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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 (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.

 

Inflation of hollow elastic structures can become unstable and exhibit a runaway phenomenon if the tension in their walls does not rise rapidly enough with increasing volume. Biological systems avoid such inflation instability for reasons that remain poorly understood. This is best exemplified by the lung, which inflates over its functional volume range without instability. The goal of this study was to determine how the constituents of lung parenchyma determine tissue stresses that protect alveoli from instability-related overdistension during inflation. We present an analytical model of a thick-walled alveolus composed of wavy elastic fibres, and investigate its pressure–volume behaviour under large deformations. Using second-harmonic generation imaging, we found that collagen waviness follows a beta distribution. Using this distribution to fit human pressure–volume curves, we estimated collagen and elastin effective stiffnesses to be 1247 kPa and 18.3 kPa, respectively. Furthermore, we demonstrate that linearly elastic but wavy collagen fibres are sufficient to achieve inflation stability within the physiological pressure range if the alveolar thickness-to-radius ratio is greater than 0.05. Our model thus identifies the constraints on alveolar geometry and collagen waviness required for inflation stability and provides a multiscale link between alveolar pressure and stresses on fibres in healthy and diseased lungs. 

Positive outcomes for colorectal cancer treatment have been linked to early detection. The difficulty in detecting early lesions is the limited contrast with surrounding mucosa and minimal definitive markers to distinguish between hyperplastic and carcinoma lesions. Colorectal cancer is the 3rd leading cancer for incidence and mortality rates which is potentially linked to missed early lesions which allow for increased growth and metastatic potential. One potential technology for early-stage lesion detection is hyperspectral imaging. Traditionally, hyperspectral imaging uses reflectance spectroscopic data to provide a component analysis, per pixel, of an image in fields such as remote sensing, agriculture, food processing and archaeology. This work aims to acquire higher signal-to-noise fluorescence spectroscopic data, harnessing the autofluorescence of tissue, adding a hyperspectral contrast to colorectal cancer detection while maintaining spatial resolution at video-rate speeds. We have previously designed a multi-furcated LED-based spectral light source to prove this concept. Our results demonstrated that the technique is feasible, but the initial prototype has a high light transmission loss (~98%) minimizing spatial resolution and slowing video acquisition. Here, we present updated results in developing an optical ray-tracing model of light source geometries to maximize irradiance throughput for excitation-scanning hyperspectral imaging. Results show combining solid light guide branches have a compounding light loss effect, however, there is potential to minimize light loss through the use of optical claddings. This simulation data will provide the necessary metrics to verify and validate future physical optical components within the hyperspectral endoscopic system for detecting colorectal cancer.