Fluorescence Lifetime Imaging Microscopy (FLIM) reveals spatial-metabolic changes in 3D breast cancer spheroids.

Cancer cells are mechanically sensitive to physical properties of the microenvironment, which can affect downstream signaling to promote malignancy, in part through the modulation of metabolic pathways. Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to measure the fluorescence lifetime of endogenous fluorophores, such as the metabolic co-factors NAD(P)H and FAD, in live samples. We used multiphoton FLIM to investigate the changes in cellular metabolism of 3D breast spheroids derived from MCF-10A and MD-MB-231 cell lines embedded in collagen with varying densities (1 vs. 4 mg/ml) over time (Day 0 vs. Day 3). MCF-10A spheroids demonstrated spatial gradients, with the cells closest to the spheroid edge exhibiting FLIM changes consistent with a shift towards oxidative phosphorylation (OXPHOS) while the spheroid core had changes consistent with a shift towards glycolysis. The MDA-MB-231 spheroids had a large shift consistent with increased OXPHOS with a more pronounced change at the higher collagen concentration. The MDA-MB-231 spheroids invaded into the collagen gel over time and cells that traveled the farthest had the largest changes consistent with a shift towards OXPHOS. Overall, these results suggest that the cells in contact with the extracellular matrix (ECM) and those that migrated the farthest had changes consistent with a metabolic shift towards OXPHOS. More generally, these results demonstrate the ability of multiphoton FLIM to characterize how spheroids metabolism and spatial metabolic gradients are modified by physical properties of the 3D ECM.

Spatial frequency domain imaging for monitoring immune-mediated chemotherapy treatment response and resistance in a murine breast cancer model.

Spatial Frequency Domain Imaging (SFDI) can provide longitudinal, label-free, and widefield hemodynamic and scattering measurements of murine tumors in vivo. Our previous work has shown that the reduced scattering coefficient (µ's) at 800 nm, as well as the wavelength dependence of scattering, both have prognostic value in tracking apoptosis and proliferation during treatment with anti-cancer therapies. However, there is limited work in validating these optical biomarkers in clinically relevant tumor models that manifest specific treatment resistance mechanisms that mimic the clinical setting. It was recently demonstrated that metronomic dosing of cyclophosphamide induces a strong anti-tumor immune response and tumor volume reduction in the E0771 murine breast cancer model. This immune activation mechanism can be blocked with an IFNAR-1 antibody, leading to treatment resistance. Here we present a longitudinal study utilizing SFDI to monitor this paired responsive-resistant model for up to 30 days of drug treatment. Mice receiving the immune modulatory metronomic cyclophosphamide schedule had a significant increase in tumor optical scattering compared to mice receiving cyclophosphamide in combination with the IFNAR-1 antibody (9% increase vs 10% decrease on day 5 of treatment, p < 0.001). The magnitude of these differences increased throughout the duration of treatment. Additionally, scattering changes on day 4 of treatment could discriminate responsive versus resistant tumors with an accuracy of 78%, while tumor volume had an accuracy of only 52%. These results validate optical scattering as a promising prognostic biomarker that can discriminate between treatment responsive and resistant tumor models.

Optical scattering as an early marker of apoptosis during chemotherapy and antiangiogenic therapy in murine models of prostate and breast cancer.

Monitoring of the in vivo tumor state to track therapeutic response in real time may help to evaluate new drug candidates, maximize treatment efficacy, and reduce the burden of overtreatment. Current preclinical tumor imaging methods have largely focused on anatomic imaging (e.g., MRI, ultrasound), functional imaging (e.g., FDG-PET), and molecular imaging with exogenous contrast agents (e.g., fluorescence optical tomography). Here we utalize spatial frequency domain imaging (SFDI), a noninvasive, label-free optical technique, for the wide-field quantification of changes in tissue optical scattering in preclinical tumor models during treatment with chemotherapy and antiangiogenic agents. Optical scattering is particularly sensitive to tissue micro-architectural changes, including those that occur during apoptosis, an early indicator of response to cytotoxicity induced by chemotherapy, thermotherapy, cryotherapy, or radiation therapy. We utilized SFDI to monitor responses of PC3/2G7 prostate tumors and E0771 mammary tumors to treatment with cyclophosphamide or the antiangiogenic agent DC101 for up to 49 days. The SFDI-derived scattering amplitude was highly correlated with cleaved caspase-3, a marker of apoptosis (ρp = 0.75), while the exponent of the scattering wavelength-dependence correlated with the cell proliferation marker PCNA (ρp = 0.69). These optical parameters outperformed tumor volume and several functional parameters (e.g., oxygen saturation and hemoglobin concentration) as an early predictive biomarker of treatment response. Quantitative diffuse optical scattering is thus a promising new early marker of treatment response, which does not require radiation or exogenous contrast agents.

Shortwave-infrared meso-patterned imaging enables label-free mapping of tissue water and lipid content.

Water and lipids are key participants in many biological processes, but there are few non-invasive methods that provide quantification of these components in vivo, and none that can isolate and quantify lipids in the blood. Here we develop a new imaging modality termed shortwave infrared meso-patterned imaging (SWIR-MPI) to provide label-free, non-contact, spatial mapping of water and lipid concentrations in tissue. The method utilizes patterned hyperspectral illumination to target chromophore absorption bands in the 900-1,300 nm wavelength range. We use SWIR-MPI to monitor clinically important physiological processes including edema, inflammation, and tumor lipid heterogeneity in preclinical models. We also show that SWIR-MPI can spatially map blood-lipids in humans, representing an example of non-invasive and contact-free measurements of in vivo blood lipids. Together, these results highlight the potential of SWIR-MPI to enable new capabilities in fundamental studies and clinical monitoring of major conditions including obesity, cancer, and cardiovascular disease.

Characterization of bony anatomic regions in pediatric and adult healthy volunteers using diffuse optical spectroscopic imaging.

SIGNIFICANCE: Diffuse optical spectroscopic imaging (DOSI) measures quantitative functional and molecular information in thick tissue in a noninvasive manner using near-infrared light. DOSI may be useful for diagnosis and prognosis of bone pathologies including osteosarcoma and Ewing's sarcoma, but little is currently known about DOSI-derived parameters in bony anatomic locations where this disease occurs. AIM: Our goal is to quantify the optical characteristics and chromophore content of bony anatomic locations of healthy volunteers and assess differences due to anatomic region, age, sex, ethnicity, race, and body fat. APPROACH: Fifty-five healthy volunteers aged 4 to 72 were enrolled in the study. The optical properties and quantitative tissue concentrations of oxyhemoglobin, deoxyhemoglobin, water, and lipids were assessed at the distal humerus, distal femur, and proximal tibia. Body fat was assessed using skinfold calipers. One volunteer underwent a more comprehensive body scan from neck to foot to explore chromophore distributions within an individual. Regression analysis was used to identify the most important sources of variation in the measured data set. RESULTS: Statistical differences between bony locations were found for scattering, water, and lipids, but not for hemoglobin. All chromophores had statistical differences with sex, but there were no significant age-related correlations. Regression analysis revealed that body fat measured with skinfold calipers was the most important predictor of oxy-, deoxy-, total hemoglobin, and lipids. Hemoglobin and lipid levels were highly correlated (ρ ≥ 0.7) over the subject population and within the single-subject body scan. CONCLUSIONS: DOSI can successfully measure bony anatomic sites where osteosarcomas and Ewing's sarcomas commonly occur. Future studies of bone pathology using DOSI should account for the variation caused by anatomic region, sex, race and ethnicity, and body fat as these cause substantial variations in DOSI-derived metrics.

Diffuse optical spectroscopic imaging reveals distinct early breast tumor hemodynamic responses to metronomic and maximum tolerated dose regimens.

BACKGROUND: Breast cancer patients with early-stage disease are increasingly administered neoadjuvant chemotherapy (NAC) to downstage their tumors prior to surgery. In this setting, approximately 31% of patients fail to respond to therapy. This demonstrates the need for techniques capable of providing personalized feedback about treatment response at the earliest stages of therapy to identify patients likely to benefit from changing treatment. Diffuse optical spectroscopic imaging (DOSI) has emerged as a promising functional imaging technique for NAC monitoring. DOSI uses non-ionizing near-infrared light to provide non-invasive measures of absolute concentrations of tissue chromophores such as oxyhemoglobin. In 2011, we reported a new DOSI prognostic marker, oxyhemoglobin flare: a transient increase in oxyhemoglobin capable of discriminating NAC responders within the first day of treatment. In this follow-up study, DOSI was used to confirm the presence of the flare as well as to investigate whether DOSI markers of NAC response are regimen dependent. METHODS: This dual-center study examined 54 breast tumors receiving NAC measured with DOSI before therapy and the first week following chemotherapy administration. Patients were treated with either a standard of care maximum tolerated dose (MTD) regimen or an investigational metronomic (MET) regimen. Changes in tumor chromophores were tracked throughout the first week and compared to pathologic response and treatment regimen at specific days utilizing generalized estimating equations (GEE). RESULTS: Within patients receiving MTD therapy, the oxyhemoglobin flare was confirmed as a prognostic DOSI marker for response appearing as soon as day 1 with post hoc GEE analysis demonstrating a difference of 48.77% between responders and non-responders (p < 0.0001). Flare was not observed in patients receiving MET therapy. Within all responding patients, the specific treatment was a significant predictor of day 1 changes in oxyhemoglobin, showing a difference of 39.45% (p = 0.0010) between patients receiving MTD and MET regimens. CONCLUSIONS: DOSI optical biomarkers are differentially sensitive to MTD and MET regimens at early timepoints suggesting the specific treatment regimen should be considered in future DOSI studies. Additionally, DOSI may help to identify regimen-specific responses in a more personalized manner, potentially providing critical feedback necessary to implement adaptive changes to the treatment strategy.

Diffuse and nonlinear imaging of multiscale vascular parameters for in vivo monitoring of preclinical mammary tumors.

Diffuse optical imaging (DOI) techniques provide a wide-field or macro assessment of the functional tumor state and have shown substantial promise for monitoring treatment efficacy in cancer. Conversely, intravital microscopy provides a high-resolution view of the tumor state and has played a key role in characterizing treatment response in the preclinical setting. There has been little prior work in investigating how the macro and micro spatial scales can be combined to develop a more comprehensive and translational view of treatment response. To address this, a new multiscale preclinical imaging technique called diffuse and nonlinear imaging (DNI) was developed. DNI combines multiphoton microscopy with spatial frequency domain imaging (SFDI) to provide multiscale data sets of tumor microvascular architecture coregistered within wide-field hemodynamic maps. A novel method was developed to match the imaging depths of both modalities by utilizing informed SFDI spatial frequency selection. An in vivo DNI study of murine mammary tumors revealed multiscale relationships between tumor oxygen saturation and microvessel diameter, and tumor oxygen saturation and microvessel length (|Pearson's ρ| ≥ 0.5, P < 0.05). Going forward, DNI will be uniquely enabling for the investigation of multiscale relationships in tumors during treatment.