Immune checkpoint inhibitors (ICIs) are among the most effective classes of cancer immunotherapies yet only a small minority of patients derive clinical benefit. We are investigating the use of multi-spectral paired-agent imaging (mPAI) to quantify available PD-1 and PD-L1 receptor concentrations for the therapeutic binding of anti-PD-1 checkpoint inhibitors.
Fluorescence paired-agent imaging (PAI) is presented as a method to rapidly screen en face margins during Mohs surgery to reduce the time required for pathological assessment of intraoperative frozen tissue sections. PAI was applied to mouse models of squamous cell carcinoma, and positive tumor burden was detected using both the mean and maximum signal intensity. It was determined that PAI BP had higher tumor detection accuracy as compared to single-agent fluorescence, while the en face margins provided detailed visualization of the tumor burden. PAI is a promising methodology for rapidly screening positive margins during Mohs surgery.
An increasing number of cancer surgery protocols are including sentinel lymph node biopsies on the day of resection to stage for non-palpable spread of cancer through tumor draining lymph nodes. The challenge is that often a tumor-positive node will make it necessary to perform an enhanced resection of the lymphatic network, and if lymph node processing is not completed within the timeframe of surgery, then patients may have to be called back for additional surgery or have to undergo amplified chemo or radiation therapy. Our group is working on a rapid lymph node staining and fluorescence tomography system that we call ADEPT to provide surgeons with lymph node biopsy results within 15 min. The aim is to minimize the number of callback surgery or amplified therapy procedures to minimize stress to patients and reduce health care costs. This work predicts, using Monte Carlo photon propagation modeling simulations, that ADEPT has the potential to yield greater than 95% accuracy in detecting the smallest amount of cancer considered clinically relevant withing 15 min of tissue processing and imaging.
Thorough tumor resection is crucial for successful treatment of squamous cell carcinomas (SCCs) because positive surgical margins are associated with poor patient prognosis. Current methods of margin analysis, however, are limited by inefficient pathological read-times that increase exponentially with tissue size. Here, a fluorescence paired-agent imaging (PAI) approach is presented to identify regions of tumor burden in whole, thick tissue margins to act as a rapid screening tool and help focus pathological evaluation. The approach was applied to mouse models of head and neck SCC, and positive tumor burden was detected and localized in deep tissue margins up to 1.3 mm thick. Serial sections with hematoxylin and eosin and EGFR-immunostaining demonstrated good correlation with binding potential (BP: proportional to targeted biomolecule concentration) maps generated from PAI fluorescence slices and confirmed the presence of positive margins suggested by high intensity regions in the whole tissue BP maps. Findings support the use of PAI as a rapid screening method for detecting regions of tumor burden in large, en face tumor margin sections.
Head and neck squamous cell carcinomas (HNSCCs) have high levels of chromosomal instability and epidermal growth factor receptor (EGFR) overexpression, both of which drive their tumorigenesis. While drug treatment targeting the extracellular domain of EGFR has shown some success, mutations and alternate intracellular pathways contribute to therapeutic resistance. Therefore, a dynamic in vivo method to monitor binding and downstream cell signaling is warranted. Previous work has demonstrated that paired-agent imaging (PAI) is a powerful tool to quantify extracellular EGFR, and so this work extends the same principles to quantify intracellular protein target engagement. Here, in ovo models were used to grow human HNSCC xenografts – eggs were windowed to reveal the chorioallantoic membrane (CAM) of chicken embryos and tumors were implanted on its surface. A fluorescent cocktail of both intracellular and extracellular, targeted and untargeted agents (four agents total) was intravenously injected and multispectral imaging was performed over two hours. To isolate the relative quantities of each agent, a spectral fitting procedure was employed that accounted for the linear contributions of each fluorescent agent and autofluorescence, and the non-linear absorbing contributions of oxy- and deoxyhemoglobin. This unmixing was performed on a pixel-by-pixel basis to generate distribution maps of each individual dye, and then motion correction was done, followed by a convolution correction to account for delivery differences. Results demonstrated successful unmixing of individual fluorophores such that a ratiometric calculation could be applied to extract both intracellular and extracellular binding potential (BP), which is proportional to EGFR concentration.
The status of lymph node is considered a critical prognostic factor for staging and guiding the future adjuvant treatment in many cancer types. The estimation of undetected micrometastases (0.2-2mm diameter) by conventional pathology was around 30-60% cases which has created a demand for the development of more fast and accurate approaches. In response, a paired-agent imaging approach is presented by employing a control imaging agent to allow rapid, quantitative mapping of microscopic cancer cells in lymph nodes to guide pathology sectioning. To identify the most feasible and effective protocol using this approach to detect micrometastases intraoperatively, swine cervical lymph nodes were used to evaluate the potential of different protocols for the agents to diffuse into and out of intact nodes. Aby-029, an anti-EGFR affibody molecule labeled with IRDye-800CW was used as targeted imaging agent, and the IRDye-700DX carboxylate was used as control agent. The time-course paired-agent fluorescence of whole lymph node were recorded to monitor the uptake and washout kinetics. Subsequently, lymph nodes were frozen-sectioned and imaged under an 85-um resolution fluorescence imaging system (Pearl, LICOR) to confirm equivalence of spatial distribution of both agents in the entire node. After much trial-and error, the intranodal infusion staining and rinsing protocol demonstrated promising results that both imaging agents shown strong correlation with each other in the absence of cancer cells (r=0.99, p<0.001). This methodology indicated the potential of using paired-agent imaging approach to allow rapid and sufficient detection of micrometastases in excised lymph nodes intraoperatively.
Filtered backprojection (FBP) reconstruction is a simple and rapid technique for reconstructing tomographic data. Optical projection tomography (OPT) for instance, makes use of this technique to facilitate three dimensional visualization of optically clear biological tissues. However, for the case of nontransparent specimens where scattering dominates, more representative algorithms are required to model the behavior of light through the sample such that the object of interest can be recreated. In this work, model-based iterative techniques are investigated for use with an angle-restricted fluorescence OPT system for the specific application of imaging lymph nodes. Through physical and simulated phantoms, the effects of model inaccuracies in Monte Carlo-generated system matrices was evaluated. Findings demonstrated the importance of accurately capturing detector response, and that sample optical properties were more influential than sample geometry in affecting the reconstructed results.
Fluorescence optical projection tomography with angular restriction is a promising technique for mesoscopic imaging of low scattering biological samples. As such, an angular domain system is being developed to address the problem of undetected micrometastases in lymph node biopsy tissues. Previous studies demonstrated its utility for lymph node applications and rigorously characterized imaging performance of the system. Through this evaluation, image artifacts were revealed in the reconstructions that limit achievable contrast and resolution. The objective of this work was to investigate the cause of those artifacts and potential remedies. Results demonstrated that an incorrect axis of rotation and detector response were the significant contributors of image artifacts, but post-acquisition calibration could account for the errors.
The ADEPT system is an angular domain optical projection tomography imaging system being developed to address the problem of undetected micrometastases in lymph node biopsy tissues. The relatively weak scattering nature of lymph nodes combined with a very low numerical aperture enables the approximation of straight-line projections that can be reconstructed into images simply with filtered back-projection (FBP). This was demonstrated in previous work where 0.2 mm diameter inclusions were detected in ~1 cm diameter lymph nodes; and while FBP was sufficient, the Radon transform is not a true representation of the imaging system. To investigate the degree of improvement that a more complex reconstruction algorithm could provide, a Monte Carlo based system matrix was generated and used to solve the inverse problem. Simulated phantoms were used to test this, and results revealed greater detection sensitivity at the periphery of samples. Such findings lend guidance in the ongoing design of the ADEPT system and so more robust evaluation of the reliability of the system matrix will be implemented in future work.
Sentinel lymph node involvement is recognized as a prognostic factor in breast cancer staging and is essential to guide optimal treatment. The possibility of missed micrometastases by using conventional methods was estimated around 20-60% cases has created a demand for the development of more accurate approaches. A paired-agent imaging approach is presented by employing a control imaging agent to allow rapid, quantitative mapping of microscopic populations of tumor cells in lymph nodes to guide pathology sectioning. To test the feasibility of this approach to identify micrometastases, lymph node micrometastases biological tissue model was developed and were stained with targeted and control imaging agent solution to evaluate the binding potential of the agents of intact nodes. ABY-029, an EGFR specific affibody was labeled with IRDye-800CW(LICOR) as targeted agent and IRDye-700DX was hydrolyzed as control agent. Lymph nodes phantoms were stained for 60 min, followed by 60 min rinsing, and the fluorescence of whole lymph node phantoms were recorded to evaluate the spatial distribution of both agents in the entire phantom. Measured binding potential of targeted agent between micrometastases and control regions were 0.652 ± 0.130 and -0.008 ± 0.042 respectively (p < 0.0001). The results demonstrate the potential to enhance the sensitivity of lymph node pathology using paired-agent imaging in a whole human lymph node.
Lymph node biopsy is a primary means of staging breast cancer, yet standard pathological techniques are time-consuming and typically sample less than 1% of the total node volume. A low-cost fluorescence optical projection tomography (OPT) protocol is demonstrated for rapid imaging of whole lymph nodes in three dimensions. The relatively low scattering properties of lymph node tissue can be leveraged to significantly improve spatial resolution of lymph node OPT by employing angular restriction of photon detection. It is demonstrated through porcine lymph node metastases models that simple filtered-backprojection reconstruction is sufficient to detect and localize 200-μm-diameter metastases (the smallest clinically significant) in 1-cm-diameter lymph nodes.
The presence of lymph node metastases played as a critical prognostic factor in breast cancer treatment and guiding the future adjuvant treatment. The possibility of missed micrometastases by conventional pathology was estimated around 20-60% cases has created a demand for the development of more accurate approaches. Here, a paired-agent imaging approach is presented that employs a control imaging agent to allow rapid, quantitative mapping of microscopic populations of tumor cells in lymph nodes to guide pathology sectioning. To test the feasibility of this approach to identify micrometastases, healthy rat and human lymph nodes were stained with targeted and control imaging agent solution to evaluate the potential for the agents to diffuse into and out of intact nodes. Erbitux, an EGFR specific antibody was labeled with IRDye-700DX(LICOR) as targeted agent and IRDye-800CW was labeled to rat IgG as control agent. Lymph nodes were stained for 60 min, followed by 30 min rinsing, and the uptake and washout of fluorescence were recorded. Subsequently, lymph nodes were frozen-sectioned and imaged under an 80- um resolution fluorescence imaging system (Pearl, LICOR) to confirm equivalence of spatial distribution of both agents in the entire node. Both imaging agents correlated well with each other(r=0.877) and the binding potential of targeted agent was found to be 0.08 ± 0.22 along the lymph node in the absence of binding. The results demonstrate this approach’s potential to enhance the sensitivity of lymph node pathology by detecting fewer than 1000 cell in a whole human lymph node.
Sentinel lymph node biopsy is a primary mean of staging cancer; however, the time-intensive nature of standard pathology limits the volume of the node that can be assessed. As a result, micrometastases can be missed, which have been shown to affect treatment decisions and therefore clinical outcomes. Optical imaging offers a potential solution for improved sensitivity and larger tissue evaluation, but an understanding of optical properties is necessary because of the high scattering nature of biological tissue. Here, time-domain optical imaging and measures of transmittance are used to characterize the optical properties of porcine lymph nodes at 685 nm and 780 nm. Results demonstrated values comparable to that of other soft biological tissue (685 nm: μa = 0.09 ± 0.01cm-1 , μs’ = 2.60 ± 0.42 cm-1 , g = 0.95; 780 nm: μa = 0.24 ± 0.10cm-1 , μs’ = 3.35 ± 0.14 cm-1 , g = 0.92). Based on these coefficients, optical properties of TiO2 were investigated so that a protocol to fabricate a lymph node tissue-mimicking phantom could be defined.
Skull base tumors are particularly difficult to visualize and access for surgeons because of the crowded environment and close proximity of vital structures, such as cranial nerves. As a result, accidental nerve damage is a significant concern and the likelihood of tumor recurrence is increased because of more conservative resections that attempt to avoid injuring these structures. In this study, a paired-agent imaging method with direct administration of fluorophores is applied to enhance cranial nerve identification. Here, a control imaging agent (ICG) accounts for non-specific uptake of the nerve-targeting agent (Oxazine 4), and ratiometric data analysis is employed to approximate binding potential (BP, a surrogate of targeted biomolecule concentration). For clinical relevance, animal experiments and simulations were conducted to identify parameters for an optimized stain and rinse protocol using the developed paired-agent method. Numerical methods were used to model the diffusive and kinetic behavior of the imaging agents in tissue, and simulation results revealed that there are various combinations of stain time and rinse number that provide improved contrast of cranial nerves, as suggested by optimal measures of BP and contrast-to-noise ratio.
Nerve preservation during surgery is critical because damage can result in significant morbidity. This remains a challenge especially for skull base surgeries where cranial nerves (CNs) are involved because visualization and access are particularly poor in that location. We present a paired-agent imaging method to enhance identification of CNs using nerve-specific fluorophores. Two myelin-targeting imaging agents were evaluated, Oxazine 4 and Rhodamine 800, and coadministered with a control agent, indocyanine green, either intravenously or topically in rats. Fluorescence imaging was performed on excised brains ex vivo, and nerve contrast was evaluated via paired-agent ratiometric data analysis. Although contrast was improved among all experimental groups using paired-agent imaging compared to conventional, solely targeted imaging, Oxazine 4 applied directly exhibited the greatest enhancement, with a minimum 3 times improvement in CNs delineation. This work highlights the importance of accounting for nonspecific signal of targeted agents, and demonstrates that paired-agent imaging is one method capable of doing so. Although staining, rinsing, and imaging protocols need to be optimized, these findings serve as a demonstration for the potential use of paired-agent imaging to improve contrast of CNs, and consequently, surgical outcome.
Brain tumors represent a leading cause of cancer death for people under the age of 40 and the probability complete surgical resection of brain tumors remains low owing to the invasive nature of these tumors and the consequences of damaging healthy brain tissue. Molecular imaging is an emerging approach that has the potential to improve the ability for surgeons to correctly discriminate between healthy and cancerous tissue; however, conventional molecular imaging approaches in brain suffer from significant background signal in healthy tissue or an inability target more invasive sections of the tumor. This work presents initial studies investigating the ability of novel dual-tracer molecular imaging strategies to be used to overcome the major limitations of conventional “single-tracer” molecular imaging. The approach is evaluated in simulations and in an in vivo mice study with animals inoculated orthotopically using fluorescent human glioma cells. An epidermal growth factor receptor (EGFR) targeted Affibody-fluorescent marker was employed as a targeted imaging agent, and the suitability of various FDA approved untargeted fluorescent tracers (e.g. fluorescein & indocyanine green) were evaluated in terms of their ability to account for nonspecific uptake and retention of the targeted imaging agent. Signal-to-background ratio was used to measure and compare the amount of reporter in the tissue between targeted and untargeted tracer. The initial findings suggest that FDA-approved fluorescent imaging agents are ill-suited to act as untargeted imaging agents for dual-tracer fluorescent guided brain surgery as they suffer from poor delivery to the healthy brain tissue and therefore cannot be used to identify nonspecific vs. specific uptake of the targeted imaging agent where current surgery is most limited.
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