Metabolic profiling of compressed stromal cells in the breast peritumoral microenvironment

Introduction: : Breast cancer represents a significant public health concern for women globally. Although the breast peritumor (i.e., the surrounding mammary tissue) is increasingly recognized for its active role in mediating breast cancer initiation, growth, progression, and treatment resistance, its biophysical relationship with the neighboring tumor is poorly understood. For example, the growing mammary tumor exerts compressive forces on the surrounding host tissue as it expands (i.e., solid stresses), potentially generating a cancer-supporting and therapy-resistant peritumoral stroma. Using optical, transcriptomic, and functional techniques, we discovered that the application of compressive stress alters the metabolic state and mitochondrial morphodynamics of stromal cells found in the breast peritumor in a cell type-specific manner in vitro. Retrospective analysis of human breast cancer samples also demonstrated that mammary stromal cells exhibit distinct, spatially-dependent metabolic states, with peritumoral cells appearing to be more glycolytic relative to those found in the normal stroma.

Materials and

Methods: : We utilized a previously established in vitro transwell compression assay to study the effects of 24 hours of breast tumor-mimicking compression on NIH3T3 fibroblasts, 3T3-L1 undifferentiated adipocytes, and 3T3-L1 differentiated (d3T3-L1) adipocytes. To characterize the metabolic state of compressed and uncompressed cells, we used fluorescence lifetime imaging microscopy (FLIM), a label-free, non-destructive imaging technique capable of detecting subtle changes in cell metabolism by measuring the autofluorescence of NADH. The fluorescence lifetime of NADH depends on its binding state, with shorter lifetimes indicating glycolysis and longer lifetimes indicating oxidative phosphorylation. In addition to FLIM, we assessed the impact of compression on stromal cell metabolism through functional enrichment analysis of bulk RNA-sequencing data. Furthermore, we evaluated the effect of mechanical stress on mitochondrial morphodynamics through qPCR and fluorescent imaging. We specifically characterized mitochondrial morphology in cells stained with MitoTracker Red CMXRos, as well as analyzed the expression of genes involved in mitophagy and of the mitochondrial fission and fusion proteins DLP1 and MFN1, respectively. Finally, we metabolically profiled fibroblasts and adipocytes across normal, peritumoral, and tumoral mammary tissue regions through fluorescence lifetime imaging of human triple-negative breast cancer tissue samples as well as by performing retrospective, single cell analysis of human breast cancer datasets. Metabolic characterization via single cell transcriptomics was accomplished using the metabolic index (MI)—an in-house metric that characterizes cell metabolism relative to the whole cell population. Importantly, MI > 0 indicates cells are more glycolytic while MI < 0 suggests they have higher levels of oxidative phosphorylation activity.

Results, Conclusions, and Discussions:: Imaging NIH3T3 fibroblasts (Fig. 1a), 3T3-L1 undifferentiated adipocytes (Fig. 1b), and d3T3-L1 differentiated adipocytes (Fig. 1c) via FLIM revealed significantly altered mean fluorescent lifetimes under breast tumor-mimicking compression. We specifically observed mean lifetime decrease in compressed NIH3T3 (Fig. 1d) and d3T3-L1 (Fig. 1f) cells, but increase in compressed 3T3-L1 cells (Fig. 1e). This suggests that compressive stress metabolically reprograms the stromal cells in a cell type-specific manner, with fibroblasts (Fig. 1g) and differentiated adipocytes (Fig. 1i) shifting to a more glycolytic state while undifferentiated adipocytes (Fig. 1h) become more oxidative. Functional enrichment analysis confirmed that these changes are reflected at the transcriptional level in compressed fibroblasts as they were observed to significantly downregulate genes related to oxidative phosphorylation and upregulate those involved in glycolysis to varying degrees.

Our assessment of mitochondrial structure in MitoTracker-stained NIH3T3 (Fig. 2a) and 3T3-L1 (Fig. 2b) cells also revealed that compression increases mitochondrial fragmentation and swelling in undifferentiated adipocytes, but not in fibroblasts. Moreover, while the expression of mitophagy transcripts is not differentially expressed in compressed fibroblasts (Fig. 2c), both Bnip3 and Park2 are upregulated in compressed undifferentiated adipocytes (Fig. 2d). By analyzing 3T3-L1 cells immunostained for MFN1 (fusion protein) and DLP1 (fission protein; Fig. 2e), we further discovered that compression significantly decreases the expression (Fig. 2f) and area fraction of (Fig. 2g) MFN1, but not of DLP1 (Fig. 2h,i). Altogether, this suggests that compression induces fragmentation of the mitochondrial network in undifferentiated adipocytes by impairing fusion dynamics, possibly facilitating increased mitophagy.

Finally, our single cell and fluorescence lifetime analysis of human breast cancer patient samples revealed that, in relation to those found in normal tissue (without compressive stresses), fibroblasts and adipocytes in the peritumor (under tumor compression) are more glycolytic. This is evidenced by decreasing lifetime and increasing MI as stromal cell proximity to the tumor decreases. Ongoing studies are aimed at determining to what degree this metabolic reprogramming is a direct consequence of external tumor compression vs. biochemical crosstalk with cancer cells. Collectively, these results may directly inform pharmacological strategies that reprogram this altered mechano-metabolism in the breast peritumor to augment anti-cancer therapies.

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