SignificanceEmerging evidence that aggressive breast tumors rely on various substrates including lipids and glucose to proliferate and recur necessitates the development of tools to track multiple metabolic and vascular endpoints concurrently in vivo.AimOur quantitative spectroscopy technique provides time-matched measurements of the three major axes of breast cancer metabolism as well as tissue vascular properties in vivo.ApproachWe leverage exogenous fluorophores to quantify oxidative phosphorylation, glucose uptake, and fatty acid oxidation, and endogenous contrast for measurements of hemoglobin and oxygen saturation. An inverse Monte Carlo algorithm corrects for aberrations resulting from tissue optical properties, allowing the unmixing of spectrally overlapping fluorophores.ResultsImplementation of our inverse Monte Carlo resulted in a linear relationship of fluorophore intensity with concentration (R2<0.99) in tissue-mimicking phantom validation studies. We next sequenced fluorophore delivery to faithfully recapitulate independent measurement of each fluorophore. The ratio of Bodipy FL C16/2-NBDG administered to a single animal is not different from that in paired animals receiving individual fluorophores (p=n.s.). Clustering of five variables was effective in distinguishing tumor from mammary tissue (sensitivity = 0.75, specificity = 0.83, and accuracy = 0.79).ConclusionsOur system can measure major axes of metabolism and associated vascular endpoints, allowing for future study of tumor metabolic flexibility.
Significance: Decreasing the oxygen consumption rate (OCR) of tumor cells is a powerful method for ameliorating tumor hypoxia. However, quantifying the change in OCR is challenging in complex experimental systems.
Aim: We present a method for quantifying the OCR of two tumor cell lines using oxygen-sensitive dual-emissive boron nanoparticles (BNPs). We hypothesize that our BNP results are equivalent to the standard Seahorse assay.
Approach: We quantified the spectral emissions of the BNP and accounted for external oxygen diffusion to quantify OCR over 24 h. The BNP-computed OCR of two breast cancer cell lines, E0771 and 4T07, were compared with their respective Seahorse assays. Both cell lines were also irradiated to quantify radiation-induced changes in the OCR.
Results: Using a Bland–Altman analysis, our BNPs OCR was equivalent to the standard Seahorse assay. Moreover, in an additional experiment in which we irradiated the cells at their 50% survival fraction, the BNPs were sensitive enough to quantify 24% reduction in OCR after irradiation.
Conclusions: Our results conclude that the BNPs are a viable alternative to the Seahorse assay for quantifying the OCR in cells. The Bland–Altman analysis showed that these two methods result in equivalent OCR measurements. Future studies will extend the OCR measurements to complex systems including 3D cultures and in vivo models, in which OCR measurements cannot currently be made.
The discovery of new treatments for cancer is imperative. Recently, we showed in a proof-of-concept study that SYnergistic IMmuno PHOtothermal NanotherapY (SYMPHONY) is a powerful treatment for metastatic bladder cancer and brain tumor in mouse models. Our work has recently demonstrated that combining immunotherapy checkpoint inhibitors and gold nanostar (AuNS) photothermal therapy (PTT) is more effective in killing primary tumors and activating the immune system to eradicate tumors at distant sites, than each individual treatment alone. When the tumor is being ablated via PTT in mice models, using low intensity heat from a near infrared laser, the dying tumor releases proteins that trigger the immune system to destroy remaining tumor cells. Immune checkpoint inhibitors prevent the tumor cells from hiding from the immune system’s mechanisms; thus, the immune system becomes capable of attacking distant secondary tumors, after the primary tumor has been eradicated using AuNS mediated PTT. The data shows that after the cured mice were rechallenged with bladder cancer cells, no tumor formation was observed after 60 days; hence these results indicate that the SYMPHONY treatment can function as a cancer vaccine and lead to long-lasting immunity.
Although nanotechnology has led to important advances in in vitro diagnostics, the development of nanosensors for in vivo molecular detection remains very challenging. Here, we demonstrated the proof‐of‐principle of in vivo detection of nucleic acid targets using a promising type of surface‐enhanced Raman scattering (SERS) nanosensor implanted in the skin of a large animal model (pig). The in vivo “smart tattoo” nanosensor used in this study employs the “inverse molecular sentinel” (iMS) detection scheme, which is a label-free homogeneous biosensing system based on a non-enzymatic DNA strand-displacement process and conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. In this study, plasmonics‐active nanostar was utilized as an efficient in vivo SERS sensing platform due to their tunable absorption bands in the near infrared region of the “tissue optical window. The results of this study illustrate the usefulness of SERS iMS nanosensors as an implantable skin‐based in vivo biosensing platform, providing a foundation for developments in continuous health status sensing, disease biomarker monitoring, and other clinical translation applications.
Gregory Palmer, Hengtao Zhang, Chen-Ting Lee, Husam Mikati, Joseph Herbert, Marlee Krieger, Jesko von Windheim, Dave Koester, Daniel Stevenson, Daniel Rocke, Ramon Esclamado, Alaatin Erkanli, Nirmala Ramanujam, Mark Dewhirst, Walter Lee
Diffuse reflectance spectroscopy (DRS) represents a quantitative, noninvasive, nondestructive means of assessing vascular oxygenation, vascularity, and structural properties. However, it is known that such measurements can be influenced by the effects of pressure, which is a major concern for reproducible and operator-independent assessment of tissues. Second, regular calibration is a necessary component of quantitative DRS to account for factors such as lamp decay and fiber bending. Without a means of reliably controlling for these factors, the accuracy of any such assessments will be reduced, and potentially biased. To address these issues, a self-calibrating, pressure-controlled DRS system is described and applied to both a patient-derived xenograft glioma model, as well as a set of healthy volunteers for assessments of oral mucosal tissues. It was shown that pressure had a significant effect on the derived optical parameters, and that the effects on the optical parameters were magnified with increasing time and pressure levels. These findings indicate that not only is it critical to integrate a pressure sensor into a DRS device, but that it is also important to do so in an automated way to trigger a measurement as soon as possible after probe contact is made to minimize the perturbation to the tissue site.
Biocompatible gold nanostars (GNS) with tip-enhanced electromagnetic and optical properties have been developed and applied for multifunctional cancer diagnostics and therapy (theranostics). Their multiple sharp branches acting like “lightning rods” can convert safely and efficiently light into heat. As with other nanoparticles, GNS sizes can be controlled so that they passively accumulate in tumors due to the enhanced permeability and retention (EPR) effect of tumor vasculature. This feature improves tumor-targeting precision and permits the use of reduced laser energy required to destroy the targeted cancer cells. The ability to selectively heat tumor areas where GNS are located while keeping surrounding healthy tissues at significantly lower temperatures offers significant advantages over other thermal therapies. GNS-mediated photothermal therapy combined with checkpoint immunotherapy was shown to reverse tumor-mediated immunosuppression, leading to the treatment of not only primary tumors but also cancer metastasis as well as inducing effective long-lasting immunity, i.e. an anticancer ‘vaccine’ effect.
We have developed a portable, breast margin assessment probe leveraging diffuse optical spectroscopy to quantify the morphological landscape of breast tumor margins during breast conserving surgery. The approach presented here leverages a custom-made 16-channel annular photodiode imaging array (arranged in a 4×4 grid), a raster-scanning imaging platform with precision pressure control, and compressive sensing with an optimized set of eight wavelengths in the visible spectral range. A scalable Monte-Carlo-based inverse model is used to generate optical property [μs′(λ) and μa(λ)] measures for each of the 16 simultaneously captured diffuse reflectance spectra. Subpixel sampling (0.75 mm) is achieved through incremental x, y raster scanning of the imaging probe, providing detailed optical parameter maps of breast margins over a 2×2 cm2 area in ∼9 min. The morphological landscape of a tumor margin is characterized using optical surrogates for the fat to fibroglandular content ratio, which has demonstrated diagnostic utility in delineating tissue subtypes in the breast.
Many studies have found that hypoxia, particularly cycling hypoxia (CH), can lead to enhanced tumor metastasis and resistance to radiation and chemotherapy. It was also reported that tumor total hemoglobin content (THb), which is directly related to tumor angiogenesis, can have significant impact on tumor’s response to radiation and neoadjuvant chemotherapy. There is a growing demand for technologies to measure tumor hypoxia and angiogenesis temporally in vivo. In this paper, a side-firing fiber optic sensor based on a multi-wavelength frequency-domain near infrared spectroscopy (FD-NIRS) instrument was used to quantify tumor oxygenation and hemoglobin concentrations in nude rats bearing human FaDu head and neck (H and N) tumors during normoxia and forced hyperoxia and cyclic hypoxia. Significant increase (with carbogen gas inhalation) or decrease (with reduced O2 supply) in tumor oxygenation was observed. The studies demonstrated the feasibility of the technology for longitudinal monitoring of H and N tumor’s response to therapy.
An inverse Monte Carlo based model has been developed to extract intrinsic fluorescence from turbid media. The goal of this work was to experimentally validate the model to extract intrinsic fluorescence of three biologically meaningful fluorophores related to metabolism from turbid media containing absorbers and scatterers. Experimental studies were first carried out on tissue-mimicking phantoms that contained individual fluorophores and their combinations, across multiple absorption, scattering, and fluorophore concentrations. The model was then tested in a murine tumor model to determine both the kinetics of fluorophore uptake as well as overall tissue fluorophore concentration through extraction of the intrinsic fluorescence of an exogenous contrast agent that reports on glucose uptake. Results show the model can be used to recover the true intrinsic fluorescence spectrum with high accuracy (R2 = 0.988) as well as accurately compute fluorophore concentration in both single and multiple fluorophores phantoms when appropriate calibration standards are available. In the murine tumor, the model-corrected intrinsic fluorescence could be used to differentiate drug dose injections between different groups. A strong linear correlation was observed between the extracted intrinsic fluorescence intensity and injected drug dose, compared with the distorted turbid tissue fluorescence.
The influence of the tumor microenvironment and hypoxia plays a significant role in determining cancer progression, treatment response, and treatment resistance. That the tumor microenvironment is highly heterogeneous with significant intratumor and intertumor variability presents a significant challenge in developing effective cancer therapies. Critical to understanding the role of the tumor microenvironment is the ability to dynamically quantify oxygen levels in the vasculature and tissue in order to elucidate the roles of oxygen supply and consumption, spatially and temporally. To this end, we describe the use of hyperspectral imaging to characterize hemoglobin absorption to quantify hemoglobin content and oxygen saturation, as well as dual emissive fluorescent/phosphorescent boron nanoparticles, which serve as ratiometric indicators of tissue oxygen tension. Applying these techniques to a window-chamber tumor model illustrates the role of fluctuations in hemoglobin saturation in driving changes in tissue oxygenation, the two being significantly correlated (r = 0.77). Finally, a green-fluorescence-protein reporter for hypoxia inducible factor-1 (HIF-1) provides an endpoint for hypoxic stress in the tumor, which is used to demonstrate a significant association between tumor hypoxia dynamics and HIF-1 activity in an in vivo demonstration of the technique.
This study demonstrates the use of optical spectroscopy for monitoring tumor oxygenation and metabolism in response to hyperoxic gas breathing. Hemoglobin saturation and redox ratio were quantified for a set of 14 and 9 mice, respectively, measured at baseline and during carbogen breathing (95% O2, 5% CO2). In particular, significant increases in hemoglobin saturation and fluorescence redox ratio were observed upon carbogen breathing. These data were compared with data obtained concurrently using an established invasive technique, the OxyLite partial oxygen pressure (pO2) system, which also showed a significant increase in pO2. It was found that the direction of changes were generally the same between all of the methods, but that the OxyLite system was much more variable in general, suggesting that optical techniques may provide a better assessment of global tumor physiology. Optical spectroscopy measurements are demonstrated to provide a reliable, reproducible indication of changes in tumor physiology in response to physiologic manipulation.
A hybrid optical device that uses a multimode fiber coupled to a tunable light source for illumination and a 2.4-mm photodiode for detection in contact with the tissue surface is developed as a first step toward our goal of developing a cost-effective, miniature spectral imaging device to map tissue optical properties in vivo. This device coupled with an inverse Monte Carlo model of reflectance is demonstrated to accurately quantify tissue absorption and scattering in tissue-like turbid synthetic phantoms with a wide range of optical properties. The overall errors for quantifying the absorption and scattering coefficients are 6.0±5.6 and 6.1±4.7%, respectively. Compared with fiber-based detection, having the detector right at the tissue surface can significantly improve light collection efficiency, thus reducing the requirement for sophisticated detectors with high sensitivity, and this design can be easily expanded into a quantitative spectral imaging system for mapping tissue optical properties in vivo.
We explore the use of Monte-Carlo-model-based approaches for the analysis of fluorescence and diffuse reflectance spectra measured ex vivo from breast tissues. These models are used to extract the absorption, scattering, and fluorescence properties of malignant and nonmalignant tissues and to diagnose breast cancer based on these intrinsic tissue properties. Absorption and scattering properties, including -carotene concentration, total hemoglobin concentration, hemoglobin saturation, and the mean reduced scattering coefficient are derived from diffuse reflectance spectra using a previously developed Monte Carlo model of diffuse reflectance. A Monte Carlo model of fluorescence described in an earlier manuscript was employed to retrieve the intrinsic fluorescence spectra. The intrinsic fluorescence spectra were decomposed into several contributing components, which we attribute to endogenous fluorophores that may present in breast tissues including collagen, NADH, and retinol/vitamin A. The model-based approaches removes any dependency on the instrument and probe geometry. The relative fluorescence contributions of individual fluorescing components, as well as -carotene concentration, hemoglobin saturation, and the mean reduced scattering coefficient display statistically significant differences between malignant and adipose breast tissues. The hemoglobin saturation and the reduced scattering coefficient display statistically significant differences between malignant and fibrous/benign breast tissues. A linear support vector machine classification using (1) fluorescence properties alone, (2) absorption and scattering properties alone, and (3) the combination of all tissue properties achieves comparable classification accuracies of 81 to 84% in sensitivity and 75 to 89% in specificity for discriminating malignant from nonmalignant breast tissues, suggesting each set of tissue properties are diagnostically useful for the discrimination of breast malignancy.
A Monte-Carlo-based model of fluorescence is developed that is capable of extracting the intrinsic fluorescence properties of tissue, which are independent of the absorption and scattering properties of tissue. This model is flexible in its applicability to different illumination-collection geometries and is also valid for a wide range of optical properties, representative of tissue in the UV-visible spectrum. This is potentially useful in a variety of biomedical applications, including cancer diagnostics and monitoring the physiological response to therapy. The model is validated using phantoms composed of hemoglobin (absorber), polystyrene spheres (scatterer), and furan-2 (fluorophore). It is found that this model is able to retrieve the intrinsic fluorescence spectra of the tissue phantoms and recover the intrinsic fluorescence intensity of furan within the phantoms to within a mean error of less than 10%.
We explore the effects of the illumination and collection geometry on optical spectroscopic diagnosis of breast cancer. Fluorescence and diffuse reflectance spectroscopy in the UV-visible spectral range are made with a multiseparation probe at three illumination-collection separations of 735, 980, and 1225 µm, respectively, from 13 malignant and 34 nonmalignant breast tissues. Statistical analysis is carried out on two types of data inputs: (1) the fluorescence and diffuse reflectance spectra recorded at each of the three illumination-collection separations and (2) the integrated fluorescence (at each excitation wavelength) or diffuse reflectance over the entire spectrum at all three illumination-collection separations. The results show that using the integrated fluorescence intensities recorded at a single excitation wavelength at all three illumination-collection separations can discriminate malignant from nonmalignant breast tissues with similar classification accuracy to that using spectral data measured at several excitation wavelengths with a single illumination-collection separation. These findings have significant implications with respect to the design of an optical system for breast cancer diagnosis. Examining the intensity attenuation at a single wavelength rather than spectral intensities at multiple wavelengths can significantly reduce the measurement and data processing time in a clinical setting as well as the cost and complexity of the optical system.
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