Mapping Metabolic Reprogramming of the Tumor Microenvironment through High-Resolution Optical Imaging of Metabolism and Vasculature

Introduction: : Tumor complexity and heterogeneity contribute to the survival of therapy-resistant tumor cell subpopulations, often leading to recurrence, a significant cause of mortality across cancer subtypes. High intra-tumoral heterogeneity is linked to poor prognosis, either due to intrinsically aggressive biology or acquired therapy resistance. This heterogeneity can be temporal, from evolutionary pressures, or spatial, reflecting phenotypic variability across tumor regions. Both forms of heterogeneity contribute to the survival of resistant cell populations, ultimately leading to recurrence, a major cause of cancer mortality.

Capturing these dynamic features in vivo with sufficient spatial and temporal resolution remains a significant challenge. Positron emission tomography (PET) offers valuable whole-body metabolic imaging but lacks the resolution to resolve heterogeneity within tumors. In contrast, optical imaging provides higher spatial resolution and molecular specificity but often sacrifices field of view or resolution. To bridge this gap, we developed a dual-mode CapCell (Capillary-Cell), a widefield-to-high-resolution optical imaging system designed to resolve spatiotemporal metabolic and vascular heterogeneity in vivo.

The dual-mode CapCell supports multiplexed fluorescence imaging of glucose uptake (2-NBDG) and mitochondrial membrane potential (TMRE), alongside label-free vascular reflectance imaging with green light illumination. The system allows longitudinal tracking of metabolic and vascular changes in the same tumor by enabling repeated imaging through a mammary window chamber in murine models. Here, we use the dual-mode CapCell to assess treatment-induced changes in the tumor microenvironment (TME) following administration of the vascular disrupting agent Combretastatin A-1 (CA1), uncovering distinct spatial metabolic profiles and their relationship to vascular structure and proximity.

Materials and

Methods: : The CapCell was configured as a dual-mode optical imaging system with interchangeable widefield and high-resolution modes. Uniform illumination was achieved using a 1:4 fan-out fiber bundle coupled to LEDs and optimized using a genetic pattern search algorithm to concentrate all of the illumination power into the target fields of view. The platform was paired with an 8-MP CMOS camera and modular optics to maximize image contrast and spatial resolution.
To assess the platform’s capabilities, murine 4T1 mammary tumors were implanted under titanium window chambers. Mice were imaged every other day starting on Day 0 and continuing through Day 8 (Days 0, 2, 4, 6, and 8). Antiangiogenic treatment (CA1, 10 mg/kg, IP) was administered on Days 1 and 5 to induce metabolic and vascular changes in the in vivo system. For each imaging session, metabolic probes (2-NBDG, TMRE) were injected systemically and imaged 60 minutes post-injection. Reflectance imaging with green light enabled label-free vascular mapping.
Image analysis included vessel segmentation using a Gabor/Dijkstra pipeline, followed by K-nearest-neighbor (KNN) analysis to quantify the distance of each tissue pixel from the nearest vessel. Metabolic endpoints were categorized into four clusters based on high or low levels of TMRE and 2-NBDG. Cluster assignment was performed on a pixel-by-pixel basis across all images and time points for both cell lines, using the mean values of each metabolic marker to define the high/low thresholds. Area fractions and spatial distributions of metabolic and vascular clusters were compared across time points using ANOVA with Tukey’s post-hoc test.

Results, Conclusions, and Discussions:: Using a validated light propagation model and global optimization (up to 50,000 evaluations), we configured four optical fibers to achieve uniform illumination over a 2.8 × 1.5 mm field of view, resulting in a 12-fold increase in optical power within the target region (Figure 1A). The simulated distribution supports both widefield and high-resolution imaging (Figure 1B–C). Reflectance imaging (Figures 1D–E) validated the system's resolution, enabling visualization of vascular features in 4T1 tumors.
To capture treatment-induced changes, we administered the vascular disrupting agent CA1 at different time points (Figure 2A). After the first CA1 administration, metabolic maps revealed a dominance of [2-NBDGLow/TMRELow] cluster, and after the second treatment, [2-NBDGHigh/TMREHigh] clusters increased. Vascular maps showed a reduction in vessel density post-treatment, with the sharpest decline after the first dose. Untreated tumors showed stable metabolic heterogeneity and vascular density.
Metabolic cluster analysis confirmed these trends: treated tumors had elevated [2-NBDGLow/TMRELow] clusters on Day 2 and a rebound of [2-NBDGHigh/TMREHigh] clusters by Day 6, indicating a transient metabolic suppression followed by partial recovery (Figure 3A–B). Untreated tumors maintained stable metabolic profiles, with no significant changes until Day 8, likely due to necrosis. Vascular analysis similarly revealed a decline in vessel area fraction and increased KNN distances after the first treatment. After the second, vessel area increased and KNN distances decreased, suggesting vascular remodeling or reperfusion, mirroring the metabolic response.
Spatial mapping revealed a significant reduction in [2-NBDGLow/TMREHigh] clusters in treated tumors, coinciding with a decreased area fraction of tissue located within 0–100 µm of vessels (Figure 3E–G, p< 0.05). Mitochondrial activity was preferentially preserved in regions with close vascular proximity, particularly in untreated tumors (p < 0.005), emphasizing the spatial dependence of metabolic function on vascular integrity.
Together, these results show that mitochondrial function is retained in well-perfused regions but collapses with vascular disruption. This dual-mode optical imaging framework elucidates how vascular integrity shapes metabolic heterogeneity and supports vascular-targeted therapy development.

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