Quantifying Spatiotemporal Heterogeneity of Tumor Metabolism and Vasculature with a Multiparametric Point-of-Investigation Microscope.

Objective: The aim of this study was to develop and apply a dual-scale Capillary-Cell (CapCell) microscope to quantify spatial and temporal heterogeneity in tumor metabolism and vasculature during anti-angiogenic therapy. Impact Statement: This study introduces a dual-scale CapCell microscope, a novel imaging system to dynamically visualize metabolic and vascular adaptations in vivo. The platform reveals subregional features associated with treatment that are often missed by bulk analyses. Introduction: Tumor recurrence is often driven by microenvironmental heterogeneity in metabolism and perfusion. Given the importance of metabolic reprogramming in treatment response, the dual-scale CapCell microscope was designed to capture widefield and high-resolution images of metabolic-vascular coupling in vivo. Methods: The dual-scale CapCell microscope was implemented to image multiple endpoints including mitochondrial membrane potential and glucose uptake (widefield and high-resolution images) that are colocalized with vessel density and distance between vessels (high resolution). The CapCell was used to image 4T1 tumors grown in an orthotopic window chamber longitudinally following treatment with Combretastatin A-1 (CA1), a vascular-disrupting agent. Imaging was performed over a period of 8 days to evaluate the effects of CA1 administered on days 1 and 5. Results: Treated tumors showed a significant decrease in metabolism and vessel fraction, and a significant increase in the distance between vessels immediately following the first treatment. Within microregional areas, elevated mitochondrial activity was associated with vascular-dense regions, whereas increased glucose uptake was more apparent in less vascularized regions. Interestingly, the second treatment on day 6 had little effect on the tumor metabolism, and in fact, metabolism at this time point recovered to baseline levels despite a persistent reduction in vessel area fraction and no corresponding recovery in vascular proximity. Conclusion: The CapCell enables dual-scale, multiparametric imaging of tumor microenvironments, capturing spatial metabolic and vascular features often linked to poor therapeutic outcomes. This platform can inform therapeutic timing and guide the development of combination strategies by resolving critical tumor subpopulations.