Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments

Abstract

The breast peritumor microenvironment (pTME) is increasingly recognized as a mediator of breast cancer progression and treatment resistance. However, if and how growth-induced tumor compressive forces (i.e., solid stresses) influence the breast pTME remains unclear. Here we show using instant fluorescence lifetime imaging microscopy (FLIM)--a frequency-domain FLIM system capable of simultaneous image acquisition and instantaneous data processing--that breast tumor-mimicking in vitro compression promotes metabolic changes in stromal cells found in the breast pTME. Namely, compression shifts NIH3T3 fibroblasts and differentiated 3T3-L1 (d3T3-L1) adipocytes toward a more glycolytic state, while it promotes increased oxidative phosphorylation in 3T3-L1 undifferentiated adipocytes. The gold-standard Seahorse extracellular flux assay fails to capture these changes, underscoring the superior sensitivity of instant FLIM in detecting metabolic shifts. We validate these phenotypic findings at the transcriptomic level via RNA sequencing, confirming that compressed fibroblasts downregulate oxidative phosphorylation and upregulate glycolysis compared to uncompressed controls. We further demonstrate that compression induces mitochondrial dysregulation in undifferentiated adipocytes, driven in part by upregulated mitophagy and disrupted fusion dynamics. Finally, we confirm that these stromal cell types recapitulate these distinct metabolic states in human breast cancer patient samples, consistent with our in vitro findings. By elucidating mechano-metabolic interactions occurring at the tumor-host interface, these results will inform the development of innovative mechano-metabolic reprogramming treatment strategies to improve breast cancer patient survival.