng/ml indicating rapid drug clearing. In all patients, the average target radiant exposure was attained as verified by irradiance sensors in the bladder. The average measured irradiance was ~ 15mW/cm^2, never exceeding 35mW/cm2 at the sensor positions. At 30 days post-treatment, all patients receiving the low PS dose tolerated the procedure well with no grade 3, 4 or 5 AEs. Three patients were then treated at the Therapeutic Dose, again with no grade 3, 4 or 5 AEs, and an identical pharmacokinetic profile to the half dose. At half dose, all patients had recurrent, but no progressive NMIBC noted at the 180-day cystoscopy. At therapeutic dose, 2 of 3 patients were tumour-free at the 180-day cystoscopy. Moderate bladder irritability was reported at full dose which primarily resolved within 90 days.

The surgical management of peritoneal metastases, whatever their origin, raises the problem of the potential involvement of the entire peritoneal serosa. Peritoneal metastases are surface lesions, sometimes numerous, extensive, and they may be theoretically accessible to photodynamic therapy (PDT). Intraperitoneal applications of PDT are limited by the photosensitizers lack of specificity, that should explain side effects and complications observed in the past, and by the difficulties to bring light into the cavity. Past clinical evaluations had demonstrated the risks associated with this technique (capillary leak syndrome, anastomotic leakages), and no trial evaluating PDT for peritoneal metastases treatment was published since 2006. Recent data may contribute to give a new breath for this technique. Indeed, new targeted molecules are being developed, and the optimization of illumination systems makes possible the implementation of PDT by minimally invasive approaches (laparoscopy). Immunotherapy also brings new arguments to promote PDT in this indication. Indeed, it is unrealistic to think that surgery alone can ensure satisfactory microscopic cytoreduction quality and the application of light uniformly over the whole peritoneal surface cannot be reasonably considered. Thus, the expected abscopal effect of PDT could enhance a systemic immune response leading to better control of residual lesions on the one hand, and also peritoneal and tissue recurrences at a distance. The objective of this review is to present the most recent data and possible approaches for the treatment of peritoneal carcinomatosis by PDT.

The clinical management of cervical low grade squamous intraepithelial lesions (low-grade squamous intraepithelial lesion, LSIL) is a hot point at present. Topical photodynamic therapy as a noninvasive and targeted therapy has been used in the treatment of lower genital tract infections disease. This study aim to investigate the clinical efficacy of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) for cervical LSIL with high risk human papillomavirus (HPV) infection.

It is estimated that up to 75% of the sexually active population is infected with this human papillomavirus virus (HPV). Condylomas acuminate are benign lesions caused by this virus. This study aims to compare photodynamic therapy (PDT) and administration of 70% trichloroacetic acid (TAA) in the condylomas. Each patient is treated weekly until total removal of the lesions, with one of treatment options. In the patients treated with PDT, a Brazilian 20% methyl aminulevulinate cream were applied in the region and incubated by 3 hours and the lesions were illuminated with the CERCa®. This Brazilian system is composed of LEDs emitting at 630 nm which was initially designed for the treatment of cervical intraepithelial neoplasia (CIN) and this study showed the need to develop new devices for illumination of the anus-genital region. During the illumination, a total dose of 150 J/cm2 is delivered over 21 minutes. 22 patients have participated of this clinical study, totaling 15 patients treated with TAA and 8 with PDT, with a complete treatment for 33% using TAA and 75% for PDT. Besides the results of complete response, it is important to highlight that 2 patients showed recurrence for TAA and 4 patients discontinued the treatment with TAA, while, for PDT, there were no cases both recurrence and withdrawal. Therefore, besides the preliminary results obtained with this project show that PDT has been more effective than TAA, it was possible to plan and develop new equipment to ensure greater range of light.

The beginnings of photodynamic therapy date from 1900 but mechanisms relating to photokilling were unknown until 1991 when Oleinick’s group first described PDT-induced apoptosis. Routes to apoptosis have been determined along with the role of sub-cellular photodamage as a factor in initiation of cell death. We have recently reported on the role of paraptosis, another death mode that can be operative in cells with an impaired apoptotic program. It appears that the sensitivity of malignant cell types to lethal photodamage can be magnified by careful selecting of sub-cellular targets. In this report, recent progress relating to potential routes to improved PDT responses is described.

Exploiting differences in photosensitizer (PS) localization and mechanisms of action with sequential or simultaneous activation protocols has been shown to improve photodynamic therapy (PDT) efficacy. Various sub-cellular, cellular and stromal components can be targeted, causing selective photodamage. Previous reports have shown that rationally targeting non-overlapping tumor compartments or sub-cellular sites considerably enhances outcomes from PDT. The current presentation describes the benefits of simultaneously targeting lysosomes and mitochondria/endoplasmic reticulum using lipid-anchored and entrapped liposomal preparations of benzoporphyrin derivative, respectively, with an emphasis on results in 3D models of ovarian cancer.

Breast cancer (BCA) in women is a leading cause of mortality and morbidity with distant metastases occurring in ~40% of cases. Here, as an alternative to ionizing radiation therapy and chemotherapy and their associated side effects, we explored a new combination approach using capecitabine (CPBN) and aminolevulinate-based photodynamic therapy (PDT). We have recently shown that a combination PDT approach in which 5-fluorouracil (5FU), a differentiation-promoting agent, increases the levels of PpIX in cancer cells when given as a neoadjuvant prior to ALA. However, 5FU can be toxic when administered systemically at high levels. We reasoned that CPBN, a known chemotherapeutic drug for BCA and less toxic than 5FU (because CPBN is metabolized to 5FU specifically within tumor tissues), might work equally well as a PDT neoadjuvant. Murine 4T1 BCA cells harboring a luciferase transgene were injected into breast fat pads of female nude mice. CPBN (600 mg/kg/day) was administered by oral gavage for 3 days followed by intraperitoneal ALA administration and PDT with red light (633 nm) on day 4. Tumor growth and regression were monitored in vivo using bioluminescence imaging. Histological changes in primary tumors and metastases were assessed after necropsy. CPBN pretreatment of 4T1 tumors increased cellular differentiation, reduced proliferation, raised PpIX levels, enhanced tumor cell death, and reduced metastatic spread (incidence and tumor load) of 4T1 cells post-PDT, relative to vehicle-only controls. The use of CPBN as a non-toxic PDT neoadjuvant for treatment of BCA represents a novel approach with significant potential for translation into the clinic.

Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to drive the production of an intracellular photosensitizer, protoporphyrin IX (PpIX), are in common clinical use. However, the tendency to accumulate PpIX is not well understood. Patients with cancer can develop recurrent metastatic disease with latency periods. This pause can be explained by cancer dormancy. Here we created uniformly sized PC-3 prostate cancer spheroids using a 3D culture plate (EZSPHERE). We demonstrated that cancer cells exhibited dormancy in a cell density-dependent manner not only in spheroids but also in 2D culture. Dormant cancer cells accumulated high PpIX levels and were sensitive to ALA-PDT. In dormant cancer cells, transporter expressions of PEPT1, ALA importer, and ABCB6, an intermediate porphyrin transporter, were upregulated and that of ABCG2, a PpIX exporter, was downregulated. PpIX accumulation and ALA-PDT cytotoxicity were enhanced by G0/G1-phase arrestors in nondormant cancer cells. Our results demonstrate that ALA-PDT would be an effective approach for dormant cancer cells and can be enhanced by combining with a cell-growth inhibitor.

The use of endoscopic optical coherence tomography (OCT) in pulmonary medicine has shown significant promise for aiding in the detection and diagnosis of airway pathology, in monitoring disease progression over time, and in assessing dynamic microstructure and function in vivo. Recent technical advances including enhanced resolution and contrast together with novel sophisticated catheter designs to improve usability are key contributing factors to the increased clinical utility of OCT in pulmonology. To increase the clinical utility of OCT for pulmonary medicine catheters have been designed to navigate the complex pulmonary anatomy, to aid in guiding biopsies, and to provide improved imaging capabilities over conventional OCT catheters. Flexible OCT needle catheters have been designed to confirm the needle position within the targeted nodule and tissue region of interest prior to biopsy. OCT image interpretation criteria to enable the differentiation of nodules from lung parenchyma and also for diagnosing pulmonary pathology relevant to lung cancer have been established and adopted by the College of American Pathologist, and clinical studies are now being conducted to evaluate the feasibility of OCT guided biopsy in vivo. While work is ongoing, we anticipate that in the future OCT guided biopsy may increase the diagnostic yield of bronchial biopsy by not only confirming that the targeted nodule has been reached, but also additionally providing the bronchoscopist with a tool to assess nodule composition and identify regions most likely to yield diagnostic tumor.

Abstract

In the area of neurosurgical tumor diagnosis, clinical research on photodynamic diagnosis using 5-aminolevulinic acid (5-ALA) has already been undertaken, and the safety of 5-ALA has also been demonstrated. 5-ALA is approved in the diagnosis of brain tumor and bladder tumor by the Ministry of Health, Labour and Welfare. 5-ALA is a natural amino acid contained in a living body and is a precursor of hemoglobin. When absorbed into the body by oral ingestion, porphyrin synthesis by heme synthesis is specifically performed in a tumor-specific manner, and accumulation of fluorescent protoporphyrin IX (pp IX) is observed. However, pp IX is metabolized and disappeared in normal tissues. When tumor tissue is irradiated with excitation light, at the site where pp IX accumulates, it exhibits red fluorescence. We applied 5-ALA for photodynamic diagnostic in thoracic malignancies.

Background: Multimodal treatment associating surgery (pleurectomy/decortication, P/D) then IV adjuvant chemotherapy (platinum/pemetrexed) is an effective therapeutic option for some selected malignant pleural mesothelioma (MPM) patients. Intra-operative pleural photodynamic therapy (iPDT) has emerged as a promising option to improve this multimodal treatment outcome (Friedberg J, Ann Thorac Surg. 2017). The MesoPDT trial (NCT02662504) aimed at assessing the feasibility of such procedure outside the only two US expert centers performing multimodal treatment including iPDT to date. Methods: A single-center pilot clinical trial was designed to assess the feasibility of iPDT protocol in Lille University Hospital. A pool of maximum six patients was expected in order to apply the iPDT protocol, and to assess its applicability and safety outside US center expert. Results: In 2016-2017, four consecutive assessable patients were included and treated per protocol, reaching the study achievement cut-off. iPDT specific procedures have been applied and managed in partnership with US experts. The safety profile was favorable. The main and most specific adverse event was acute lung injury occurring within 72 hours after iPDT, which may lead to reversible respiratory distress, manageable with adequate intensive care. The 4 patients achieved the full scheduled protocol. Conclusion: The iPDT multimodal treatment for MPM is applicable and manageable in a European expert center, involving local skills and dedicated teams. The safety profile of the iPDT in Lille center was favorable, as validated by an external board. Median overall survival was promising (≈28 months), similar to previous US results. Our center is expected to join soon a large phase II randomized, multicentric US trial assessing MPM multimodal treatment (P/D, chemotherapy) ±iPDT (NCT02153229; UPENN, USA).

The report of patients with lung cancer who could receive PDT combined with other modalities like surgery and chemotherapy is relatively rare. Combination of PDT and surgery is useful for multiple lung cancers (MPLC) or minimally invasive procedure to reduce resection line to peripheral site of superficial invasion in advanced lung cancer. Also, PDT combined with chemotherapy for advanced lung cancer with central airway stenosis seems to be usefull for local control and improvement of patient’s QOL. MPLCs were noted in 22 (34.4%) of 64 patients treated with PDT using Laserphyrin (synchronous:10, and metachronous: 12). Among them, 10 patients (synch:2, meta:8) underwent surgery (lobectomy: 9, pneumonectomy: 1) for peripheral-type lung cancer as their first primary lesion followed by PDT for central type early stage lung cancer. CRs were achieved after PDT in all patients, and all patients were alive. We performed induction PDT to reduce resection line for 28 patents. Among them, histological type revealed squamous cell ca. in 24. Superficial invasion was recognized in trachea: 2, carina: 3, main bronchus or 2nd carina: 22. Reduction surgery could be successfully performed in 23 of 28 patients (82%). PDT combined with chemotherapy was performed for 12 consecutive patients with 13 advanced NSCLC whose stages were IIIA–IV. The median stenosis rates before treatment, 1 week, and 1 month after treatment were 60%, 15%, and 15%, respectively. All patients improved symptoms and QOL after treatment. The mean survival time was 5.9 months, and the overall one-year survival rate was 30.0%.PDT combined with other modalities may be a promising strategy in lung cancer treatment.

Operation, chemotherapy and radiotherapy are typical treatments of lung cancer today. Curative tumor resections are still the mainstream treatment for early-stage small peripheral lung cancer. However, non-surgical treatments such as trans-bronchoscopy ablation, including bronchoscopy microwave ablation and bronchoscopy PDT, are also potential options. Several ongoing clinical trials have revealed that using electro-magnesium bronchoscopy (EMB) system in hybrid OR suit is feasible when performing PDT. Although having technique difficulties, repeating bronchoscopy PDT per operation is also conceivable for larger tumor. However, the diameter of light penetration is still around 1.5 cm, which limits the widely use of PDT treatment for peripheral lung tumor. In 2009, Dr. Friedberg injected castor oil into the bronchus and the result was very similar as using optic fiber. But castor oil is not suitable for clinical practice due to severe lipoid pneumonia complication. It is found in our study that Lipiodol (FDA-approved) could replace Castor oil as the light diffusor agent. The refractive index (1.49) is similar to castor oil (1.47). We also found that dissolving Indole-3-acetic acid (IAA) in Lipiodol could create a new drug, which can be used as light scattering agent and oxidant as a novel photodynamic therapy method. We have run a pig model to test this theory. The living pig was sacrificed after 1 month and lung injury condition pathology examination prove that no significant injury is noted even at the maxima light energy level(800J). The feasibility of our novel method has been proved and needed further studies.

Photochemical internalization (PCI) is a technology to enhance intracellular drug delivery by light-induced translocation of endocytosed therapeutics into the cytosol. The aim of this study was to explore the efficacy of PCI-based delivery of bleomycin and the impact on systemic anti-tumor immunity. Mouse colon carcinoma cells (CT26.CL25), stably expressing the bacterial β-galactosidase, were inoculated into the legs of athymic or immuno-competent BALB/c mice strains. The mice were injected with the photosensitizer AlPcS2a and bleomycin (BLM) prior to tumor light exposure from a 670 nm diode laser. Photochemical activation of BLM was found to induce synergistic inhibition of tumor growth as compared to the sum of the individual treatments. However, a curative effect was not observed in the athymic mice exposed to 30 J/cm2 of light while more than 90% of the thymic mice were cured after exposure to only 15 J/cm2 light. Cured thymic mice, re-challenged with CT26.CL25 tumor cells on the contralateral leg, rejected 57-100% of the tumor cells inoculated immediately and up to 2 months after the photochemical treatment. T-cells from the spleen of PCI-treated mice were found to inhibit the growth of CT26.CL25 cells in naïve thymic mice with a 60% rejection rate. The results show that treatment of CT26.CL25 tumors in thymic mice by PCI of BLM induces a systemic anti-tumor immunity.

Photodynamic therapy (PDT) has been used clinically for the treatment of head and neck cancer. The effectiveness of PDT is often strongly dependent on fluence rate. Targeted photo-immunotherapy (PIT) may reduce the adverse effects of non-targeted PDT. The in-vivo distribution of the anti-EGFR targeted conjugate Cetuximab-IRDye700DX was investigated. Vascular and tumor responses were determined with respect to fluence rate. Intra-vital confocal microscopy of skin-fold chambers with the EGFR-overexpressing OSC-19 tumor showed peak tumor fluorescence 24 hrs after administration. Tumor to normal ratio was 3.1±1.6 (n=8). Tumor vascular responses were determined by imaging rhodamine-dextran extravasation. Two hrs after illumination (24 hr DLI, 100 J.cm-2 at 50 mW.cm-2) showed no leakage in 3 of 4 animals and stasis in 1. Normal vasculature showed mild to severe constriction of larger vessels up to 48 hrs after illumination. Subcutaneous OSC-19 tumors were transdermally illuminated with 100 J.cm-2 at 20, 50 and 150 mW.cm-2. Control tumors took 5.3±1.1 days to grow to 200%. All animals treated with 20 mW.cm-2 showed no tumor up to 90 days post treatment (n=4) compared to 1 of 4 in the 50 and 150 mW.cm-2 groups. The remaining tumors reached 200% after 17.9±5.2 and 19.5±7.4 days. Crust formation of the overlying skin was observed at low fluence rate. Cetuximab-IRDye700DX showed significant tumor to normal ratio. Normal tissue responses like vascular effects and crust formation of the skin was observed and may be caused by conjugate still present in the circulation. The effect of targeted-PIT is strongly dependent on fluence rate.

Aminolevulinate-based photodynamic therapy (ALA-PDT) is effective for actinic keratosis (AK), but when administered in a conventional regimen, PDT can elicit pain during illumination that is very uncomfortable for patients. In conventional PDT, a 1-4 hr preincubation period occurs after ALA application, prior to the start of illumination. In a new regimen called metronomic PDT (mPDT), which simulates daylight PDT, blue light (405 nm) is delivered concurrently with ALA application and thereby provides a treatment that is not only painless but also nearly as effective as conventional PDT for AK lesion clearance. In this investigation, a murine AK model, generated by repeated UVB exposure, was treated with either mPDT or PDT. Lesion clearance was monitored via area measurements, and samples were harvested for assessment of biological mechanisms. Compared to pretreatment (100%), the average lesion area decreased to 47% and 32% in PDT, and to 57% and 40% in mPDT at 1 and 2 weeks after PDT, respectively. Relative to untreated controls, we observed enhanced cell death (by H&E staining, apoptosis, and TUNEL assay), generation of Reactive Oxygen Species (ROS, by CM-H2DCFDA staining), and autophagy (by Atg 5 and Atg 7 expression) in both the PDT and mPDT samples. Cleaved (activated) Caspase-3 was specifically observed only in PDT samples. Immunomodulation by inflammatory cells was indicated by enhanced infiltration/retention of neutrophils and macrophages in mPDT samples. Our results suggest that mPDT can be just as effective as conventional PDT for treatment of AK, but the mechanisms may be quite different.

The lack of tumor specificity by photosensitizers often hampers the clinical application of photodynamic therapy (PDT). Delivery of photosensitizer to tumor based entirely on the enhanced permeability and retention effect could lead to nonspecific uptake of photosensitizer by normal tissue, causing adverse effect in patients. Accumulating evidence showed that the combination of antibody immunotherapy and PDT can complement each other to produce effective cancer-targeted photoimmunotherapy. IRDye 700DX (IR700) is a water-soluble phthalocyanine-based photosensitizer that can produce reactive oxygen species (ROS) efficiently upon light irradiation. Cadherin-17 (CDH17) is a cell surface antigen commonly overexpressed in gastrointestinal cancers. By conjugating IR700 to anti-CDH17 humanized monoclonal antibody (ARB102), the conjugate could target CDH17 positive cancer cells and achieve selective accumulation to induce potent cytotoxicity upon light irradiation.

Clinical protoporphyrin IX (PpIX) fluorescence imaging was conducted using a pre-validated, non-invasive imaging system (Dyaderm, Biocam, Germany) during routine methyl aminolevulinate (MAL)-PDT treatment of 172 patients with licensed dermatological indications (37.2% actinic keratosis, 27.3% superficial basal cell carcinoma and 35.5% Bowen’s disease). Linear and logistic regressions were employed to model any relationships between variables that may have affected PpIX accumulation and/or PpIX photobleaching during irradiation and thus clinical outcome at three months. Patient age was found to be associated with lower PpIX accumulation and photobleaching, however only a reduction in PpIX photobleaching appeared to consistently adversely affect treatment efficacy. Clinical clearance was reduced in lesions located on the limbs, hands and feet with lower PpIX accumulation and subsequent photobleaching adversely affecting the outcome achieved (OR: 0.5 (0.2, 0.9; p<0.05). If air cooling pain relief was employed during light irradiation, PpIX photobleaching was significantly reduced (p < 0.05) and this resulted in an approximate three-fold reduction in the likelihood of achieving clinical clearance (OR: 0.4 (0.2, 0.7; p<0.01). PpIX accumulation and photobleaching are therefore concluded to be important indicators of dermatological MAL-PDT treatment success and anything that adversely effects them has the potential to reduce treatment efficacy. PpIX photobleaching during the first treatment was found to be an excellent predictor of clinical outcome across all lesion types and non-invasive imaging of PpIX fluorescence during MAL-PDT continues to provide important treatment insights that can be utilised to improve treatment protocols and thus clinical outcomes.

Photodynamic therapy (PDT) is an established treatment for actinic keratosis (AK). The conventional approved PDT protocol in Europe (C-PDT) involves red-light photoactivation at irradiances higher than 60 mW/cm² , making the treatment painful. Several clinical studies have reported similar efficacy and better tolerability when using red-light photoactivation at lower irradiances. The aim of the study was to investigate whether there is a minimum irradiance threshold for red-light photoactivation above which there is no further improvement in efficacy. A photodiode sensor connected to a power meter was used to measure the irradiance delivered to 114 AKs on the scalp and forehead of 19 patients during C-PDT using the Aktilite CL 128 (Galderma SA, Switzerland). The widely ranging measured irradiances, resulting from the heterogeneous photoactivation over the treatment area provided by the Aktilite CL 128, were cross-referenced with the clinically evaluated complete responses (CR) at 3 months. The 66 AKs in CR at 3 months received an average irradiance of 30.9 mW/cm² (standard deviation: 16.7 mW/cm² ) compared to 33.3 mW/cm2 (standard deviation: 17.9 mW/cm² ) for the 48 AKs in incomplete response. No significant effect of the irradiance on the CR at 3 months was found (odds ratio for a 6 mW/cm² -unit change, 0.96; 95% confidence interval, 0.83 to 1.10; p=0.53). No minimum irradiance threshold could therefore be determined in the considered irradiance range. A red-light device enabling homogeneous irradiation at a lower irradiance than the Aktilite CL 128 may therefore provide similar efficacy and higher treatment tolerability than C-PDT.

Port-wine stains (PWS) are congenital vascular malformations, consisting of ectatic capillaries and post-capillary venules in the papillary and mid-reticular layers of the dermis. PWS progressed slowly increased with age in 70% patients, the color deepened, the thickness increased, the plaques and nodules formed. PWS mainly occurs in the face and neck regions, which may influence psychological well-being and physical impact. Pulsed dye laser (PDL) is the standard treatment for PWS with excellent cosmetic outcome without a scar. Although red PWS shows good therapeutic effect with PDL, purple and hypertrophic PWS show tolerance which is associated with depth and diameter of vessels. At present, subjective evaluation method show little stable, commonly used to evaluate the efficacy of the treatment of PWS by different therapy. Spectrophotometric can detect skin color changes before and after the treatment, naming bleaching rate calculation. Reflectance confocal microscopy RCM, optical coherence tomography (OCT) can be used to observe the change of blood vessels, but most of departments do not have spectrophotometric, RCM and OCT. We used dermascopy, VISIA, doppler ultrasound and two-dimensional (2D) ultrasound to evaluate treatment efficacy of PWS. Hypertrophic PWS showed reduction of blood flow by doppler ultrasound and attenuation of dermis and subcutaneous tissue by 2D ultrasound. Upon dermascopic examination, we found different types of PWS showed reduction of vessels and faded in color after treatment. In addition, VISIA could be used to calculate the mutative area of erythema by treatment. Using non-invasive evaluation technique could be effective and scientific to evaluate the curative effect.

The biologic mechanism of tissue ablation with Tookad soluble vascular targeted photodynamic therapy (TS-VTP) is well suited for treating tumors in sensitive organs such as the human genitourinary tract. Preclinical collaborative studies conducted at the Weizmann Institute and Memorial Sloan Kettering have provided insight for the development of TS-VTP as a treatment modality for prostate, kidney and urothelial cancers that are now being tested in prospective clinical trials. MSK trial 17-070 is a Phase 2b study of the efficacy of TS-VTP for the treatment of intermediate risk, unilateral prostate cancer. Men with suitable cancers are being treated with VTP therapy by interstitial technique through fiberoptic diffusers placed into the prostate gland under ultrasound guidance in order to illuminate a volume of targeted tissue based on tumor localization from prior biopsy. Tissue effects, tolerability and serum biomarkers of response are being monitored as important secondary endpoints. MSK protocol 18-140 is a Phase 1 clinical trial investigating the safety and tolerability of TS-VTP for the treatment of urothelial cancer involving the bladder and upper urinary tract. In this study, patients with transitional cell carcinoma found in the bladder, ureter or renal pelvis of the kidney are treated by endoluminal technique with fiberoptic diffusers placed in proximity to the tumors to achieve tumor ablative effects. The identification of adverse events is the primary goal of the study however tissue effects, tolerability and urinary biomarkers of response are also being monitored. Preliminary findings from both of these studies as well as other clinical trials in TS-VTP treatment will be presented.

Background: Most patients with esophageal and esophago-gastric junction cancers present with locally advanced or advanced disease. Dysphagia is the predominant symptom in 80-96% of patients and 42-46% experience significant weight loss due to luminal obstruction caused by the tumor. Esophageal stenting permits rapid relief of dysphagia, but is associated with pain, bleeding, migration, and food-impaction and has no impact on overall survival. Photodynamic therapy with porfimer sodium is approved in the USA for palliation of obstructing esophageal cancer but is rarely used due to prolonged skin photosensitivity. A new form of PDT using a Bacteriochlorophyll derivative (WST11) has rapid clearance, resulting in reduced skin photosensitivity and has gained interest because of animal studies suggesting that tumor destruction is associated with immune effects that can be augmented with selective immunosuppressants and checkpoint inhibitors. Methodology: We initiated a Phase 1 study to determine the maximally tolerated laser light fluence rate (mW/cm) of light exposure for VTP treatment of obstructing esophagogastric cancer using a fixed drug dose of WST11. This is a preliminary assessment of results. Results: Eight patients have been treated thus far, 3 at a fluence rate of 150 mW/cm, 3 at 200 mW/cm and 2 at 250 mW/cm. There have been no dose limiting toxicities thus far and only 3 serious adverse events consisting of 2 patients with tumor associated bleeding requiring blood transfusions, and one patient with chest pain and nausea requiring hospitalization and medical therapy for 24 hours. Visible signs of tumor necrosis have been observed at each fluence rate, with objective improvement in dysphagia in one patient at 150 mW/cm, 2 patients at 200 mW/cm and 1 of 2 patients treated at 250 mW/cm. Conclusion: Although it is still early in the study, luminal therapy with WST11 VTP seems to be safe and may provide partial tumor destruction with short-term relief of dysphagia in patients with obstructing esophago-gastric cancer.

Vascular targeted photodynamic therapy with TOOKAD-from local ablation to systemic cancer treatment Background: Local therapies that allow for safe ablation of primary lesions and trigger anti-tumor immunity with minimal side effects, may be particularly effective in management of early-stage cancer. Vascular-targeted photodynamic therapy (VTP) with TOOKAD®, recently granted EMA approval as a first-line treatment for localized prostate cancer, has been shown by us to also trigger anti-tumor immunity. Our recent preclinical and current clinical studies aim to test the hypothesis that synchronizing TOOKAD®VTP with immune-modulation that attenuates the pro-tumor immunity, will result in systemic micrometastases annihilation and a high cure rate. Methods: 4T1-Luc, adenosquamous JA and Met-Lu cells were orthotopically grafted in the mammary pad, esophagus and prostate of immune-competent mice and rats, respectively. Seven days postgrafting, tumors were treated with TOOKAD®VTP, with or without immune-modulators. Immune profiles were examined by 10-X genomics, FACS analysis and immunohistochemistry. Animal survival and lung and lower abdomen metastases counts were followed up for 3 months. Clinical protocols were composed based on the preclinical data and utilized in the clinical studies that will be described in other lectures of this session. Results: TOOKAD®VTP triggered anti-tumor immunity sufficient to achieve 60-90% cure in various cancer models. However, primary lesion annihilation did not prevent disease progression in the more aggressive cancers such as 4T1-Luc breast cancer. TOOKAD®VTP synchronized with metronomic administration of immune-modulators was associated with a 60-90% disease-free animals and resistance to re-challenge. The rate of success was highly pending on the exact synchronization of the immune modulating agents with the VTP timing. The required modulators match the deciphered immune profiles. This combined immuno-VTP therapy concept has been translated to clinical trials currently ongoing at Memorial Sloan Kettering Cancer Center. Conclusion: Application of TOOKAD®VTP to localized tumors combined with immune modulation appears to provide novel means to treat early disseminated cancers.

Vascular photodynamic therapy (PDT) with redaporfin was shown to destroy the primary tumor and to reduce the development of distant cancer lesions in animal models, thus suggesting anti-tumor immunity. Vascular-PDT with redaporfin triggers an acute local inflammation, neutrophilia and leads to an increase in CD4+/CD8+ T cells ratio, which has been correlated with increased survival rates in cancer patients, and further corroborates the activation of the adaptive immune system a few hours post-PDT. Previous experiments also confirmed the importance of some cell populations that are increased in response to PDT (CD4+ and CD8+ T cells expressing IFNγ,). Specific inhibition with monoclonal antibodies revealed significant decreases in percentage of survival of CD8+ T cells depleted animals, in comparison with PDT alone. To evaluate if combinatorial therapies in animal model would led to synergies between PDT and immunotherapy, we evaluated the therapeutic outcome of the combination between redaporfin-PDT and immune checkpoint blockers (ICB). ICB’s function as boosters of tumor immunity through reversing T-cell exhaustion and are an emerging immunotherapeutic modality. Our experiments with mice bearing CT26WT tumors revealed an improvement of redaporfin-PDT outcome when combined with anti-CTLA-4 antibody therapy, but no significant differences were observed with anti-PD-1. On-going experiments intend to clarify about the immune infiltrates variations in the tumor bed after combinatorial approaches, as well as to validate these results in a second animal model.

Photosensitizer Photolon, a complex of chlorin e6 and PVP, was originally developed by RUE “Belmedprepa, and registered in Belarus, Russian Federation, Kazakhstan and Ukraine. During the last 18 years Photolon was used clinically and proven to be effective in photodynamic diagnostics and treatment of different cancerous and benign conditions. Pre–marketing clinical trials of Photolon were conducted at 4 major oncology centers in Minsk, Obninsk, and Moscow and total of 112 patients were included. Open-label randomized non-comparative clinical trial confirmed safety and efficiency of PDT with Photolon for 154 patients with CIN grade I-III. Clinical trial of PDT with Photolon was carried out in 68 patients with disseminated skin melanoma. Clinical trial was conducted in 37 patients with central lung carcinoma. In 2006 we started an open-label randomized controlled clinical trial of Photolon applied intraoperatively in patients with primary and metastatic brain tumors, 102 patients were treated. In 2006 an open-label multicenter clinical trial of Photolon for treatment of choroidal neovascularization (CN) in age-related macular degeneration and myopic maculopathy was complete. 50 patients with CN were treated and positive therapeutic effect was maintained in 100% of patients within 1 year. Since 2001 more then 35 000 doses of Photolon were produced, and about 20 000 patients were treated. During this time there were no reports of adverse events or failed treatment with Photolon. Photolon was proven to be an extremely effective photosensitizer for PTD for multiple medical conditions and has minimal side effects.

Despite the advances of therapeutic approaches, morbidity and mortality rate of oral squamous cell carcinoma (OSCC) have not improved significantly during the last 30 years. Photodynamic therapy (PDT) represents a potential treatment modality for locally recurrent OSCC. However, treatment resistance attributed to PDT-induced epidermal growth factor receptor (EGFR) up-regulation was reported. Here, we showed that vandetanib, a multi-target tyrosine kinase inhibitor that inhibits EGFR and vascular endothelial growth factor receptor-2 (VEGFR-2) enhance the efficacy of PDT. First, we observed increased cytotoxicity in the combinatorial treatment that is attributed to impaired DNA double strand break (DSB) repair. We revealed significant down-regulation DNA-dependent protein kinase catalytic subunit (DNA-PKcs) expression to be the downstream target following inhibition of nuclear EGFR accumulation, therefore impairing non-homologous end-joining (NHEJ) following PDT. Next, the combinatorial treatment-mediated tumour vasculature shutdown and normalisation, coupled with reduction of PDT-induced EGFR activation corresponded to the most pronounced tumour growth delay in vivo. Interestingly, we observed the restoration of tumour cell proliferation and vascularisation, coupled with ERK1/2 activation in the recurrent tumours post-vandetanib+PDT treatment. This corroborated the importance of the modulation of the tumour microenvironment for the observed synergistic efficacy of vandetanib+PDT combinatorial treatment. Collectively, our data suggests that vandetanib works synergistically with PDT through the impairment of EGFR-dependent DNA repair machinery and modulation of tumour microenvironment.

The short lifetime and thereby the diffusion length of singlet oxygen as well as the correlation between photosensitizer (PS) fluorescence and treatment effects made it attractive in the 80-ties to analyze the intracellular localization of PSs to reveal the initial intracellular hit in PDT. Some PSs were found intracellularly in fluorescing granules that was found to be lysosomes and endosomes. Such vesicles were rupture upon exposure of the cells to light and the PS relocated to other compartments in the cells. About 40 different hydrolytic enzymes are found in late endosomes and lysosomes. Release of lysosomal hydrolases into cytosol has been documented to be a cytotoxic event. This suicide sac hypothesis originally postulated by De Duve suggests that released hydrolases lead to degradation of cellular constituents to such an extent that cellular functions are impaired and the cells subsequently die. However, it was found that a large fraction of the vesicles containing PSs could be ruptured without inducing substantial cytotoxicity. The scientific basis for this surprising observation as well as the potential utilization of the photochemical rupture of endosomes and lysosomes for intracellular delivery of various therapeutics, named photochemical internalization (PCI), will be discussed. During the last 20-30 years macromolecular therapeutics have become increasingly attractive for treatment of cancer due to their improved specificity and reduced side effects. Intracellular delivery of macromolecular therapeutics has however so far showed only limited success, a limitation that may be circumvented by PCI. The historic and scientific basis for PCI will be presented.

The Photochemical internalization (PCI) PCI technology can be used for enhancing the intracellular delivery of many different types of molecules with therapeutic activity or therapeutic potential. Clinical studies are on-going with the use of PCI for improving the effect of a cancer chemotherapeutic agent, as well as for evaluation of the potential of PCI for enhancing the effect of vaccination with peptide and protein antigens. The application of PCI in these areas will be discussed. In addition to the applications already in clinical development the PCI technology also has a large potential for the use in other therapeutic areas. Thus, in preclinical studies very interesting results have been achieved using PCI for the delivery of various types of nucleic acids, with especially interesting results for mRNA delivery. mRNA are molecules with a very interesting therapeutic potential if the current delivery problems could be solved, and pre-clinical results indicate that PCI may contribute to solving these problems for several potential uses of mRNA-based therapeutics. PCI has also shown very promising results with protein-based cytotoxic agents such as protein toxins and immunotoxins, as well as for drug delivery mediated by various types of nanoparticles. Recently, it has also been shown that PCI can enhance the intracellular delivery of certain antibiotic agents, with a potential to treat local infections with intracellular pathogens. Key pre-clinical findings in these areas will be presented, and current and potential clinical uses of the PCI technology will be discussed.

Cholangiocarcinoma (CCA) is a rare cancer, usually silent and presenting at late stage. Most common location is extrahepatic, perihilar or distal biliary adenocarcinoma. Liver function is critically affected, and biliary duct patency maintenance key for survival and life quality. Standard-of-care (SoC) in inoperable CCA (> 75%) is gemcitabine/cisplatin in 8 cycles with a response rate of <25%, and median progression free survival (PFS) and overall survival (OS) of approximately 8 and 12 months, respectively. The PCI approach is well suited in CCA; gemcitabine effects are clearly enhanced in preclinical models, and PhotoChemical Internalization can be triggered by targeted illumination during endoscopy (ERCP). In a multicenter phase I dose escalation trial with 16 patients in 4 dose cohorts (light energy and photosensitizer (fimaporfin; Amphinex®) + gemcitabine), the PCI procedure preceded standard gem/cis therapy up to 8 cycles. No dose-limiting toxicities or unexpected serious adverse reactions (SAE) emerged. The predominant SAE, cholangitis, had similar frequency, incidence pattern and severity reported previously. The two highest dose cohorts had >20% reduction in 17/19 target lesions, with 12 lesions becoming undetectable. The Duration of Response was > 12 and >15 months in the full study and highest dose cohort respectively; corresponding median OS ended at 14.4 and 21.7 months. An Extension study including a second PCI at cycle 5 demonstrated a similar safety profile. Based on these encouraging data, the RELEASE trial, a pivotal randomized trial comparing SoC chemotherapy +/- up to two PCI procedures in Europe and the United States is now starting recruitment.

The synergistic effects of PDT and chemotherapy are well established. however, this effect can be more than simple synergy if the correct balance of photosensitiser and chemotherapy agent is selected. We have also shown that by the careful selection of sensitiser, chemotherapy agent and light dose, there is minimal neurotoxicity which is a clear advantage over surgery in cancer therapy The concept of photochemical internalisation is explored in the previous talk. Here the clinical application is explored in a first in human study of PCI. These results plus some results of a follow up study are presented.

A central challenge in the treatment of different diseases is the delivery of therapeutic agents to a specific cellular site. Liposomes that can release their cargo upon an externally controlled trigger are attractive candidates for localized drug release. Light as external trigger can be controlled temporal and spatial with high precision. In this study, we investigate the potential of light sensitive liposomes with four different photosensitizers for light-induced release. To demonstrate permeabilization of the liposomes, we encapsulated calcein in high concentration inside liposomes, that calcein fluorescence is quenched. If calcein is released from the liposome, quenching is diminished and the fluorescence increases. We demonstrated that liposomes with the sensitizers Benzoporphyrine derivative monoacid (BPD), chlorine e6 (Ce6), Al(III) Phthalocyanine chloride disulfonic acid (AlPcS2) and a di-hydroxyphenyl porphyrine (5,10-DiOH) release cargo effectively after irradiation. Liposomes with 5,10-DiOH showed a quicker release compared to the other sensitizers. Further we observed through fractionated irradiation, that most of the release took place during light irradiation, while the permeability of the liposome decreased shortly after light exposure.

Photoactivatable Ru complexes are multifunctional compounds: (i) they have similar anticancer effects as cisplatin; (ii) they are photocleavable; (iii) they can sensitize the generation of singlet oxygen for photodynamic therapy. However, they are easily cleaned from blood stream, instable in biological environments, and may not efficiently inhibit tumor growth in hypoxic environments. To solve these problems, we design Ru-containing polymers for deep-tissue phototherapy. In this talk, I will present the synthesis of block copolymers (BCPs) that contain photoactivatable Ru moieties and their photo-controlled anticancer effect. First, we synthesized Ru-containing BCPs, in which Ru moieties act as photocleavable moieties, singlet oxygen sensitizers, and anticancer agents. Depending on the molecular weights and chemical structures, the BCPs assembled into micelles, vesicles, large compound micelles and other nanostructures. The BCP assemblies can circulate in blood stream, accumulate at tumor sites via the EPR effect, and be taken up by cancer cells. Red light passed through tissue and activated the Ru-containing BCP assemblies. The photoactivated Ru complexes and generated singlet oxygen inhibited the growth of cancer cells. We demonstrated anticancer activities of the photoactivatable Ru-containing BCPs in vitro and in vivo. In addition, we designed Ru-containing BCPs against hypoxic tumors. The BCPs, which contain photocleavable drug-Ru complex conjugates, self-assembled into micelles. The micelles are biocompatible and carried the conjugates into tumor cells. Subsequent red light irradiation induced intracellular release of the drug-Ru complex conjugates. Because the photoinduced release is oxygen-independent, the novel polymer provides a new platform for phototherapy against hypoxic tumors in vivo.

Hypothesis: ruthenium-based photo activated chemotherapy compounds are activated by photo substitution, which is independent from the presence of dioxygen. Hence phytotoxicity is kept in hypoxic cancer cells. Method: we have developed a setup to shine light on living cancer cells in a controlled atmosphere, with the O2 concentration comprised between 1 and 21%. We use this setup to study the influence of hypoxia on the dose-response curve of a series of ruthenium-based photo activated compounds. Results: with different ruthenium compounds the photo index remains indeed independent on the concentration of O2. However, the IC50 in the dark and after light irradiation are often higher under hypoxia, compared to normoxia. Conclusion: photo activated chemotherapy compounds are indeed promising for killing hypoxic cancer cells, but synergistic effects may be needed to obtain high photo indexes under hypoxia.

Primary Extramammary Paget’s disease (EMPD) is a rare cancer that mainly affects the genital region including vulvar and perianal areas. Without treatment, vulvar EMPD progresses and presents always more erythematous and pruritic plaques, which may become ulcerated and erosive. To control disease progression and symptoms usually experienced by patients, surgical excision is the mainstay of treatment. Unfortunately, even after large surgical excision with intra-operative margin control, recurrences are common [1]. For recurrent patients which undergo multiple resections, severe functional and sexual alterations are described. Only few data are available on the efficacy of alternative conservative treatments, such as laser ablation, radiotherapy, topical chemotherapy and photodynamic therapy (PDT) [2]. To date, none of them can be considered as a solid alternative to surgical excision yet [3]. Nonetheless, multiple clinical cases suggest that PDT could provide the opportunity to treat subclinical lesions, and give some relief on patient’s symptoms of the disease [4-6]. Unfortunately, the benefits of using photodynamic therapy for vulvar EMPD remains a challenge to demonstrate, because of the inhomogeneous illumination of vulvar and perianal areas, and the extreme pain that patients usually experienced during the illumination procedure that may lead to premature end of treatment [7, 8]. Resulting from the knowledge of previous works on actinic keratosis of the scalp treatment with PDT, light emitting fabrics (LEF) technology could address both of the hereinbefore described issues [9-12]. A new medical device based on LEF named PAGETEX dedicated to illumination of vulvar and perianal areas has been developed. The device delivers a homogeneous red light (635 nm) with a low irradiance during 2h30, for a total fluence of 12 J/cm². The PAGETEX device is being assessed in a clinical study (NCT03713203) which aims to establish PAGETEX- PDT efficacy and tolerability.

Light-responsive compounds can be utilized to control spatially and temporally the initiation of biochemical processes. This has the potential to improve the treatment of diseases, such as cancers, by controlling the location of drug activity at the site of disease rather than the drug acting systemically. This would reduce side-effects as well as the overall systemic dose of anticancer agents. Until recently light-responsive molecules that released active compounds in a stoichiometric manner required the use of short wavelength, high energy ultraviolet light (UV). These compounds cannot be used to treat disease because the required light wavelengths are readily absorbed by biological molecules preventing the light from penetrating tissue to an appreciable extent. Recently, we have a developed a platform technology that combines Vitamin B12 and a near infrared (NIR) absorbing fluorophore that converts an inactivated drug into an active form when exposed to NIR. In contrast to UV, NIR is poorly absorbed by biological tissues. Therefore, NIR penetrates tissue and can be used for photochemotherapeutic treatment of disease. In addition to targeting diseased tissue based on controlling drug activity by regulating light exposure, these compounds target cancer cells due to the Vitamin B12 moiety because rapidly dividing cancer cells have an increased demand for Vitamin B12 in comparison to normal, healthy cells. The reported technology could improve treatment of certain by diseases by affording effective treatment while reducing side effects.

During the past decades, a growing body of evidence clearly indicates that type I IFNs (IFN-1) play a pivotal role in naturally occurring and therapy induced immune responses to cancer. In this context, we describe here a novel effect of photodynamic therapy (PDT): besides its potential to induce apoptosis, PDT elicited an autocrine/paracrine activation of IFN-1 pathway. In the current work, B16-OVA cells were sensitized with Me-ALA-induced protoporphyrin IX (PpIX) which preferentially localized in the endoplasmic reticulum prior to irradiation. Subsequent photoactivation of PpIX with red-light irradiation ignificantly stimulated tumor cells to induce autocrine IFN-1 transcription, concurrently with IRF-3 phosphorylation, at levels that were capable of activating STAT1 and enhancing ligand receptor (cGAS) and ISGs (CXCL10, MX1, ISG15) expression. Among the cellular and molecular pathways identified so far, type I IFNs are critical components for the host immune response against tumor, more specifically for the dendritic cell (DC) compartment. In this sense, PDT-treated melanoma cells induced paracrine IFN-1-dependent phenotypic maturation of monocyte-derived dendritic cells (DCs) by enhancing co-stimulatory signals (CD80, MHC-II) and tumor-directed chemotaxis (transwell migration assay). Collectively, our findings strongly demonstrate the effects of a novel danger signal released by cancer cells undergoing PDT on the maturation and activation of DCs, highlighting the potential added value of PDT in adoptive immunotherapy protocols.

Therapeutic cancer vaccines work by boosting the immune system’s ability to defend the body from existing oncologic growth. Because of the well established characteristics of photodynamic therapy (PDT) to promote the immune response against targeted tumor, the standard direct PDT treatment of lesions in patients is often referred to as the vaccination in situ as the vaccine is generated in vivo exploiting antigens available at the tumor site. An alternative approach is the ex vivo generation of PDT vaccine, where surgically removed tumor treated by PDT in vitro serves as the vaccine material that is administered to the original host to eradicate residual malignant deposits. This second approach has certain advantages since it avoids certain inflammatory and other negative aspects of host response to PDT. Recent research progress has brought significant advances in the understanding of the nature of interaction of PDT with immune system. It is now clear that the critical determinant is the engagement of stress signaling networks by PDT-induced insult in targeted cancer cells. This ensures the expression of immunogenic cell death (ICD), abundant exposure of tumor antigens (including neoantigens) and espousal of damage-associated molecular patterns (DAMPs). Important advancements towards clinical translation of PDT vaccines include the identification of highly effective adjunct immunoadjuvants and strategies of controlling immunoregulatory activity. Both these elements are critical for optimizing the strength of vaccine-elicited antitumor immune response and its duration in order to ensure clinical efficacy.

In the fimaVacc technology photochemical internalization (PCI) is employed for enhancing cytotoxic T-cell responses to vaccination. Thus, in pre-clinical studies fimaVacc has been shown to increase MHC class I antigen presentation, leading to strongly enhanced cytotoxic- and helper T-cell responses to various types of vaccines. On the basis of promising preclinical results, a phase I clinical study with fimaVacc has been performed in healthy volunteers. FimaVacc involves formulating the vaccine with a photosensitising compound (fimaporfin) and a toll-like receptor (TLR) agonist. The vaccine is given as intradermal injections followed by illumination of the vaccination site. Results from a phase I clinical study in healthy volunteers will be presented. The subjects were vaccinated with models for peptide- and protein-based vaccines; HPV16 E7 peptide antigens and Keyhole Limpet Hemocyanin. Both antigens were formulated with the TLR3 agonist poly-ICLC (Hiltonol), and up to three vaccinations were be given. Local and systemic adverse effects will be assessed, and cellular and humoral immune responses were analysed by ELISPOT and ELISA assays, and by flow cytometry.

Nanobody-targeted photodynamic therapy (NB-PDT) has been developed as a potent and more tumor-specific approach compared to conventional PDT [1]. Interestingly, conventional PDT is able to induce immunogenic cell death, characterized by the exposure/release of damage associated molecular patterns (DAMPs) from dying cells, which can result in an anti-tumor immune response. In this study, we aim at understanding the first steps towards immune-modulation triggered by NB-PDT. For this, the EGFR-targeted NB 7D12 was conjugated to the photosensitizer IRDye700DX and used to perform NB-PDT on EGFR-overexpressing A431 tumor cells, exposed to 30 minute incubation with the conjugates and followed by a light dose of 10 J/cm2. The cytoplasmic DAMP HSP70 was detected on the cell membrane after mild NB-PDT (1 nM conjugate), while it was detected in the medium after highly toxic NB-PDT (25 nM conjugate). The nuclear DAMP HMGB1 was found in the cell cytoplasm under both NB-PDT conditions. Furthermore, cells treated with highly toxic NB-PDT showed an increased release of the pro-inflammatory cytokines IL-1β and IL-6, and a decreased release of pro-tumoral IL-8. Lastly, medium collected from tumor cells treated with highly toxic NB-PDT was able to induce the phenotypic maturation of a dendritic cell line, as indicated by the upregulation of CD40, CD86 and MHCII. Altogether, these results are the first to indicate immune-modulation by NB-PDT, which can be exploited to increase NB-PDT efficacy even further.

Even though a patient has a good immune system, tumors are shielded from it, because tumors grow by suppressing the host’s immune-response by various mechanisms. They are keeping their local microenvironment immune suppressed by producing immune suppressive cytokines like IL-10 and TGF-b, express immune checkpoint ligands like Programmed Death Ligand 1 (PDL1), and harbor immune suppressive cells like Tregs and MDSCs. To overcome these barriers a stronger anti-tumour immune-response is essential. We evaluated a whole cell vaccine with extracorporeal Rutherrin®-PDT treated cancer cells (RuVaCareTM) to break the suppressive barrier in the RG2-glioblastoma model. Rutherrin®-PDT induced strong immunogenic cell death (ICD) in glioblastoma cells in-vitro. RuVaCareTM supernatants showed significantly higher level of extracellular ATP, which is known to induce recruitment of antigen presenting cells (APCs) and their activation by eliciting an effective anti-tumour immune-response. Extracellular calreticulin (CRT) is one of the hallmarks of ICD; its expression went up in more than 85% cells undergoing Rutherrin®-PDT mediated cell death. There was a close to 10 times increase in expression of HSP 70 in RuVaCareTM. Immunostimulatory cytokines IFNa, IL-1b and GMCSF expression is high in the RuVaCareTM. In-vivo efficacy of the RuVaCare™ was evaluated in orthotopic RG2 rat glioblastoma model. There was a significant increase (~43% with 2-time vaccine and 87% in the 6-time vaccine) in survival in the RuVaCare™ vaccinated groups compared to unvaccinated controls. Increased intratumoral CD8+T-cell numbers are shown to be correlated with increased survival in glioblastoma rats, with RuVaCare™ there was a significant increase in the number of CD8+T-cells.

Many parts of the world do not have healthcare infrastructure and many people die from a lack of medical attention. Photodynamic therapy is portable, inexpensive and effective in the treatment of many malignant conditions in the head and neck and skin (among others). With my coauthors, we have rolled out PDT in a number of settings, initially in Indonesia and susequently in South America and India. There is great potential for this therapy in a number of settings and this will be highlighted in this presentation.

Purpose: The current standard of care for advanced stage head and neck cancer is neck dissection with removal of relevant lymph nodes. However comprehensive cervical lymphadenectomy can be associated with significant morbidity and poor quality of life. This study evaluated the sensitivity and specificity of cetuximab-IRDye800CW to identify metastatic disease in patients with head and neck cancer. Experimental Design: Consenting patients scheduled for curative resection were enrolled in a clinical trial to evaluate the safety and specificity of cetuximab-IRDye800CW. Patients (n=12) received escalating doses of the study drug. Where indicated, cervical lymphadenectomy accompanied primary tumor resection, which occurred 3-7days following intravenous infusion of cetuximab-IRDye800CW. All 471 dissected lymph nodes were imaged with a closed-field, near-infrared imaging device during gross processing of the fresh specimens. Intraoperative imaging of exposed neck levels was performed with an open-field fluorescence-imaging device. Blinded assessments of the fluorescence data were compared to histopathology to calculate sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV). Results: Of the 35 nodes diagnosed pathologically positive, 34 were correctly identified with fluorescence imaging, yielding a sensitivity of 97.2%. Of the 435 pathologically negative nodes, 401 were correctly assessed using fluorescence imaging, yielding a specificity of 92.7%. The NPV was determined to be 99.7%, and the PPV was 50.7%. When 37 fluorescently false-positive nodes were sectioned deeper (1mm) into their respective blocks, metastatic cancer was found in 8.1% of the re-cut nodal specimens, which altered staging in two of those cases. Conclusions: Fluorescence imaging of lymph nodes after systemic cetuximab-IRDye800CW had a high sensitivity and was able to identify additional positive nodes on deep sectioning, which suggests a role for this technology for accurate assessment of regional metastatic disease.

Standard of care in the management of cancer of the oral tongue is complete extirpation with clear margins. There is a direct correlation between the radial margin distance and local failure and death from disease. Surgical resection of this site is balanced between resection of enough tissue to ensure clear margins and excessive resection that would result in unnecessary oral dysfunction. The optimal margin distance is 5 mm on permanent fixed histological assessment Mucosal margins are obtained primarily using visual inspection with a reasonable degree of accuracy. More difficult is the deep margin which cannot be seen during the resection, where surgeons must rely on manual palpation to estimate the depth invasion and the needed deep margin tissue thickness. Over the last three years we have utilized intraoperative ultrasound to improve the likelihood of a clear deep margin. Preliminary analysis has shown that using this technique we are able to reliably clear the deep margin with an improved rate of local control. Drawing upon our preliminary findings using intraoperative ultrasound for surgical navigation, we have designed a study to employ dual functional antibody conjugates as a theranostic tool to allow molecular based optical imaging of tumor margins, improving precision of the resection and utilizing a benzoporphyrin derivative to provide for a tumor specific photodynamic therapy treatment at the time of resection. Techniques and preliminary oncologic and margin outcomes of intraoperative ultrasound as well as the concepts of the use of dual functional antibody conjugates in surgical navigation will be discussed.

Oral cancers are among the most prevalent malignancies in males. In developing countries like India where chewing tobacco and other carcinogen containing mixtures is common, incidence of oral cancers is high. Photodynamic therapy is emerging as a promising method for non-disfiguring treatment for early malignant lesions of the buccal mucosa with relatively few effects and potential for implementation in settings with limited medical infrastructure We evaluate ultrasonography as an approach for assessment of PDT response in oral malignancies.In our study we selected stage 1 malignancy of the buccal mucosa using both ultrasonography followed by histopathology.The criterion for selection on USG was lesion length of 2cm or less and depth of 5mm confirmed by biopsy and histopathology analysis. Subsequently, the patients found positive on biopsy were treated with ALA PDT with a LED light source. Following treatment these patients were reassessed with ultrasound at day 10. The images obtained were compared and evaluated for change in the size of the lesion, their echopattern, vascularity, mucosal status etc. It was found, lesions completely hypoechoic signifying necrosis came out negative on biopsy every single time, confirming hypoechogenecity as the single best indicator for success of PDT treatment. However, those lesions which did not develop necrosis but were negative on biopsy, the indirect markers were small initial size of the lesion, a lack of vascularity within the lesion and surrounding inflammation. To conclude, ultrasonography is a convenient, reliable and radiation free method for post PDT evaluation of lesions of buccal mucosa.

Atropisomerism is a frequently overlooked source of structural diversity in drug development. Photosensitizer development for photodynamic therapy (PDT) is not an exception. The present work aimed at investigating the efficacy of redaporfin (and precursor porphyrin) atropisomers for use in PDT. Redaporfin is a synthetic sulphonamide fluorinated bacteriochlorin with enhanced photostability, strong absorption at 750 nm and high ROS yields which is currently in clinical trials for head and neck cancer (NCT02070432). This bacteriochlorin and related porphyrin have phenyl-macrocyclic single bonds with hindered rotations that can generate a different spatial distribution of the sulphonamide groups in the meta positions. The present work demonstrated that it is possible to separate the four atropisomers of redaporfin, and related porphyrins and chlorins. They are designated as: α4 when all the sulphonamides of the phenyl groups are on the same side of the macrocycle plane; α3β when three of the sulphonamides are in the same side of the plane and one is on the other side of the plane; α2β2 when two sulphonamide groups are on each side and adjacent to each other and finally, αβαβ when two sulphonamides are on each side but alternate in the positions with respect to the macrocycle. Although the photo and physicochemical properties of the four atropisomers are similar, their therapeutic efficacies are dramatically different: the α4 atropisomer is orders of magnitude more toxic than the less photoactive αβαβ atropoisomer. Similar results were found for bacteriochlorins and porphyrins.

Multifunctional hybrid nanoparticles are being developed to carry a wide variety of therapeutic and imaging agents for multiple biomedical applications. Polysilsesquioxane (PSilQ) nanoparticles is a promising hybrid platform with numerous advantages to be used as delivery system for photodynamic therapy. In this work, we developed a redox-responsive PSilQ-based platform to transport and deliver simultaneously protoporphyrin IX (PpIX) and curcumin inside human cells. These PSilQ nanoparticles contain a high loading of PpIX (24.4 ± 2.5 %wt) and curcumin (7.6 ± 1.5 %wt), and are biodegraded inside cancer cells due to the high reducing environment. This multimodal delivery system shows a synergistic performance for the combined photo- and chemotherapy of the triple-negative breast cancer (TNBC) MDA-MB-231 cells. The safety and phototherapeutic efficacy of this PSilQ-based platform was evaluated in an orthotopic mice model of TNBC. The PSilQ nanoparticles are completely biodegraded and excreted from mice without any side effect. The efficacy data show that the PSilQ nanoparticles efficiently reduce tumor growth in the orthotopic mice model of TNBC. This work demonstrates that PSilQ nanoparticle-based platform is an excellent alternative for the combined photo- and chemotherapy of TNBC.

Photosensitizers can be integrated with drug delivery vehicles to develop chemophototherapy agents with anti-tumor synergy between chemo- and photo- components. Long-circulating doxorubicin (Dox) in porphyrin-phospholipid (PoP) liposomes (LC-Dox-PoP) incorporates a phospholipid-like photosensitizer (2 mole %) in the bilayer of Dox-loaded stealth liposomes. Hematological effects of endotoxin-minimized LC-Dox-PoP were characterized via standardized assays. In vitro interaction with erythrocytes, platelets, and plasma coagulation cascade were generally unremarkable while complement activation was found to be similar to that of commercial Doxil. Blood partitioning suggested both the Dox and PoP components of LC-Dox-PoP were stably entrapped or incorporated in liposomes. This was further confirmed with pharmacokinetic studies in Fischer rats, which showed the PoP and Dox components of the liposomes both had nearly identical, long circulation half-lives (25-26 hours). In a large orthotopic mammary tumor model in Fischer rats, following intravenous dosing (2 mg/kg Dox), the depth of enhanced Dox delivery in response to 665 nm laser irradiation was over 1 cm. LC-Dox-PoP with laser treatment cured or potently suppressed tumor growth, with greater efficacy observed in tumors 0.8-1.2 cm compared to larger ones. The skin at the treatment site healed within approximately 30 days. Taken together, these data provide insight into nanocharacterization and photo-ablation parameters for a chemophototherapy agent.

Pancreatic ductal adenocarcinoma remains one of the worst types of cancers mainly due to its late diagnosis, lack of effective therapies for advance disease and high chemoresistance. Novel therapeutic options that could improve patient quality of life and overall survival are therefore imperative. In this study, we describe the use of an original strategy based on photochemical internalisation (PCI) technology for pancreatic cancer treatment. Subcellular localisation of the photosensitiser meso-tetraphenylporphine-disulfonate (TPPS2a) was performed in PANC-1 cells, showing its preferential accumulation in lysosomes. Treatments with increasing concentrations of the ribosome-inactivating protein saporin or TPPS2a alone were compared with PCI-saporin. Metabolic activity and cell viability of PANC-1 cells were determined 96h post-illumination by MTT and trypan blue assays, respectively. Our results show that PCI using the photosensitiser TPPS2a, synergistically enhances the cytotoxic effects of saporin in PANC- 1 cells and could offer more effective treatment options for pancreatic cancer.

Photodynamic therapy (PDT) of tumors relies on the delivery of a photosensitizer to the tumor site followed by external laser activation. The effectiveness of the therapy is determined by the fluence and irradiation of the laser, intratumoral photosensitizer retention, and availability of molecular oxygen. We hypothesized that retention and resulting therapeutic response may be improved with local hyperthermia. Tumors were grown in the rear limb then locally heated for 1 hour at 42.5 °C immediately following i.v. injection of the photosensitizer in an immunocompetent murine model. Laser exposure was applied at 2 h or 24 h after the heating session. Administration of heating caused a significant growth delay when a single laser treatment was applied only 2 hours following injection. However, in animals given photosensitizer only and irradiated 2 h later there was no measurable anti-tumor effect observed. The marked anti-tumor effects obtained with prior local hyperthermia were observed even as the dose was lowered from 10 mg/kg to 1.3 mg/kg. Additionally, histological analysis of our intial studies revealed that the majority of the tumor tissue (~75%) was necrotic after two days when heat was combined with PDT, while PDT alone resulted in only ~25% necrotic tissue. Additionally, a significant, though less notable, increase in the efficacy was observed if the laser treatment was applied 24 hours after hyperthermia and photosensitizer administration. This increase is in part ascribed to the increased retention of the photosensitizer in the tumor tissue and likely lasting effects on tumor blood flow and oxygenation in the heated vs. control groups.

ABSTRACT The photodynamic antimicrobial process is a multi-target method to inactivate pathogenic microorganisms by exciting a photoantimicrobial agent with visible light of appropriate wavelength in the presence of molecular oxygen (3O2). There are two major pathways by which reactive oxygen species (ROS) are produced. In type-1 reactions, radicals such as superoxide (O2•−) and hydroxyl radicals (•OH) are generated by electron transfer. In type-2 reactions, highly reactive singlet oxygen (1O2) is produced by direct energy transfer. This study investigated the efficiency of the photodynamic antimicrobial process in Escherichia coli wild type (EC WT) and the mutant Escherichia coli PN134 (EC PN134) which is not able to produce SOD A and SOD B, by means of two different photoantimicrobials from different chemical classes with different 1O2 quantum yields: methylene blue (MB) and 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP). Mutants, which lack antioxidant enzymes, were particularly susceptible towards type-1 reactions. When using light-activated MB, quenching agents such as superoxide dismutase (SOD) and catalase (CAT) were sufficient for protecting both the wild type and the mutant, whereas they were not able to prevent bacterial killing sufficiently using light-activated TMPyP. Summary The susceptibility of EC PN134 and EC WT differed towards photodynamic inactivation via the type-1 mechanism of action. Thus, already existing defense mechanisms against ROS in bacteria might influence the susceptibility against type-1 photodynamic mechanism of action, while this was not the case using type-2 photoantimicrobials.

Antimicrobial photodynamic therapy (APDT) combines the use of light with a photosensitizer (PS) and oxygen to kill microbial cells. Even though this technique was first reported in the beginning of the 20th century, APDT never took off as antimicrobial chemotherapy did. However, microbial resistance to chemotherapy is currently expanding in faster rates than drug discovery. Therefore, introduction of therapeutic alternatives that bypass mechanisms of drug resistance now presents an urgent status. Fortunately, the scientific and technological development related to APDT made it far more feasible for mainstream clinical applications. Our research group has been working on mechanisms and applications of APDT for almost 20 years. We have already reported that successful APDT results depend on a number of factors, such as PS and light parameters, cell type, and oxygen abundance, among others. We have also demonstrated that APDT is an effective adjuvant in endodontics and periodontics and can be a non-invasive treatment for caries, candidiasis and cutaneous leishmaniasis. In Veterinary Medicine, we have reported effective treatment for penguin pododermatitis, snake stomatitis and dog otitis. This presentation will give an integrated perspective from the basic APDT mechanisms, preclinical and clinical trials to protocol optimization and future perspectives.

Antimicrobial Photodynamic Therapy (APDT) is being increasingly used for treatment of acute infections. The cause of upper respiratory tract infections represent a large part of the diseases caused by drug-resistant microorganisms. Acute pharyngotonsillitis caused by bacteria represent many cases that are admitted to hospital emergency dialy. Antibiotics are the first line treatment for bacterial pharyngotonsillitis. However, drug failure may occur by antibiotic therapy, which can cause recurrent pharyngonsilitis. Pharyngotonsillitis treatment has been studied by CEPOF for the last five years. The studies were focused on the following tests: determination of a formulation used in tonsils; development of lighting device for tonsils; incorporation of the photosensitizer by the main pathogenic microorganisms, microbial behavior in successive PDT sessions; study of microbial virulence of surviving bacteria after PDT sessions; development of clinical study phase I and II. The results have shown that under specified the PDT can be used for the treatment of pharyngotonsillitis. It was possible to observe the increase of incorporation of photosensitizer depending of formulation composition; the microbial resistance behavior in relation to successive PDT sessions; the safety of the technique in clinical phase I study, and clinical results, not yet completed, in phase II showed the PDT efficient against different types of pathogenic microorganisms in adults.

Antimicrobial photodynamic therapy (APDT) is trending as a powerful therapeutic platform to minimize the negative impacts of microbial drug resistance. The mechanisms of action of APDT are imposed as a generalized oxidation of all cellular structures. Therefore, is assumed that the development of resistance to APDT is very unlikely to occur due to its multitarget oxidative effects. Even though the instant effects of APDT may be interesting to several situations, the few microbial cells left alive after irradiation procedure may be enough to allow tissue recolonization. Therefore, to ensure higher effectiveness of APDT protocols should also rely on therapeutic combinations with longer lasting effects. In this study, we report the statistical correlation of bacterial inactivation rate with the degradation rate of lipids, proteins and DNA. We observed that APDT mediated by methylene blue (MB) and red light can induce degradation of enzymes associated with drug resistance. Thus, cells exposed to sublethal doses of MB-APDT may recover sensitivity to antibiotics they were previously resistant. This fact may lead to a time interval where highly resistant pathogens become sensitive to most standard drugs, such as penicillin. Additionally, we observed that drug-resistance genes present in bacterial DNA are severely damaged. Hence, drug resistance gene expression and/or dissemination to other cells should also be impaired. In summary, we can conclude that APDT also challenges drug resistance by degradation of related enzymes and DNA.

The human epidermal growth factor receptor (EGFR) is a ~180 kDa transmembrane glycoprotein that contains an extracellular domain, a transmembrane domain and an intracellular tyrosine kinase domain. We have recently prepared porphyrin-, phthalocyanine- and boron BODIPY-peptide conjugates that target the extracellular domain of EGFR. These compounds are promising theranostic agents for the diagnosis and treatment of cancers that overexpress EGFR, including breast, ovarian, prostate, and colorectal cancers.1-4 We will present our recent studies on the preparation of these photosensitizer-peptide conjugates and investigations of their EGFR-targeting ability using surface plasmon resonance, molecular modeling, cell culture assays, and animal studies. References 1. B. G. Ongarora, K. R. Fontenot, X. Hu, I. Sehgal, S. D. Jois and M. G. H. Vicente. Journal of Medicinal Chemistry 2012, 55, 3725-3738. 2. K. R. Fontenot, B. G. Ongarora, L. E. LeBlanc, Z. Zhou, S. D. Jois and M. G. H. Vicente. Journal of Porphyrins and Phthalocyanines 2016, 20, 352-366. 3. N. Zhao, T. M. Williams, Z. Zhou, F. R. Fronczek, M. Sibrian-Vazquez, S. D. Jois and M. G. H. Vicente. Bioconjugate Chemistry 2017, 28, 1566–1579. 4. T. M. Williams, R. Sable, S. Singh, M. G. H. Vicente and S. D. Jois. Chemical Biology & Drug Design 2018, 91, 605-619. Acknowledgements: The authors thank the US National Institutes of Health and the US National Science Foundation for supporting this research.

Targeted photodynamic therapy (PDT) combined with image-guided surgical resection is a promising strategy for precision cancer treatment. Targeting the prostate-specific membrane antigen (PSMA) has drawn significant attention due to its marked overexpression in a variety of malignant tissues, most notably in prostate cancer. Recently, we reported the design of a pyropheophorbide-based long-circulating PSMA-targeted phototheranostic agent for multimodal PET/fluorescence imaging and potent PDT of prostate cancer. While this agent is effective in a subcutaneous mouse model, the non-optimal optical properties of pyropheophorbide (Q_y absorption maximum at 671 nm) pose a limitation for treatment of deep-seated solid tumors. To further advance PSMA-targeted PDT and enable effective treatment to the deeper layers of tumor tissue, we developed a bacteriochlorophyll-based PSMA-targeted photosensitizer (BPP) which consists of three building blocks: 1) a urea-based PSMA-affinity ligand, 2) a peptide linker to prolong plasma circulation time, and 3) a bacteriochlorophyll photosensitizer for NIR fluorescence imaging (Q_y absorption maximum at 750 nm). BPP demonstrated effective cell internalization as well as PDT activity in PSMA-expressing PC3-PIP cells. Furthermore, this agent possesses excellent targeting selectivity in vivo as demonstrated in a dual PSMA-positive and PSMA-negative subcutaneous tumor model. The peptide linker in BPP allowed for its long plasma circulation time (12.65 hours), which enabled its effective tumor accumulation. Overall, bright NIR fluorescence of BPP enables effective image guidance for surgical resection, while the combination of its superb targeting and strong PDT activity allows for potent and precise photodynamic treatment of deep-seated tumors.

Methods: G-chlorin e6 was synthesized with a core photosensitizer chlorin e6 conjugated to glucose. 1); The half maximal inhibitory concentration (IC50) was measured to compare the PDT effects of G-chlorin e6 and TS. 2); Flow cytometry was performed to examine the accumulation of G-chlorin e6 in cancer cells. We also compared the accumulation of G-chlorin e6 between normal immortalized esophageal epithelial cells and esophageal cancer cells. 3); In vivo using mice evaluation, the radio activated carbon 14 labeled G-chlorin e6 was manufactured for evaluation of the excretion from the body. 4); Antitumor effects of G-chlorin e6 PDT were analyzed in allograft tumor mouse models. Results: 1); PDT in vitro using G-chlorin e6 elicited 9, 000-34,000 times stronger antitumor effects than TS. 2); there was 70-190 times more G-chlorin e6 accumulated than TS by flow cytometry. G-chlorin e6 accumulated more selectively in esophageal cancer cells than in esophageal immortalized epithelial cells. 3); The excretion of G-chlorin e6 from plasma to bile juice was much faster than that of TS. 4); In an allograft model, PDT with G-chlorin e6 showed very strong antitumor effects and a 40% complete response (CR) rate.

For evaluating and optimization of photodynamic therapy (PDT) efficacy, there is a strong need for imaging modalities that can provide (bio)markers fast, frequently and non-invasively. By combining multiple techniques, optical imaging can simultaneously quantify several PDT-response biomarkers including blood flow, oxygenation and photosensitizer (PS) fluorescence concentration during PDT. Additionally, fluorescence imaging can provide high contrast for visualization for PDT planning and dose optimization. I will present monitoring and predicting the PDT response of oral cancer at both preclinical and clinical settings. The results indicate that real-time blood flow measurements can provide useful feedback for PDT optimization in preclinical models, and that multi-parameter analysis of blood flow, PS fluorescence concentration and oxygen saturation can predict the response of oral cancer patients at the operating room. In the final part of the talk, I will present a novel, dual-channel, dual-modal theranostic endoscope that allows imaging, therapeutic light delivery, and light-triggered release of doxorubicin (Dox) from liposomes to optimize chemophotoherapy, the combination of PDT and chemo. The feasibility of noninvasive, continuous monitoring and optimization based on quantitative Dox/PS concentration distributions will be presented in an ovarian cancer model.

Introduction. Multiple myeloma (MM) is a cancer of terminally differentiated plasma B-cells that originates in the hematopoietic bone marrow and accounts for 15-20% of all hematologic malignancies. Combination therapies are commonly prescribed to patients with relapsed/refractory MM, which necessitates the stratification of responding patients to minimize toxicities and improve quality of life. Very late antigen-4 (VLA-4) and cluster of differentiation 38 (CD38) are over-expressed proteins on MM cells. Here, we will demonstrate VLA-4 and CD38 targeted molecular imaging as potential surrogates for targeted therapy. Materials and Methods: Daratumumab (Janssen Biotech), CD38 targeted antibody, was provided by the Siteman Cancer Center pharmacy. Sulfo-Cyanine5 (Cy5) NHS ester (Lumiprobe) was conjugated resulting in a 5:1 dye to antibody ratio. In vivo fluorescence imaging was performed on fox chase severe combined immunodeficient beige mice bearing MM1.S-GFP subcutaneous myeloma tumors (n = 7). VLA-4 imaging was performed using a highly selective peptidomimetic ligand (LLP2A) for the activated form of VLA-4 in 5TGM1-GFP syngeneic mouse model. Results and Conclusion. Here we used selective near-infrared fluorescent probes LLP2A-Cy5 and daratumumab-Cy5 to target α4β1 and CD38 expression respectively in vitro and in vivo in relevant myeloma models. Specificity for α4β1 over expressing cells was maintained in vivo in medullar MM with 7-fold and 2-fold increase in LLP2A-Cy5 uptake into tumor-bearing bone marrow and spleen, respectively. Tumor uptake of the fluorescently-labelled daratumumab increased through the imaging period with tumor to blood ratio 7 days post-contrast nearly doubling relative to pre-contrast. We are validating molecular imaging approaches for targeted therapy.

Photodynamic therapy (PDT) is a modality with promising results for the treatment of various cancers. PDT is increasingly included in the standard of care for different pathologies. The role of medical imaging in this context is crucial to better understand how and where to deliver the therapy but also to observe the different mechanisms involved in the effects on tumors. At different stages of delivery, PDT requires imaging to plan, evaluate and monitor treatment. In this paper, we review the contribution of Magnetic Resonance Imaging or Positron Emission Tomography for planning and therapeutic monitoring purposes. Several solutions have been proposed to plan PDT from imaging. MRI and dedicated segmentation algorithm have been recently proposed to plan interstitial PDT with stereotactic localization and light diffusion simulation capabilities. Additionally, photosensitizer biodistribution has been evaluated with radiolabeled photosensitizers. The effects of PDT delivery have also been explored with specific Magnetic Resonance Imaging or Positron Emission Tomography radiopharmaceuticals to evaluate the effects on cells (apoptosis, necrosis, proliferation, metabolism) or vascular damage. The contribution of medical imaging in the context of photodynamic therapies is important and continues to increase. Using morphological or molecular imaging has to be considered for future developments of PDT.

Introduction: Pancreatic cancer often recurs following surgery suggesting new operative approaches are required. Fluorescence-guided surgery aims to assist surgeons in identifying tumour intraoperatively to facilitate complete resection. However, the ideal contrast agent for this purpose is not yet determined. The Rose criterion states that accurate imageguided surgery requires a Tumour-to-Background Ratio of contrast agent greater than 5. We investigated the potential of 5-aminolevulinic acid (ALA) for this purpose. Methods: Pancreatic cancer cell lines CFPAC-1 and PANC-1 were compared with the control pancreatic ductal cell line H6c7. Cells were seeded on day 1 and fluorescence measured on day 4 following 4, 8, 24 or 48 hours incubation with 0.25, 0.50, 0.75 or 1.00mM ALA. Fluorescence was measured using a plate reader and microscopy. Results: The maximum ALA-induced fluorescence for CFPAC-1 and PANC-1 was achieved after 48 hours incubation with 0.50mM ALA. Compared to cells incubated without ALA, a relative fluorescence increase of 39.4-fold in CFPAC-1 and 2.7-fold in PANC-1 was seen. ALA concentrations above 0.50mM did not result in higher fluorescence. In contrast, the control cell line H6c7 showed progressively increasing fluorescence with increasing ALA concentrations. The highest cancer/control cell fluorescence ratios for ALA were after 48 hours incubation with 0.25mM ALA; 122.9 in CFPAC-1 and 9.7 in PANC-1. Conclusion: ALA-induced fluorescence in CFPAC-1 is significantly higher than the control cell line H6c7. PANC-1 achieved only mildly increased fluorescence compared to H6c7. ALA has the potential to provide an adequate level of fluorescence for image-guided pancreatic surgery in ALA-susceptible cancers.

Recurrent Non-Muscle Invasive Bladder Cancer (NMIBC) is a diffuse disease, and patients have failed standard BCG therapy face prophylactic cystectomy. PDT fell out of favour due to its variable outcome, and high morbidity. To overcome PDT associate toxicity to the bladder’s muscle layer, the use of shorter wavelength and instillation of the photosensitizer were suggested. While either approach was shown to improve the outcome in animal models they have not previously combined in human studies. Additionally, the effects of highly variable tissue optical properties of the bladder and its shape have not been studied. Here, we present surface dose histograms derived from light propagation simulation in 6 human bladders using CT images for anatomical detail and the FullMonte software package. The ability of a single light sensor versus 3 or 12 light sensors to measure the average irradiance on the bladder surface was evaluated as a function of the bladder wall’s tissue optical properties. Results show that the irradiance in non-spherical bladders can vary over an order of magnitude, but the irradiance histograms are affected little by displacement of the emitter inside the bladder void. As the surface area monitored by a single sensor depends strongly on the bladder shape, the responsivity of a single sensor to the average bladder irradiance can vary equally. Twelve light sensors monitor the entire bladder surface almost complete and hence their average responsivity is constant to the average irradiance on the bladder largely independent of shape. The dependency of the sensor’s response on the tissue optical properties is also lower.

Pretreatment Planning and Light Dosimetry for Interstitial PDT of Locally Advanced Cancer Gal Shafirstein¹, David Bellnier¹, Emily Oakley¹, Michael Habitzruther¹, Sasheen Hamilton¹, Hannah Cooper¹, Sarah Chamberlain1, Alan Hutson2, Sandra Sexton3, Leslie Curtin3, Joe Spernyak4, Steven Turowski4, Hassan Arshad5, Lawrence Tworek¹, Matthew Mallory¹ and Barbara Henderson1. ¹ Photodynamic Therapy Center at the Department of Cell Stress Biology, and ² Department of Biostatistics and Bioinformatics, 3Laboratory Animals Shared Resources, 4Translational Imaging Shared Resource, 5Department of Head and Neck Surgery. Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA Objective: To highlight the critical need for pretreatment planning and light dosimetry in administering interstitial photodynamic therapy (I-PDT) in the treatment of locally advanced cancer (LAC). Background: There is no effective standard treatment for LAC that failed to respond to surgery and chemo-radiation. The newest immunotherapies are effective in only 5-16% of the cases. I-PDT can be considered if the target tumor does not invade a major blood vessel, and the tumor is accessible to fiber-optic placement. Multiple fibers are required for interstitial illumination of the target tumor and margins. Image-based pretreatment planning is used to determine the number and proposed location of fibers. Light dosimetry is employed to measure intratumoral light fluence and irradiance. To date, it was acceptable that a threshold intratumoral light fluence is required for adequate response. Whereas, we demonstrated that intratumoral light irradiance impacts tumor response [1]. Overview: This talk will present an image-based finite element method (FEM) approach for guiding I-PDT. We will discuss the importance of delivering a threshold intratumoral light irradiance in addition to threshold fluence. We will demonstrate that even if a threshold fluence is delivered, an inadequate intratumoral irradiance will result in poor response. We will reveal that adequate light irradiance and fluence can yield up to 90% cure rate in mice and excellent local control in rabbits with LAC. We will present our image-based FEM and light dosimetry approach to translate the preclinical findings to test I-PDT in the treatment of patients with locally advanced head and neck cancer. References 1.Shafirstein G, Bellnier DA, Oakley E, Hamilton S, Habitzruther M, Tworek L, Hutson A, Spernyak JA, Sexton S, Curtin L, Turowski SG, Arshad H, Henderson B. Irradiance controls photodynamic efficacy and tissue heating in experimental tumours: implication for interstitial PDT of locally advanced cancer. Br J Cancer. 2018;119(10):1191-9. Acknowledgements: Supported in part by NCI/NIH R01 CA193610 to GS, P01CA55791 to Sandra Gollnick, and by Roswell Park Comprehensive Cancer Center Support Grant P30CA16056. We thank Diane Filippini and Raymond Wasielewski for their assistance in obtaining the CT scans and Dr. Craig Hendler MD for conducting diagnosis of the CT scans. We thank the staff of Laboratory Animal Shared Resource at Roswell Park. We thank Concordia Laboratories Inc. for providing the Photofrin® at no cost.

Previous dosimetric studies during PDT of superficial cavitary lesions such as Barret’s esophagus, trachea and nasopharyngeal malignancies demonstrated significant intra- and inter-patient variations in fluence rate build up as a result of tissue surface re-emitted and reflected photons. The total fluence rate at the cavity surface consists of a direct incident term, i.e. photons directly from the source entering the tissue and a scatter term and will therefore affect the response to PDT as a result of the number of photons available for photosensitizer absorption. The build-up factor depends on the optical properties and geometry. Recently a Foscan™ mediated PDT study of tumors in the paranasal sinuses after partial maxillectomy and/or endoscopic sinus surgery was initiated. These geometries are complex in shape, and have spatially varying optical properties, therefor pre-planning and in-vivo dosimetry to ensure a steady and effective light dose delivered to the tumor while securing the vital structures is required. For this purpose, two 3D mesh based light transport models were developed. An empirical model that utilizes a linear function that determines the build-up factor as function of the source to surface distance, and an analytical model based on Lambert’s cosine law and an average diffuse reflectance. Both models were evaluated by means of 3D printed tissue optical phantoms, and a porcine tissue model. The models are within ± 20% accurate and have the potential to determine the optimal source location (OSL) along with the output power settings. EM navigation can be used to guide the source to the OSL.

While the prevalence of central bronchial tumours is declining, that of peripheral lung tumours is increasing. Peripheral lung tumours present either as individual index lesion or as field cancerization, requiring for the former targeting of particular confined volumes of lung tissue versus a therapy for an entire lung or particular lobes thereof. Using FullMonte, a Monte Carlo code; the ability to achieve a tumour selective PDT by transbronchial light source placement was simulated for 525, 665 and 808 nm wavelength. Simulations were executed utilizing in silica models with up to 10 generations of the bronchial tree, tissue photosensitizer concentrations taken from literature or measure in preclinical model systems and tissue optical properties measured with alive ex vivo pig and human lungs perfused with either blood or a transparent low cellular (STEEN) fluid. The measured effective attenuation coefficients [cm-1] at the three wavelengths for ventilated lungs with either blood 1.26±1.07, 1.93±0.534, 1.09±0.93 or STEEN fluid 1.01±0.873, 0.901±0.318, 0.641±0.31 used as perfusate. When modelling the PDT dose distribution in the lung’s the bronchial air ducts up to the eight generations perturb the fluence considerably. In all simulations, a dose sufficient to cause necrosis in 98% of the target volume placement of 3 source fibres albeit with various extent of normal lung tumour damage. Full coverage of an entire lung lobe with only three source fibres placed does not provide for effective coverage of the diffuse disease unless a very high selective uptake of the photosensitizer in malignant tissues can be achieved.

For safe and effective interstitial PDT (iPDT), treatment planning is required since the efficacy depends on optical irradiation conditions including the number of inserted optical diffusers, the insertion positions, their powers, and so on. We are developing a treatment planning method based on a numerical model of singlet oxygen generation during light irradiation for iPDT. In the model, photosensitizer photobleaching, which occurs usually during light irradiation for iPDT, is taken into consideration. In this paper, we implement a numerical simulation of the efficacy of iPDT of malignant brain tumor with 5ALA according to desired optical irradiation conditions and evaluate quantitatively the photobleaching effect on the efficacy simulation. In the simulation, to calculate light propagation into biotissues, a 3D brain model segmented into tissue types, brain normal and tumor tissues, was constructed using an MRI data of a patient with a brain tumor (tumor volume: 34 cm3). Supposing that 8 optical diffusers (light power density of the diffuser surface: 580 mW/cm2, length: 40 mm, diameter: 1.1 mm) were inserted into the tumor region, the light distribution was calculated. Then, the photobleached ALA and the accumulated singlet oxygen distribution were calculated to obtain the treated volumes depending on singlet-oxygen concentration threshold values to induce cell death. Compared to the estimated treatment volume without the photobleaching effect, the estimated efficacy with the proposed model was decreased. This result indicates that the model with photobleaching effect will lead to improvement of the efficacy prediction of iPDT.

Background and Objectives: The focus of this study was to demonstrate the application of our finite element method (FEM) to guide light dosimetry for I-PDT of locally advanced VX2 carcinoma. Methods: VX2 carcinoma was surgically implanted in either the sternomastoid muscle of the neck (n = 6) or the biceps femoris of the thigh (n = 8) of New Zealand White rabbits. Periodic computerized tomography scans were used to assess tumor growth. Once the VX2 tumors reached a volume of approximately 3-15 cm^3, our image-based FEM was used to develop individualized treatment plans for I-PDT to deliver our recently suggested threshold intratumoral light irradiance and fluence (1). I-PDT was performed with 630-nm light that was administered 24 hours after injection of 5 mg/kg porfimer sodium. In several of the VX2 tumors, multiple sessions of consecutive light illumination were required to deliver the threshold light irradiance and the FEM was adjusted to simulate these treatments. Light dosimetry measurements were taken during treatment to monitor the intratumoral irradiance and fluence. Results: Six rabbits had no tumor recurrence within 6-12 weeks post I-PDT. Eight rabbits experienced local control, but were euthanized before the end of the study period due to either metastasis or infection. Tumor metastasis was often observed prior to treatment. Normal tissue damage was observed and managed in some rabbits. There were no treatment related deaths. Conclusions: Our results suggest that our FEM-based treatment planning can be applied in the safe and effective delivery of I-PDT to locally advanced cancers. This work is currently being translated into a clinical study. References 1.Shafirstein G, Bellnier DA, Oakley E, Hamilton S, Habitzruther M, Tworek L, Hutson A, Spernyak JA, Sexton S, Curtin L, Turowski SG, Arshad H, Henderson B. Irradiance controls photodynamic efficacy and tissue heating in experimental tumours: implication for interstitial PDT of locally advanced cancer. Br J Cancer. 2018;119(10):1191-9. Acknowledgements: Supported in part by NCI/NIH R01 CA193610 to GS. We thank Diane Filippini and Raymond Wasielewski for their assistance in obtaining the CT scans and Dr. Craig Hendler MD for conducting diagnosis of the CT scans. We thank the staff of Laboratory Animal Shared Resource at Roswell Park. We thank Concordia Laboratories Inc. for providing the Photofrin® at no cost.

The burden of cancer predominantly falls on developing countries where there are inadequate healthcare facilities. Traditional cancer treatments are surgery, radiotherapy and chemotherapy - all of which are expensive to set up and deliver. The WHO suggests 4 linear accelerators are required to meet the needs of 1 million population. In many parts of the world, there may be only a fraction of this number with a consequence that there are long waiting times and the strong possibility that even if a course of radiotherapy is started, it is quite likely that it will be delivered in an optimal time. In practical terms, a delay of 1 month results in a volume doubling (in head and neck cancer for example) and as waiting times are often of the order of 3 - 6 months, volumes can increase dramatically. Photodymanic therapy provides the opportunity for effective inexpensive treatment. It has been shown to be as good as surgery in early disease and at the very least offers significant palliation of advanced disease. The elements required are a laser and drug and as long as these can be produced at modest cost, treatment could be widely available for surface lesions and with a modest investment in ultrasound, even deeply seated lesions are a treatment target. Adoption of PDT faces many challenges, however the potential in cancer as well as infection is an opportunity to be explored.

Main clinical PDT application is still on non-melanoma skin cancer, using topical photosensitization with ALA-based compounds. For systemic oncological PDT, head and neck cancers are the main indication. In Latin America, several researchers have been working to develop protocols and light sources to improve the treatment success and to allow a lower cost procedure, relevant factors especially when considering low setting facilities and large populations. In this talk, we will present examples of instrumentation development and clinical protocols. For topical PDT, different strategies have been proposed to enhance the transdermal delivery of the ALA, considering its depth penetration and the homogeneity of PpIX production. ALA delivery methods using a dermography device and soluble microneedles patch have been under clinical investigation. We will also present the proposal of the Single session MAL-PDT for basal cell carcinoma, where both PDT applications are performed at the same day. The conventional protocol of 2 PDT sessions one week apart is an important limitation for patients that have to travel long distances to receive medical care. Another relevant medical problem that PDT has also been showing promising results is the cervical intraneoplasia. A Brazilian-Mexican device and different fiber-based protocols have been tested in a clinical study for CIN 1-3. The results will be presented, also discussing the strategies for improving irradiation of the lesions.

The role of PDT in nasopharyngeal cancer in Indonesia I.B. Tan NPC is a rare disease in the Western world, but is endemic in certain parts of south East Asia and China. The last decades the outcome of the treatment has improved considerable. Concurrent chemo-radiation has become the treatment of choice in advanced NPC. Five years survival figures of 80-90% for T1-T2 and 60-80% for T3-T4 are not exceptional. However, figures in low income countries are less favorable. Under capacity of radiotherapy units and poor compliance to treatment are the main concerns. A study in a tertiary university hospital in Yogyakarta Indonesia showed 3yrs survival of 30% with many patients with residual disease. Photo Dynamic Therapy (PDT) has been shown to be effective in residual and recurrent nasopharyngeal cancer with 3 year survival rates of over 65%. It’s noninvasive and only limited infra-structure is needed. Future research should focus on innovative treatment strategies like immunotherapy in combination with PDT as an induction therapy to reduce the tumor load during the waiting time.

Oral cancers are the 8th most common cancer among males. In India, this corresponds to almost 80,000 new cases per year. With such a disease burden, oral cancers are a prime cause of morbidity and functional disability. Conventional treatment for oral cancers is surgery and radiation therapy which are fraught with physical and functional side effects, and the high cost and infrastructure requirements present barriers to timely intervention for patients in rural and/or resource-limited areas. Motivated by these considerations we sought to evaluate photodynamic therapy (PDT) as an approach which is inherently conducive to adaptation for resource-limited settings and has previously shown promising clinical results for early stage oral lesions. In this study we evaluate a low-cost platform for aminolevulinic acid (ALA)-based PDT consisting of a portable 635nm fiber-coupled LED light source integrated with 3D printed applicators for stable intraoral light delivery and smartphone-based fluorescence imaging for treatment guidance. Using this technology, we treated 18 subjects with histologically confirmed T1N0M0 lesions of the buccal mucosa with a mean diameter of 1.38 cm2 and micro-invasive (≤ 5mm depth) disease. A total light dose of 100 J/cm2 was delivered in 3 to 5 fractions to the buccal mucosa after oral administration of 60mg/kg ALA (in 3 aliquots of 20mg/kg). The post-PDT investigations showed a 72% success rate (no residual malignancy in follow-up biopsy). The treatment was very well tolerated and has potential for broader dissemination into primary care sites and as an early intervention for pre-malignant conditions including submucous fibrosis and leucoplakia.

Photosensitized oxidations are the key pharmacological reactions in Photodynamic Therapy (PDT). Several tissues and cellular targets have been shown to be responsible for the PDT response, but damage in membranes is key to modulate the mechanism as well as the overall efficiency of cell death.1 There are two major mechanisms of photosensitized oxidations, the direct-contact reactions with biological targets and the oxidations mediated by diffusing species, such as singlet oxygen. In a recent publication, we demonstrated that for a PS to fully compromise membrane function, it needs to be sacrificed through contact-dependent reactions, forming lipid-truncated aldehydes.2 In this presentation we will report data obtained with a series of Mg(II) porphyrazines (MgPzs), whose mechanism of photobleaching is not related with oxidation by singlet oxygen. By comparing the efficiency of membrane rupture by PSs with different electron-deficient fluorinated side groups, we showed that the higher the rate of photobleaching, which occurs because of a redox reaction with the lipid double bond, the faster the rate of membrane leakage. Therefore, our results indicate that the efficiency of causing membrane damage correlates with the efficiency of PS photobleaching, and consequently, PS regeneration should be exploited as an effective tool to developed improved PDT photosensitizers. 1. I Bacellar, et al. Photodynamic efficiency: from molecular photochemistry to cell death. Int. J. Mol. Sci. 2015, 16, 20523. 2. I Bacellar, et al. Photosensitized membrane permeabilization requires contact-dependent reactions between photosensitizer and lipids, J. Am. Chem. Soc. 2018, 140, 9606.

Primary breast cancer while increasing in incidence has been successfully treated with a combination of surgery and adjuvant therapies in the majority of patients. Novel treatments for primary breast cancer need to show additional benefits to existing treatments with equivalent or improved efficacy for niche groups. Photodynamic therapy (PDT) is a potential novel treatment and a Phase I/IIA, open label, non-randomised, single site trial of photodynamic therapy for the treatment of primary breast cancer was conducted. The primary aim was to identify the light dose required for 12 mm of tumour necrosis (or a plateau of necrosis) assessed by histopathology. Post-dose MRI correlation with histopathology findings in treated tumours and in normal breast tissue was sought. In addition adverse events were recorded and comparison of outcome made with matched controls. Results of the first human clinical trial with 12 patients with median follow-up of 39 months showed PDT was well tolerated, with no adverse effects and comparable outcome to control populations. Tumour necrosis increased with incremental increases in light dose, however some patients showed a poor response even at the highest light dose. Analysis suggests that there may be predictive factors for good and poor response. PDT in primary breast cancer requires further investigation to identify which patients would most benefit from this therapy.

Homogeneous, flexible light sources with pure, deep red color and sufficiently high power densities are necessary for more effective and widely used photodynamic therapy (PDT), but have been difficult to achieve with lasers or LED arrays at reasonably low cost. Quantum dot light emitting diodes (QLEDs) have outstanding wavelength tunability, ideal color purity, sufficient power density and unique form factors as thin, flexible, light weight and uniformly large area light sources, which will meet the pressing needs for PDT. Here we report QLEDs fabricated with emission wavelengths precisely tuned to match the absorption peaks of several FDA approved photosensitizers. Preliminary in-vitro studies with rigid on-glass QLEDs as photosensitizer activators demonstrate they can kill cancerous A431 cells or Methicillin-resistant Staphylococcus aureus (MRSA) with efficiency comparable or better than control LED sources, indicating their potential for PDT treatments of cancers or infections. Computer simulation of light propagation in a tissue mimicking phantom suggests that about 50% of the QLED power can be delivered to a depth of about 4 mm from the treated surface. Recent progress on the fabrication of low-cost flexible QLEDs will be presented. Finally, the perspectives of using these devices for PDT to address medical conditions such as cancer treatment, wound repair or cosmetic dermatology will be discussed.

Photodynamic therapy (PDT) is used for cancer treatment based on the interaction of a photosensitizer (PS), light, and oxygen. The photodynamic interaction is termed type I and II depending on whether the cytotoxic oxygen species is through an electron transfer, producing oxygen superoxide and its secondary reactive oxygen species (ROS) or an energy transfer, producing singlet oxygen (1O2). Most photosensitizer exhibit both type I and II interactions. Explicit dosimetry of light fluence rate (ϕ), PS concentration ([PS]), and oxygen concentration ([3O2]) has been developed for clinic use, however, it is important to integrate these explicit quantities to a reacted ROS concentration, [ROS]rx. A mathematical model has been developed to incorporate the macroscopic kinetic equations for [ROS] generation, photosensitizers in ground and triplet states, 3O2, and tissue acceptors along with the Monte Carlo simulation for light transport in tissue. In this study, the ROSED model has been applied to type I (e.g., WST09) and several type II (e.g., HPPH, BPD, Photofrin) photosensitizers. Cure index was computed from the rate of tumor regrowth after treatment and was compared against three calculated dose metrics: total light fluence, PDT dose (product of light fluence and PS concentration), and measured and calculated reacted [ROS]rx. The tumor growth study demonstrates that [ROS]rx serves as a better dosimetric quantity for predicting treatment outcome, as a clinically relevant tumor growth endpoint. Values of threshold dose of [ROS]rx for type I and II interactions are discussed.

The development of low-cost diagnostic tools is essential for translation into various clinical and low-resource settings. Current advances in solid-state lighting, smart-phone capabilities, and 3D printing provide an ideal environment for the development of low-cost imagers for point-of-care diagnostic applications. Protoporphyrin IX (PpIX) based photodynamic therapy (PDT) for actinic keratosis (AK) and non-melanoma skin cancers (NMSC) is widespread, yet no diagnostic point-of-care tools are commonly used to account for the heterogeneity of PpIX accumulation between patients and between lesions. These heterogeneities are correlated with treatment outcomes, such that PpIX dosimetry can provide guidance in PDT and can be used to direct critical decisions about treatment time, retreatment, or further skin preparation. Here, we report on a low-cost smart-phone based dosimetry system designed for use in individualized PDT treatment planning. This hand-held dosimetry system uses an iPhone 6s smartphone alongside a custom app for streamlined image capture and analysis, custom LED board and electronics for PpIX excitation, and a 3D printed base for system integration and measurement standardization. The system has been tested on mice incubated with Ameluz for 10-400 minutes, imaging every 10 minutes. The images showed monotonic response over time with saturation occurring around 3hrs. Linearity of PpIX vs. measured fluorescence was confirmed with 1% intralipid phantom imaging, with a lowest detectable level of 0.05 μM and highest non-saturated level of 4 μM. With the data to date confirming the viability of the system to measure the production of PpIX relevant for skin PDT, preparations for human clinical imaging are underway.

India has one of the highest rates of oral cancer incidence in the world, with an estimated 80,000 new cases per year, accounting for 30% of reported cancers. In rural areas, a lack of adequate medical infrastructure contributes to unchecked disease progression and dismal mortality rates. PDT emerges as a potential modality which can be implemented in resource limited settings, while photosensitizer fluorescence can be leveraged for treatment guidance. Here, as part of an ongoing clinical study evaluating low-cost technology for ALA PDT treatment, we evaluated the capability of a simple smartphone-based device for imaging ALA-induced PpIX fluorescence. The imaging device itself consists of an annulus of 405nm LEDs for PpIX excitation with emission filter in the center mounted over the phone camera. 18 subjects having <2 cm diameter (mean size; ~1.38 cm2) lesions with micro-invasive (≤5 mm. depth) moderately/well-differentiated squamous cell carcinoma were administered 60 mg/kg ALA in oral solution and imaged before and after delivery of 100 J/cm2 total light dose to the lesion surface. We will present comparative analysis of pre-and post-treatment fluorescence, white light, and ultrasound images. In general, PpIX fluorescence images obtained prior to therapeutic light delivery are able to resolve lesion margins while dramatic photobleaching in post-treatment images confirms the irradiated zone. Overall this approach is able to generate sufficient fluorescence contrast for treatment guidance and monitoring photobleaching while the use of a smartphone-based device provides a low-cost, widely available platform with potential for telemedicine integration.

Objective: Despite its approval in 2004 PDT has not well established in the palliative tumour therapy of head neck cancers (HNC). Furthermore we have no diagnostic and therapeutic standards for this procedure. Material and methods: We performed a retrospective analysis of the treated HNC patients of ORL department Nordhausen between 11-2016 and 10-2018 in order to develop practical standards for daily practice, and to describe new projects of research. We analyzed the data of 35 patients with histological confirmed squamous cell carcinoma. The patients have got 1 mg/kg Chlorin E6 IV. Then we performed PD in optical and/or spectroscopical (405 nm) technique. The application was corrected, and the PDT was done by a red-light system (670 nm). Results: We treated 9 malignancies of the tongue/mouth, 9 laryngeal cancer, 7 malignancies of nose/paranasal sinus, 4 cancer of palatal and buccal region, and 3 pharyngeal cancers. Aims of PDT were 33times tumour reduction, and twice decrease of symptom burden. The follow-up interval was median 5 month (range 1-30); 24 patients are alive, 8 patients died, 3 were lost in follow up. We performed median 2 PDT per patient (range 1-19). The application technique was interstitial in 12 patients, frontal in 10 patients and cylindric in 13 patients. PDT was performed as only therapy in 15 patients, in multimodal combinations in 20 patients. We have registered 14 CR, 10 PR, 10 SD and 1 PD. Conclusion: PDT is an effective procedure to get local control in palliative situation of HNC.

Photodynamic therapy (PDT) for actinic keratosis (AK) and certain non-melanoma skin cancers (NMSC) is performed with either blue light (415nm Lamp), red light (630nm LED Lamps) or with sunlight. The differences in PDT efficacy can be high when using the latter broad-spectrum activation of PpIX. The purpose of this work was to establish a predictive treatment plan approach to daylight PDT which incorporated both local weather forecast information as well as prediction of wavelength-depth dependence upon the efficacy. The concept of PpIX-weighted light fluence is now well established as a way to compare effective light doses from daylight PDT to traditional lamp PDT, but there is limited work to date considering the effects of tissue optical properties or the dynamic distribution of PpIX at depths into the tissue. Using a Monte Carlo model of light fluence in a multi-layer skin geometry we estimate the effective fluence at depth in tissue. The result of these simulations in combination with a model for PpIX production and photobleaching at a range of depths are used to generate lookup tables for the time needed to reach a specific photodynamic dose at a predicted lesion thickness. These tables are then used as the foundation of a web-based application that will better inform the dermatology team of light dosing options. GPS-derived location is used to retrieve forecasted and historical weather patterns automatically, and used as an additional input to further refine prescribed dosing of daylight-PDT. The application is currently being tested in conjunction with fluorescence dosimetry, as a method to verify and alleviate clinical variation in lesion clearing from daylight PDT.

Low-intensity (< 0.1 mW/cm2) yet long-term (> 2-3 days) photodynamic therapy(PDT), termed metronomic photodynamic therapy(mPDT), is attracting attention because of its superior selectivity for malignant tumors and safety for the surrounding normal tissues. Because mPDT requires only a feeble light, the light source can be miniaturized and thus fully implantable in the human body by using the technology of wireless electric power supply. These advantages suggest that mPDT can be applied to the tumors in internal cavities such as the brain, chest, and abdomen. We investigated the anti-tumor effect of mPDT using wirelessly powered fingernail size LED device which was sandwiched by tissue-adhesive nanosheets for suture-free fixation onto the tissue. The devices were implanted subcutaneously beneath small intradermal tumors on the back of mice. Mice moved freely in the cage which was placed on the antenna board for 12 days experimental period. We used photofrin as the photosensitizer, and applied two separate colors of LED devices, red(λ=630nm) and green(λ=530nm), according to light absorbances by photofrin. After ten days of treatment, mouse receiving mPDT achieved significant growth suppression of the tumor compared to the control group which carried non-functional device along with being placed on the antenna board. Unlike conventional PDT which is confined to use red light by optical fibers, mPDT employing green light led even stronger anti-tumor effect than red. These results showed that mPDT using implantable optoelectronic device could be applied to cancers in internal organs as a new treatment strategy.

In order to improve the PDT efficacy, different strategies are under development such as the use of targeted photosensitizers or nanoparticles to enhance the tumor selectivity, and also the design of light devices to better monitor the light dose. Due to the limited light tissue penetration depth, another way to improve the efficacy of PDT destruction of deep tumors is the use of upconversion nanoparticles or bi-photon absorption compounds. A relatively new approach, named PDTX, is the use of X-ray instead of UV-visible to overcome the poor penetration depth of visible light into tissue. Upon exposure to X-ray ionizing radiation nanoscintillators transfer their energy to the photosensitizers resulting in their activation and leading to the destruction of targeted cells. This novel PDTX approach could allow the deep-tissue PSs activation by X-ray radiation for deep-seated tumor PDT, using lower X-ray radiation dose than conventional radiotherapy. An overview of what has been tested in the past and what is currently done in this research field will be presented. On that occasion, we will also be describing some examples from our own expertise concerning terbium oxide NPs or gadolinium-based NPs coupled to porphyrin, chlorin or phthalocyanine-type photosensitizers.

Cherenkov excitation of molecular probes in tissue can be used for photodynamic effect or diagnostic luminescence imaging. Excitation of dendritic platinum-based metalloporphyrn molecule complexes as an oxygen sensor (PtG4) has been shown to allow oxygen measurement in vivo with spatial resolution defined by the exciting x-ray beam. By sweeping the beamlet shapes and geometries, and detecting the emitted luminscence lifetime, a highly accurate pO2 distribution can be reconstructed of the tissue volume. This has been shown in tumors as well as in normal tissues, and now for implanted microtubes. Additional imaging of luminescent agents based upon Cherenkov or direct radio-excitation could be used for molecular sensing. The combination of depth, concentration, and radiation dose have been analyzed for the tradeoffs in signal to noise, and the signal to background optimization has been completed. The use of this approach can be applied to any clinical radiation therapy linac.

Photodynamic therapy (PDT) is intrinsically restricted by the low penetration depth of light in tissue and is therefore mostly used to treat superficial or optical-fiber accessible lesions. An elegant non-invasive approach to overcome this limitation is to conjugate the photosensitizers to radioluminescent nanomaterials, also called nanoscintillators, and to activate these with radiation therapy. Upon X-ray irradiation, nanoscintillators are “switched on” and emit light that can subsequently excite the photosensitizer and induce PDT. As X-rays penetrate deeply in tissues, radioluminescence can activate PDT non-invasively at depth and without being restricted by large tumor volumes and optical shielding by blood vessels. The feasibility of exciting photosensitizers using nanoscintillators has been demonstrated, but the effects of this complex concept may stem from several therapeutic contributions that remain under-investigated. In this presentation, we report on the investigation of two confirmed contributions: 1) a potential synergy between low dose PDT and radiation therapy, and 2) a radiation dose enhancement effect stemming from increased radiation absorption by nanoscintillators composed of high-Z elements. The combination between low dose PDT and radiation therapy was assessed on heterocellular spheroid models of pancreatic cancer. The ability of nanoscintillators to induce radiation dose enhancement was experimentally assessed on 3D models of glioma, using synchrotron radiation to deliver radiation therapy. For this research, synchrotron radiation offers the unique opportunity to monochromatize the beam and tune its energy to an optimal value.

X-Ray activated pharmaceutical therapy is highly sought after as it provides deep tissue, synergistic method of treating cancers in which the standard method of care involves radiotherapy. Traditional drugs utilized as neoadjuvant chemotherapy have significant side effects and a lack of selectivity, leaving a dire need for targeted drug delivery methods. We have recently developed a unique delivery platform whereby the drug is conjugated to an alkylcobalamin vitamin B12 scaffold, and these alkylcobalamins are actively transported into cells by transcobalamin receptors (TCblR). A large number of cancer types have enhanced expression of these receptors; therefore, the drug-cobalamin conjugate could be effectively ferried into the tumor selectively via the TCblR pathway. This delivery system provides light-activatable release of chemotherapeutics. Due to the drug becoming active at the specific site that it is needed, such as a tumor, the potential side effects of that drug in organs at risk are mitigated. As a proof of concept, we have found that a fluorescent cobalamin derivative localized within xenograft tumors in mice, demonstrating the effectiveness of the vitamin B12 scaffold as a theranostic targeting agent. In addition, this derivative is also activated with clinical X-ray doses from a linear accelerator. We explored the ability of a variety of cobalamin drug conjugates to be used in combination with radiotherapy to elicit an enhanced reduction in tumor margins in pancreatic adenocarcinoma models.

A homogeneous and reproducible fluence rate delivery during clinical PDT plays a determinant role in preventing underor overtreatment. In Dermatology, topical PDT has been carried out with a wide variety of light sources delivering a broad range of light doses. However, these light sources do not deliver a uniform light distribution on the skin due to their structure and morphology and the complexities of the human anatomy. The development of a flexible light source able to generate uniform light on all its surface would considerably improve the homogeneity of light delivery. The integration of plastic optical fibers (POF) into textile structures offers an interesting alternative. The homogeneous light side-emission from the fabric is obtained by controlling the bending angles of POF inside the LEF due to specific architecture generated by knitting of textile structure. LEF of different surfaces can be easily manufactured (up to 500cm² The LEF thickness is less than 1 mm. The mean irradiance is typically 2.5 mW.cm^-2. W-1 with heterogeneity of 12.5% at any point of the LEF. The temperature elevation remains below 1°C. These LEF were evaluated in Dermatology for the treatment of Actinic Keratosis. Two clinical evaluation were performed. The first one was a monocentric, randomized, controlled, phase II clinical study (ClinicalTrials.gov Identifier: NCT03076918). Twenty five (25) patients with grade I-II actinic keratosis (AK) of the forehead and scalp were treated with methyl aminolevulinate photodynamic therapy in two symmetrical areas. One area was treated with the conventional LED panel (154 AK), whereas the other area was treated with the LEF device (156 AK). The second clinical was performed in 2 centers. This new LEF device was a more ergonomic and compact version of the original system developed for FLEXIPDT. In this clinical study (ClinicalTrials.gov Identifier: NCT03076892), the irradiance has been reduced from 12.3 mW/cm² to 1.3 mW/cm² and the light dose from 37 J/cm² to 12 J/cm² . Compared to Conventional PDT, the 2 protocols clearly shown that LEF are equivalent and even superior in terms of efficacy for treating AK of the forehead and scalp. However, the use of LEF resulted in much lower pain scores and fewer adverse effects. In conclusion, thanks to LEF, PDT of AK can be conducted in all weather conditions, in any geographic location, year-round and benefits from the optimal adaptability of the flexible, light-emitting, fabrics to the treatment area. At last, LEF can be easily can be easily manufactured in large series.

Uniform delivery of light fluence is an important goal for photodynamic therapy. We present summary results for an infrared (IR) navigation system to deliver light dose uniformly during intracavitory PDT by tracking the movement of the light source and providing real-time feedback on the light fluence rate on the entire cavity surface area. In the current intrapleural PDT protocol, 8 detectors placed in selected locations in the pleural cavity monitor the light doses. To improve the delivery of light dose uniformity, an IR camera system is used to track the motion of the light source as well as the surface contour of the pleural cavity. A MATLAB-based GUI program is developed to display the light dose in real-time during PDT to guide the PDT treatment delivery to improve the uniformity of the light dose. We have developed an improved model for direct light calculation that accounts for the anisotropy of the light from the light sources. A comprehensive analysis of the distribution of light fluence during HPPH and Photofrin PDT is presented in clinical cases and the improvements between the two protocol are discussed.

Whether preclinical studies either involve a cell or animal model, the distribution of light plays a determinant role in the reproducibility of results of photodynamic therapy (PDT) studies. Unfortunately, only few illumination devices dedicated to preclinical studies are available and are for the most, very expensive. Most research teams use home-made solutions that may not always be reproducible because of undefined light distribution, additive thermal emission, or unsuitable for shapes and volumes to illuminate. To address these issues, we developed illumination devices dedicated to our preclinical studies, which embed knitted light emitting fabrics (LEF) technology. LEF technology offers a homogeneous light distribution, without thermal emission and can be coupled with various light sources allowing investigation of several PDT modalities (irradiance, wavelength, illumination duration/mode). For in-vitro studies, we designed light plates, each allowing illumination of up to four 96-cells plates. For in-vivo studies, we designed mice boxes allowing three animals placement in prone position, equally surrounded by LEF and ensuring homogeneous extracorporeal illumination. Optical validation was performed and reproducibility of both preclinical systems were assessed. Both systems can deliver homogeneous light with an irradiance that can reach several mW/cm2, with varying durations and wavelengths. First results of preclinical studies demonstrate a high reproducibility, with an easy setup, and a great adaptability of illumination modalities with these devices based on light emitting fabrics.

A range of cellular, architectural, and physical cues in the tumor microenvironment influence the intrinsic and acquired resistance mechanisms that lead to treatment failure. Strategies that leverage photodynamic therapy (PDT), a photochemistry-based biophysical treatment modality, to regionally target and prime stubborn tumor populations may be essential to realizing durable improvements in cancer management while minimizing toxicity from traditional agents. Capturing these attributes in rationally-designed combinations has shown promise by synergistically reducing tumor area in 3D models, and durably controlling tumor burden in vivo. Among the areas that remain understudied is the influence of mechanical forces, such as hydrodynamic shear stress, on resistance, and the development of 3D tumor models and in vivo models that account for physical stress. To evaluate and optimize PDT regimens, and PDT-based combinations, designed to overcome resistance to conventional therapies due to physical stress, a multi-faceted approach is needed. Here the impact of hydrodynamic stress is evaluated in bioengineered 3D tumor models in the context of ovarian cancer. The potential value of using biologically inspired in vitro models to guide customized, rationally-designed PDT-based combination regimens will be presented.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease marked by poor response to virtually all available treatments. Clinical trials of chemo, targeted therapies, and radiation, have resulted in minimal advances, and survival after diagnosis is generally less than a year. In the pursuit of new avenues for therapeutic development for this lethal disease, a number of studies have implicated the role of tumor-promoting microRNAs with altered expression PDAC tissues. Here, using in vitro 3D organoids we explore the strategy of combining photodynamic therapy (PDT) with a novel microRNA therapeutic platform to target miR21, an established onco-miR that targets multiple tumor suppressors. This approach is motivated by recent clinical studies establishing the technical feasibility of light delivery to the pancreas, combined by evidence that PDT may disrupt or loosen stroma and potentially increase delivery of anti-miR agents. To evaluate this hypothesis, we use in vitro co-culture models of PDAC cells and stromal fibroblasts that recapitulate the dense fibrotic stroma of PDAC tumors to evaluate response to treatment in verteporfin PDT and anti-miR combination and monotherapy treatment arms. Using stably-transfected miR-21 sponge expressing we also perform mechanistic studies to investigate the role of miR 21 in response to PDT and chemotherapy in 3D cultures with and without stromal partners.

It is estimated that metastasis is responsible for about 90% of cancer deaths. Therefore, the effective control of tumor progression and metastasis is crucial to decrease the patient mortality rate. The searching for effective regimens has become one of the hot research topics. Photodynamic therapy (PDT) has shown very promising in local control of pancreatic tumors, but had little impact in reducing tumor metastasis. We have been actively investigating the PDT-based combination treatment efficacies in orthotopic mouse models of human pancreatic cancers, and had previously found and reported that PDT followed by 4-time administrations with chemotherapeutic adjuvant abraxane could dramatically shrink primary pancreatic tumors, some of which even disappeared from the pancreas. In this study, we focused on the evaluation of treatment responses on metastasis inhibition with the same treatment regimen after two periods of time: short-term (24 days) and mid-term (60 days). The treatment regimen had the most suppressive effect on metastasis as compared with other control groups: 1) PBS, 2) Gemcitabine, 3) PDT, 4) abraxane, and 5) Gemcitabine + abraxane in both AsPC-1 and MIA PaCa-2 cell line models and for the short- and mid-term experimental courses. However, the triple combination therapy (this treatment regimen plus Gemcitabine) could not further increase the metastasis inhibition. Regression analysis for tumor weights and metastasis burden in any of the corresponding treatment groups suggested that there might not be a correlation between tumor size and metastasis, and the metastasis decrease might not be the results of tumor decrease upon the treatments.

Including hilar cholangiocarcinoma (C.C.), extrahepatic C.C. is a relatively common cancer in Far East Asia and its overall incidence is on the increase. The majority of patients are found to have an unresectable tumor on presentation and their survival is approximately 3 months without intervention and 4–6 months with biliary Intervention. In patients with unresectable C.C., photodynamic therapy (PDT) with biliary stents and adjuvant chemotherapy is used for palliation of jaundice and improving survival. Most therapeutic effects are aimed at delaying bile duct obstruction rather than decreasing the tumor burden. On the based experiences of 200 cases for 10 years in single center, introducing new concepts of PDT compare to previous reports regarding to techniques for improving benefits according to each essential stages of PDT procedure such as pre-PDT, during PDT, and post PDT stage.

Gastrointestinal cancer is a major public health problem. Gastrointestinal cancer is the third most common cancer in Western countries and Korea. Detection of early neoplastic lesions in gastrointestinal tract is essential for cure, because prognosis and survival are related to the size and stage of malignant lesions. Endoscopic screening and treatment of pre-malignant lesions prevents approximately 80% of gastrointestinal cancer. However, white-light endoscopy is an imperfect technology, since missed rates of up to 25% have been reported in colonoscopic evaluation. Furthermore, polyps without malignant potential were treated without benefit but with additional costs and risks to the patient. There are several known predictors of an inadequate endoscopic examination. These include patient characteristics or skills of the endoscopists. Therefore, a variety of advanced technologies has been attempted to overcome these issues. Molecular imaging has emerged as a new discipline in gastrointestinal endoscopy. Molecular imaging is defined as visualization and measurement of biological processes at the cellular and subcellular levels within living systems. Targeted molecular imaging can quantify the target expression, which can be used for diagnosing and managing diseases. The current targeting ligands include peptide, aptamer, protein or antibody. Endoscopic molecular imaging can be defined as the visualization of molecular characteristics with endoscopy. These innovations will allow us not only to locate a tumor or dysplastic lesion but also to visualize its molecular characteristics and the activity of specific molecules and biological processes that affect tumor behavior and/or its response to therapy. Molecular imaging techniques have been developed for a more precise classification of mucosal alterations with selection of patients for invasive therapy or surveillance. Further, molecular and functional imaging techniques could identify novel targets for therapies and new prospects to access response to therapies. The future of endoscopic diagnosis is likely to be impacted by a combination of biomarkers and technology.

Background and Aims: In a single-center, prospective, dose-escalation phase 1 study, patients with treatment-naïve locally advanced pancreatic cancer (LAPC) received intravenous porfimer sodium followed 2 days later by EUS-PDT. EUS-PDT was performed by puncture with a 19-gauge needle and insertion of a 1.0-cm light diffuser and illumination with a 630-nm light. A CT scan 18 days after PDT was done to assess for change in pancreatic necrosis. Nab-paclitaxel and gemcitabine were initiated 7 days after CT. Results: Twelve patients (mean age, 67 ± 6 years; 8 male) with tumors (mean diameter, 45.2 ± 12.9 mm) in the head and/or neck (8) or body and/or tail (4) underwent EUS-PDT. Compared with baseline imaging, increased volume and percentage of tumor necrosis were observed in 6 of 12 patients (50%) after EUS-PDT. No serious adverse events from PDT occurred. Conclusion: EUS-PDT for LAPC is technically feasible. Phase II studies are warranted.

The main goal of this study is to analyze photochemical and molecular mechanisms of PDT with the set of polarity-tunable bacteriochlorins. The relationship of the structure and biological activity of the tested bacteriochlorins (F2BOH, F2BMet, F2BPrc, Cl2BHep) was determined, including (i) physicochemical, spectroscopic and photophysical characterization; (ii) the analysis of in vitro activity (cytotoxicity, subcellular localization, cell death modes); and (iii) the photodynamic efficacy in vivo (vascular versus cellular targeting). A detailed analysis of PDT-induced inflammation and a full characterization of molecular mechanisms were performed. The differences in the efficacy of the chosen phototherapeutic protocols (V-PDT, E-PDT, C-PDT) with optimized PDT conditions (Ps formulation, DLI, light dose) are demonstrated. To elucidate these differences, a Luminex technology was applied to detect a number of cytokines in the tumor and in plasma of PDT-treated mice. Among a wide range of cytokines (IL-6, IL-10, IL-13, IL-15) and chemokines (KC, MIP1, MIP2) released after PDT, an important role is assigned to IL-6. Moreover, expression of recombinant cytokines such as GM-CSF and TNFα significantly enhance antitumor response, whereas blocking anti-inflammatory cytokines such as IL-10 or VEGF improves the cure rates after PDT. Bacteriochlorin-mediated PDT generates specific immune response capable of inducing immunological memory that enables mice to reject a tumor rechallenge. Moreover, we have demonstrated that PDT activates innate and adaptive immunity that result in the eradication of NIR-irradiated primary tumors and the inhibition of untreated distant tumors by generating a systemic tumor-specific cytotoxic T-cell response.