Z45 - Metabolic Heterogeneity in Breast Cancer: Differential Expression of ACAT1 and SR-B1 Across the Epithelial to Mesenchymal Transition

Introduction: : Breast cancer metastasis remains a significant challenge in oncology, with intratumoral heterogeneity driving disease progression and treatment resistance. Dysregulated cellular metabolism is a hallmark of cancer, yet its role in metastasis remains poorly understood. The epithelial-to-mesenchymal transition (EMT) is a key biological process that enables epithelial cells to acquire migratory and invasive capabilities, facilitating cancer dissemination. This study explores how Acyl-CoA:cholesterol acyltransferase (ACAT1) and scavenger receptor class B type 1 (SR-B1), key regulators of cholesterol metabolism, vary across the EMT spectrum. We find that clonal triple-negative breast cancer cells from the 4T1 cell line exhibit distinct patterns of protein expression and localization. Specifically, ACAT1 and SR-B1 levels appear to increase with EMT and are associated with more aggressive subpopulations. Our findings suggest that differential regulation of cholesterol metabolism may influence the biochemical landscape of breast cancer cells, contributing to EMT and invasive phenotypes. While this study focuses on breast cancer, we expect these findings may extend to other malignancies, potentially informing how metabolic reprogramming could be targeted as an anticancer therapeutic strategy.

Materials and

Methods: : We isolated distinct clonal subpopulations from heterogeneous 4T1 triple-negative breast cancer cells based on phenotypic traits spanning the epithelial to mesenchymal spectrum by performing limiting dilution cloning. Clonal cell subpopulations were seeded on collagen-coated glass coverslips and fixed after 72 hours of culture. Immunostaining was then performed to visualize ACAT1 and SR-B1 proteins, with Hoechst used as a nuclear stain. Images were acquired using an epifluorescence microscope to analyze protein expression levels and subcellular localization patterns. Fluorescence images were segmented at the individual cell level using Python to quantify ACAT1 and SR-B1 expression. Whole-cell protein expression was quantified by measuring the integrated fluorescence intensity per cell. Custom code was developed to detect intracellular punctate expression, applying a difference of Gaussians filter to identify a wide range of localized protein sizes, from small puncta to larger aggregates. Metrics assessed included the number of puncta per cell, individual puncta sizes, and their spatial distribution across nuclear and cytoplasmic compartments.

Results, Conclusions, and Discussions:: The epithelial-to-mesenchymal transition (EMT) drives phenotypic heterogeneity in breast cancer, enabling cells to acquire invasive and therapy-resistant traits. Given the emerging links between metabolic reprogramming and cancer progression, we investigated how the expression and localization of cholesterol regulatory proteins—ACAT1 and SR-B1—vary across distinct clonal subpopulations of 4T1 breast cancer cells spanning the epithelial to mesenchymal spectrum.

Distinct expression and localization patterns of ACAT1 and SR-B1 were observed across clonal subpopulations of 4T1 breast cancer cells, highlighting metabolic heterogeneity that mirrors phenotypic diversity along the epithelial to mesenchymal transition (EMT). Invasive phenotypes—specifically mesenchymal (M) and amoeboid (A) clones—exhibited significantly higher expression levels of both ACAT1 and SR-B1 compared to epithelial-like clones (E1 and E2), as shown by integrated fluorescence intensity measurements. These results suggest an upregulation of cholesterol regulatory pathways in more aggressive breast cancer subpopulations. Puncta analysis further underscored this metabolic heterogeneity. Both the number of puncta per cell and the average puncta size were elevated in invasive clones relative to epithelial clones. Interestingly, an inverse relationship between puncta size and number was observed, suggesting that invasive phenotypes may balance numerous small cholesterol storage sites with larger aggregates to support dynamic metabolic needs. Subcellular localization analyses revealed additional differences. Both ACAT1 and SR-B1 exhibited a shift from cytoplasmic toward nuclear localization in mesenchymal and amoeboid clones, suggesting potential roles in nuclear lipid metabolism or lipid-mediated regulation of gene expression in invasive cells. These findings indicate that cholesterol trafficking and compartmentalization are reprogrammed as breast cancer cells transition toward more aggressive phenotypes.

Together, these results demonstrate that ACAT1 and SR-B1 expression, presentation, and compartmentalization vary across clonal subpopulations of breast cancer cells, underscoring the role of cholesterol metabolism in supporting phenotypic plasticity and invasive potential. These proteins may serve as functional markers of invasive phenotypes and could represent candidate therapeutic targets for suppressing heterogeneity-driven metastasis in triple-negative breast cancer.

Acknowledgements and/or References (Optional)::