Portable optical spectroscopic assay for non-destructive measurement of key metabolic parameters on in vitro cancer cells and organotypic fresh tumor slices.

To enable non-destructive metabolic characterizations on in vitro cancer cells and organotypic tumor models for therapeutic studies in an easy-to-access way, we report a highly portable optical spectroscopic assay for simultaneous measurement of glucose uptake and mitochondrial function on various cancer models with high sensitivity. Well-established breast cancer cell lines (MCF-7 and MDA-MB-231) were used to validate the optical spectroscopic assay for metabolic characterizations, while fresh tumor samples harvested from both animals and human cancer patients were used to test the feasibility of our optical metabolic assay for non-destructive measurement of key metabolic parameters on organotypic tumor slices. Our optical metabolic assay captured that MCF-7 cells had higher mitochondrial metabolism, but lower glucose uptake compared to the MDA-MB-231 cells, which is consistent with our microscopy imaging and flow cytometry data, as well as the published Seahorse Assay data. Moreover, we demonstrated that our optical assay could non-destructively measure both glucose uptake and mitochondrial metabolism on the same cancer cell samples at one time, which remains challenging by existing metabolic tools. Our pilot tests on thin fresh tumor slices showed that our optical assay captured increased metabolic activities in tumors compared to normal tissues. Our non-destructive optical metabolic assay provides a cost-effective way for future longitudinal therapeutic studies using patient-derived organotypic fresh tumor slices through the lens of tumor energetics, which will significantly advance translational cancer research.

Non-contact optical spectroscopy for tumor-sensitive diffuse reflectance and fluorescence measurements on murine subcutaneous tissue models: Monte Carlo modeling and experimental validations.

Fiber-optic probes are commonly used in biomedical optical spectroscopy platforms for light delivery and collection. At the same time, it was reported that the inconsistent probe-sample contact could induce significant distortions in measured optical signals, which consequently cause large analysis errors. To address this challenge, non-contact optical spectroscopy has been explored for tissue characterizations. However, existing non-contact optical spectroscopy platforms primarily focused on diffuse reflectance measurements and may still use a fiber probe in which the probe was imaged onto the tissue surface using a lens, which serves as a non-contact probe for the measurements. Here, we report a fiber-probe-free, dark-field-based, non-contact optical spectroscopy for both diffuse reflectance and fluorescence measurements on turbid medium and tissues. To optimize the system design, we developed a novel Monte Carlo method to simulate such a non-contact setup for both diffuse reflectance and fluorescence measurements on murine subcutaneous tissue models with a spherical tumor-like target. We performed Monte Carlo simulations to identify the most tumor-sensitive configurations, from which we found that both the depth of the light focal point in tissue and the lens numerical aperture would dramatically affect the system's tumor detection sensitivity. We then conducted tissue-mimicking phantom studies to solidify these findings. Our reported Monte Carlo technique can be a useful computational tool for designing non-contact optical spectroscopy systems. Our non-contact optical setup and experimental findings will potentially offer a new approach for sensitive optical monitoring of tumor physiology in biological models using a non-contact optical spectroscopy platform to advance cancer research.

Low-cost compact optical spectroscopy and novel spectroscopic algorithm for point-of-care real-time monitoring of nanoparticle delivery in biological tissue models.

Objective: Real-time monitoring of nanoparticle delivery in biological models is essential to optimize nanoparticle-mediated therapies. However, few techniques are available for convenient real-time monitoring of nanoparticle concentrations in tissue samples. This work reported novel optical spectroscopic approaches for low-cost point-of-care real-time quantification of nanoparticle concentrations in biological tissue samples. Methods: Fiber probe measured diffuse reflectance can be described with a simple analytical model by introducing an explicit dependence on the reduced scattering coefficient. Relying on this, the changes on the inverse of diffuse reflectance are proportional to absorption change when the scattering perturbation is negligible. We developed this model with proper wavelength pairs and implemented it with both a standard optical spectroscopy platform and a low-cost compact spectroscopy device for near real-time quantification of nanoparticle concentrations in biological tissue models. Results: Both tissue-mimicking phantom and ex vivo tissue sample studies showed that our optical spectroscopic techniques could quantify nanoparticle concentrations in near real-time with high accuracies (less than 5% error) using only a pair of narrow wavelengths (530 nm and 630 nm). Conclusion: Novel low-cost point-of-care optical spectroscopic techniques were demonstrated for rapid accurate quantification of nanoparticle concentrations in tissue-mimicking medium and ex vivo tissue samples using optical signals measured at a pair of narrow wavelengths. Significance: Our methods will potentially facilitate real-time monitoring of nanoparticle delivery in biological models using low-cost point-of-care optical spectroscopy platforms, which will significantly advance nanomedicine in cancer research.

Single cell metabolism: current and future trends.

Single cell metabolomics is an emerging and rapidly developing field that complements developments in single cell analysis by genomics and proteomics. Major goals include mapping and quantifying the metabolome in sufficient detail to provide useful information about cellular function in highly heterogeneous systems such as tissue, ultimately with spatial resolution at the individual cell level. The chemical diversity and dynamic range of metabolites poses particular challenges for detection, identification and quantification. In this review we discuss both significant technical issues of measurement and interpretation, and progress toward addressing them, with recent examples from diverse biological systems. We provide a framework for further directions aimed at improving workflow and robustness so that such analyses may become commonly applied, especially in combination with metabolic imaging and single cell transcriptomics and proteomics.

[18F]Fluoro-DCP, a first generation PET radiotracer for monitoring protein sulfenylation in vivo.

Redox metabolism plays essential functions in the pathology of cancer and many other diseases. While several radiotracers for imaging redox metabolism have been developed, there are no reports of radiotracers for in vivo imaging of protein oxidation. Here we take the first step towards this goal and describe the synthesis and kinetic properties of a new positron emission tomography (PET) [18F]Fluoro-DCP radiotracer for in vivo imaging of protein sulfenylation. Time course biodistribution and PET/CT studies using xenograft animal models of Head and Neck Squamous Cell Cancer (HNSCC) demonstrate its capability to distinguish between tumors with radiation sensitive and resistant phenotypes consistent with previous reports of decreased protein sulfenylation in clinical specimens of radiation resistant HNSCC. We envision further development of this technology to aid research efforts towards improving diagnosis of patients with radiation resistant tumors.

Understanding the sources of errors in ex vivo Hsp90 molecular imaging for rapid-on-site breast cancer diagnosis.

Overexpression of heat shock protein 90 (Hsp90) on the surface of breast cancer cells makes it an attractive molecular biomarker for breast cancer diagnosis. Before a ubiquitous diagnostic method can be established, an understanding of the systematic errors in Hsp90-based imaging is essential. In this study, we investigated three factors that may influence the sensitivity of ex vivo Hsp90 molecular imaging: time-dependent tissue viability, nonspecific diffusion of an Hsp90 specific probe (HS-27), and contact-based imaging. These three factors will be important considerations when designing any diagnostic imaging strategy based on fluorescence imaging of a molecular target on tissue samples.

Empirical method for rapid quantification of intrinsic fluorescence signals of key metabolic probes from optical spectra measured on tissue-mimicking turbid medium.

SIGNIFICANCE: Optical fluorescence spectroscopy technique has been explored extensively to quantify both glucose uptake and mitochondrial metabolism with proper fluorescent probes in small tumor models in vivo. However, it remains a great challenge to rapidly quantify the intrinsic metabolic fluorophores from the optically measured fluorescence spectra that contain significant distortions due to tissue absorption and scattering. AIM: To enable rapid spectral data processing and quantify the in vivo metabolic parameters in real-time, we present an empirical ratio-metric method for rapid fluorescence spectra attenuation correction with high accuracy. APPROACH: A first-order approximation of intrinsic fluorescence spectra can be obtained by dividing the fluorescence spectra by diffuse reflectance spectra with some variable powers. We further developed this approximation for rapid extraction of intrinsic key metabolic probes (2-NBDG for glucose uptake and TMRE for mitochondrial function) by dividing the distorted fluorescence spectra by diffuse reflectance intensities recorded at excitation and emission peak with a pair of system-dependent powers. Tissue-mimicking phantom studies were conducted to evaluate the method. RESULTS: The tissue-mimicking phantom studies demonstrated that our empirical method could quantify the key intrinsic metabolic probes in near real-time with an average percent error of ∼5 % . CONCLUSIONS: An empirical method was demonstrated for rapid quantification of key metabolic probes from fluorescence spectra measured on a tissue-mimicking turbid medium. The proposed method will potentially facilitate real-time monitoring of key metabolic parameters of tumor models in vivo using optical spectroscopy, which will significantly advance translational cancer research.

In Vivo Optical Metabolic Imaging of Long-Chain Fatty Acid Uptake in Orthotopic Models of Triple-Negative Breast Cancer.

Targeting a tumor's metabolic dependencies is a clinically actionable therapeutic approach; however, identifying subtypes of tumors likely to respond remains difficult. The use of lipids as a nutrient source is of particular importance, especially in breast cancer. Imaging techniques offer the opportunity to quantify nutrient use in preclinical tumor models to guide development of new drugs that restrict uptake or utilization of these nutrients. We describe a fast and dynamic approach to image fatty acid uptake in vivo and demonstrate its relevance to study both tumor metabolic reprogramming directly, as well as the effectiveness of drugs targeting lipid metabolism. Specifically, we developed a quantitative optical approach to spatially and longitudinally map the kinetics of long-chain fatty acid uptake in in vivo murine models of breast cancer using a fluorescently labeled palmitate molecule, Bodipy FL c16. We chose intra-vital microscopy of mammary tumor windows to validate our approach in two orthotopic breast cancer models: a MYC-overexpressing, transgenic, triple-negative breast cancer (TNBC) model and a murine model of the 4T1 family. Following injection, Bodipy FL c16 fluorescence increased and reached its maximum after approximately 30 min, with the signal remaining stable during the 30-80 min post-injection period. We used the fluorescence at 60 min (Bodipy60), the mid-point in the plateau region, as a summary parameter to quantify Bodipy FL c16 fluorescence in subsequent experiments. Using our imaging platform, we observed a two- to four-fold decrease in fatty acid uptake in response to the downregulation of the MYC oncogene, consistent with findings from in vitro metabolic assays. In contrast, our imaging studies report an increase in fatty acid uptake with tumor aggressiveness (6NR, 4T07, and 4T1), and uptake was significantly decreased after treatment with a fatty acid transport inhibitor, perphenazine, in both normal mammary pads and in the most aggressive 4T1 tumor model. Our approach fills an important gap between in vitro assays providing rich metabolic information at static time points and imaging approaches visualizing metabolism in whole organs at a reduced resolution.

Numerical investigation of depth-sensitive diffuse reflectance and fluorescence measurements on murine subcutaneous tissue with growing solid tumors.

In most biomedical optical spectroscopy platforms, a fiber-probe consisting of single or multiple illumination and collection fibers was commonly used for the delivery of illuminating light and the collection of emitted light. Typically, the signals from all collection fibers were combined and then sampled to characterize tissue samples. Such simple averaged optical measurements may induce significant errors for in vivo tumor characterization, especially in longitudinal studies where the tumor size and location vary with tumor stages. In this study, we utilized the Monte Carlo technique to optimize the fiber-probe geometries of a spectroscopy platform to enable tumor-sensitive diffuse reflectance and fluorescence measurements on murine subcutaneous tissues with growing solid tumors that have different sizes and depths. Our data showed that depth-sensitive techniques offer improved sensitivity in tumor detection compared to the simple averaged approach in both reflectance and fluorescence measurements. Through the numerical studies, we optimized the source-detector distances, fiber diameters, and numerical apertures for sensitive measurement of small solid tumors with varying size and depth buried in murine subcutaneous tissues. Our study will advance the design of a fiber-probe in an optical spectroscopy system that can be used for longitudinal tumor metabolism and vasculature monitoring.

Optical Imaging of Glucose Uptake and Mitochondrial Membrane Potential to Characterize Her2 Breast Tumor Metabolic Phenotypes.

With the large number of women diagnosed and treated for breast cancer each year, the importance of studying recurrence has become evident due to most deaths from breast cancer resulting from tumor recurrence following therapy. To mitigate this, cellular and molecular pathways used by residual disease prior to recurrence must be studied. An altered metabolism has long been considered a hallmark of cancer, and several recent studies have gone further to report metabolic dysfunction and alterations as key to understanding the underlying behavior of dormant and recurrent cancer cells. Our group has used two probes, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG) and tetramethyl rhodamine ethyl ester (TMRE), to image glucose uptake and mitochondrial membrane potential, respectively, to report changes in metabolism between primary tumors, regression, residual disease, and after regrowth in genetically engineered mouse (GEM)-derived mammospheres. Imaging revealed unique metabolic phenotypes across the stages of tumor development. Although primary mammospheres overexpressing Her2 maintained increased glucose uptake ("Warburg effect"), after Her2 downregulation, during regression and residual disease, mammospheres appeared to switch to oxidative phosphorylation. Interestingly, in mammospheres where Her2 overexpression was turned back on to model recurrence, glucose uptake was lowest, indicating a potential change in substrate preference following the reactivation of Her2, reeliciting growth. Our findings highlight the importance of imaging metabolic adaptions to gain insight into the fundamental behaviors of residual and recurrent disease. IMPLICATIONS: This study demonstrates these functional fluorescent probes' ability to report metabolic adaptations during primary tumor growth, regression, residual disease, and regrowth in Her2 breast tumors.

Simultaneous in vivo optical quantification of key metabolic and vascular endpoints reveals tumor metabolic diversity in murine breast tumor models.

Therapeutically exploiting vascular and metabolic endpoints becomes critical to translational cancer studies because altered vascularity and deregulated metabolism are two important cancer hallmarks. The metabolic and vascular phenotypes of three sibling breast tumor lines with different metastatic potential are investigated in vivo with a newly developed quantitative spectroscopy system. All tumor lines have different metabolic and vascular characteristics compared to normal tissues, and there are strong positive correlations between metabolic (glucose uptake and mitochondrial membrane potential) and vascular (oxygen saturations and hemoglobin concentrations) parameters for metastatic (4T1) tumors but not for micrometastatic (4T07) and nonmetastatic (67NR) tumors. A longitudinal study shows that both vascular and metabolic endpoints of 4T1 tumors increased up to a specific tumor size threshold beyond which these parameters decreased. The synchronous changes between metabolic and vascular parameters, along with the strong positive correlations between these endpoints suggest that 4T1 tumors rely on strong oxidative phosphorylation in addition to glycolysis. This study illustrates the great potential of our optical technique to provide valuable dynamic information about the interplay between the metabolic and vascular status of tumors, with important implications for translational cancer investigations.

Near-simultaneous quantification of glucose uptake, mitochondrial membrane potential, and vascular parameters in murine flank tumors using quantitative diffuse reflectance and fluorescence spectroscopy.

The shifting metabolic landscape of aggressive tumors, with fluctuating oxygenation conditions and temporal changes in glycolysis and mitochondrial metabolism, is a critical phenomenon to study in order to understand negative treatment outcomes. Recently, we have demonstrated near-simultaneous optical imaging of mitochondrial membrane potential (MMP) and glucose uptake in non-tumor window chambers, using the fluorescent probes tetramethylrhodamine ethyl ester (TMRE) and 2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Here, we demonstrate a complementary technique to perform near-simultaneous in vivo optical spectroscopy of tissue vascular parameters, glucose uptake, and MMP in a solid tumor model that is most often used for therapeutic studies. Our study demonstrates the potential of optical spectroscopy as an effective tool to quantify the vascular and metabolic characteristics of a tumor, which is an important step towards understanding the mechanisms underlying cancer progression, metastasis, and resistance to therapies.

Metaboloptics: Visualization of the tumor functional landscape via metabolic and vascular imaging.

Many cancers adeptly modulate metabolism to thrive in fluctuating oxygen conditions; however, current tools fail to image metabolic and vascular endpoints at spatial resolutions needed to visualize these adaptations in vivo. We demonstrate a high-resolution intravital microscopy technique to quantify glucose uptake, mitochondrial membrane potential (MMP), and SO2 to characterize the in vivo phentoypes of three distinct murine breast cancer lines. Tetramethyl rhodamine, ethyl ester (TMRE) was thoroughly validated to report on MMP in normal and tumor-bearing mice. Imaging MMP or glucose uptake together with vascular endpoints revealed that metastatic 4T1 tumors maintained increased glucose uptake across all SO2 ("Warburg effect"), and also showed increased MMP relative to normal tissue. Non-metastatic 67NR and 4T07 tumor lines both displayed increased MMP, but comparable glucose uptake, relative to normal tissue. The 4T1 peritumoral areas also showed a significant glycolytic shift relative to the tumor regions. During a hypoxic stress test, 4T1 tumors showed significant increases in MMP with corresponding significant drops in SO2, indicative of intensified mitochondrial metabolism. Conversely, 4T07 and 67NR tumors shifted toward glycolysis during hypoxia. Our findings underscore the importance of imaging metabolic endpoints within the context of a living microenvironment to gain insight into a tumor's adaptive behavior.

Scattering-independent glucose absorption measurement using a spectrally resolved reflectance setup with specialized variable source-detector separations.

We report a novel approach for the accurate measurement of glucose absorption in turbid media using a spectrally resolved reflectance setup. Our proposed reflectance setup with specialized variable source-detector separations enables scattering-independent absorption measurement, which is critical to in vivo long-term glucose concentration monitoring. Starting from the first-order approximation of the radiative transfer equation (RTE), we developed a scattering-independent glucose absorption measurement method and then evaluated this approach by Monte Carlo simulations as well as tissue-mimicking phantom studies in which glucose concentration was accurately measured. Our study demonstrates the potential of our proposed scattering-independent absorption measurement technique as an effective tool to quantify glucose levels in turbid media, which is an important step towards future in vivo long-term glucose concentration monitoring in human subjects.

Near-simultaneous intravital microscopy of glucose uptake and mitochondrial membrane potential, key endpoints that reflect major metabolic axes in cancer.

While the demand for metabolic imaging has increased in recent years, simultaneous in vivo measurement of multiple metabolic endpoints remains challenging. Here we report on a novel technique that provides in vivo high-resolution simultaneous imaging of glucose uptake and mitochondrial metabolism within a dynamic tissue microenvironment. Two indicators were leveraged; 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) reports on glucose uptake and Tetramethylrhodamine ethyl ester (TMRE) reports on mitochondrial membrane potential. Although we demonstrated that there was neither optical nor chemical crosstalk between 2-NBDG and TMRE, TMRE uptake was significantly inhibited by simultaneous injection with 2-NBDG in vivo. A staggered delivery scheme of the two agents (TMRE injection was followed by 2-NBDG injection after a 10-minute delay) permitted near-simultaneous in vivo microscopy of 2-NBDG and TMRE at the same tissue site by mitigating the interference of 2-NBDG with normal glucose usage. The staggered delivery strategy was evaluated under both normoxic and hypoxic conditions in normal tissues as well as in a murine breast cancer model. The results were consistent with those expected for independent imaging of 2-NBDG and TMRE. This optical imaging technique allows for monitoring of key metabolic endpoints with the unique benefit of repeated, non-destructive imaging within an intact microenvironment.

Spectral diffuse reflectance and autofluorescence imaging can perform early prediction of blood vessel occlusion in skin flaps.

Flap transfer has become a common technique in reconstructive surgery. However, a significant number of compromised skin flaps are not successfully salvaged because the current clinical method for flap assessment relies heavily on the clinician's experience. Vascular occlusion is the major reason for flap failure, thus the accurate and objective early prediction of blood vessel occlusion is vitally important. Our parallel point measurement study has demonstrated the great potential of joint diffuse reflectance and autofluorescence spectroscopy in the early detection and differentiation of venous and arterial occlusion in skin flaps. Unfortunately, the technique of point measurements is not suitable to examine a large skin flap when a high spatial resolution is required. In this study, we attempted to overcome this problem by performing spectral diffuse reflectance and autofluorescence imaging on a rat skin flap model. Both imaging data and reconstructed spectra were used to statistically differentiate control flaps, arterially occluded flaps and venously occluded flaps. Our preliminary results suggest that the technique of joint diffuse reflectance and autofluorescence spectroscopic imaging can achieve high classification accuracy thus could be used to detect and differentiate flaps with venous and arterial occlusion accurately at an early time point in a large skin flap. Typical reconstructed spectra of (a) diffuse reflectance and (b) autofluorescence after normalization.

Dark field optical imaging reveals vascular changes in an inducible hamster cheek pouch model during carcinogenesis.

In this study, we propose a low-cost cross-polarized dark field microscopy system for in vivo vascular imaging to detect head and neck cancer. A simple-to-use Gabor-filter-based image processing technique was developed to objectively and automatically quantify several important vascular features, including tortuosity, length, diameter and area fraction, from vascular images. Simulations were performed to evaluate the accuracies of vessel segmentation and feature extraction for our algorithm. Sensitivity and specificity for vessel segmentation of the Gabor masks both remained above 80% at all contrast levels when compared to gold-standard masks. Errors for vascular feature extraction were under 5%. Moreover, vascular contrast and vessel diameter were identified to be the two primary factors which affected the segmentation accuracies. After our algorithm was validated, we monitored the blood vessels in an inducible hamster cheek pouch carcinogen model over 17 weeks and quantified vascular features during carcinogenesis. A significant increase in vascular tortuosity and a significant decrease in vessel length were observed during carcinogenesis.

Early detection and differentiation of venous and arterial occlusion in skin flaps using visible diffuse reflectance spectroscopy and autofluorescence spectroscopy.

Our previous preclinical study demonstrated that both visible diffuse reflectance and autofluorescence spectroscopy, each of which yields a different set of physiological information, can predict skin flap viability with high accuracy in a MacFarlane rat dorsal skin flap model. In this report, we further evaluated our technique for the early detection and differentiation of venous occlusion and arterial occlusion in a rat groin flap model. We performed both diffuse reflectance and autofluorescence measurements on the skin flap model and statistically differentiated between flaps with and without occlusions as well as between flaps with venous occlusion and those with arterial occlusion based on these non-invasive optical measurements. Our preliminary results suggested that visible diffuse reflectance and autofluorescence spectroscopy can be potentially used clinically to detect both venous and arterial occlusion and differentiate one from the other accurately at an early time point.

Non-invasive controlled release from gold nanoparticle integrated photo-responsive liposomes through pulse laser induced microbubble cavitation.

Drug-carriers, capable of releasing the drug at the target sites upon external stimuli, are attractive for theranostic applications. In recent years, photo-responsive nanoparticles (NPs) have received considerable attention because of their potentials in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the process of development and are unprocurable by the clinics. Here, we demonstrated facile fabrication of these photo-responsive NPs by loading hydrophilic gold NPs within thermo-responsive liposomes. Calcein was used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Furthermore, we characterized their size, morphology, phase transition temperature and stability. Finally, we demonstrated that this photo-triggered release might be due to the membrane disruption caused by microbubble cavitation.

Sequential weighted Wiener estimation for extraction of key tissue parameters in color imaging: a phantom study.

Key tissue parameters, e.g., total hemoglobin concentration and tissue oxygenation, are important biomarkers in clinical diagnosis for various diseases. Although point measurement techniques based on diffuse reflectance spectroscopy can accurately recover these tissue parameters, they are not suitable for the examination of a large tissue region due to slow data acquisition. The previous imaging studies have shown that hemoglobin concentration and oxygenation can be estimated from color measurements with the assumption of known scattering properties, which is impractical in clinical applications. To overcome this limitation and speed-up image processing, we propose a method of sequential weighted Wiener estimation (WE) to quickly extract key tissue parameters, including total hemoglobin concentration (CtHb), hemoglobin oxygenation (StO2), scatterer density (α), and scattering power (ß), from wide-band color measurements. This method takes advantage of the fact that each parameter is sensitive to the color measurements in a different way and attempts to maximize the contribution of those color measurements likely to generate correct results in WE. The method was evaluated on skin phantoms with varying CtHb, StO2, and scattering properties. The results demonstrate excellent agreement between the estimated tissue parameters and the corresponding reference values. Compared with traditional WE, the sequential weighted WE shows significant improvement in the estimation accuracy. This method could be used to monitor tissue parameters in an imaging setup in real time.