In vivo imaging of metabolic heterogeneity across three endpoints relevant to aggressive breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis and a high likelihood of recurrence. Residual disease after therapy is a key predictor of recurrence, often driven by intratumoral metabolic heterogeneity. Accumulating evidence indicates that tumors are able to shift between glycolysis and oxidative metabolism and alter nutrient preferences to sustain growth and resist therapy. We have developed a in vivo microscope that enables near-simultaneous measurements of fluorescent metabolic surrogates of glucose, fatty acids, and oxidative phosphorylation through a combination of spectral separation and sequential delivery schemes. Widefield imaging with uniform illumination across the entire tumor landscape (5 mm × 5 mm) informs on the spatial distribution of these metabolic probes. We used this technology to investigate metabolic heterogeneity of a murine model of TNBC (4T1 tumor line) and normal mammary tissues that have distinctly different metabolic pathways. Mammary tissues relied primarily on oxidative metabolism and showed high levels of glucose and fatty acid uptake across the entire imaging area reflecting a single metabolic phenotype. Though tumors were predominantly glycolytic, they displayed a heterogeneous distribution of nutrient preferences with regions dominated by either fatty acid uptake, glucose uptake, or both. Taken together, this work highlights the importance of not only capturing multiple metabolic endpoints but also investigating their spatial relationships to understand heterogeneity in key substrates and metabolic pathways for energy production in vivo.