Introduction: Obesity represents a global health concern, leading to the development of several types of malignancy, including breast cancer (BC). In the obese state, adipocytes become hypertrophic, releasing elevated amounts of fatty acids, adipokines and pro-inflammatory cytokines, which support BC progression. The secreted factors from adipocytes establish paracrine network with BC cells and the components of the tumor microenvironment (TME), creating a pro-tumoral milieu. Cancer-associated fibroblasts (CAFs) represent the main population within the TME. However, whether their phenotype might be influenced by the secreted factors from adipocytes is not fully understood. Here, we aimed to investigate whether the conditioned medium of mature 3T3-L1 adipocytes (3T3-L1A) influences human breast CAF phenotype.

Methods: CAFs were isolated from breast tumor biopsies. Conditioned medium (CM) from adipocytes was collected incubating mature murine adipocytes (3T3-L1A) in 3% charcoal-stripped fetal bovine serum for 36-48 hours and used in co-culture systems with CAFs. MTT, Boyden Chamber Migration, Contraction, Phalloidin Staining Assays and Multiplex Bead-Based Immunoassay were performed to assess the phenotypic features of 3T3-L1A CM-treated CAFs.

Results: CM from 3T3-L1A enhanced CAF viability and migration compared to untreated control. CAF contractility was markedly increased after incubation with 3T3-L1A CM for 48 h and 72 h. Consistent with this result, the stress fiber formation was higher in 3T3-L1A CM-treated CAFs. Multiplex analysis revealed that 3T3-L1A CM induced the upregulation of several pro-tumoral secreted factors in CAFs, with RANTES among the most markedly upregulated.

Conclusion: Our findings demonstrated that the secreted factors from adipocytes affect the phenotypic features of human breast CAFs, highlighting the relevance of paracrine interactions within the TME.