K11 - Unraveling the mechanism of skin metastasis through CAF activation and ECM remodeling induced by triple-negative breast cancer

Introduction: : Breast cancer, particularly triple-negative breast cancer (TNBC), is characterized by high rates of metastasis and recurrence, underscoring the urgent need for novel therapeutic strategies due to the limited efficacy of current treatments. Recent studies highlight the critical role of extracellular matrix (ECM) stiffness within the tumor microenvironment (TME) in modulating cancer invasiveness and metastasis. TNBC is known to induce cutaneous metastasis and activate cancerassociated fibroblasts (CAFs), which remodel the ECM and increase matrix stiffness, thereby promoting tumor progression. This study aims to investigate the effects of TNBC-derived conditioned medium (CM) on CAF activation and ECM stiffness, with the goal of exploring the potential for skin tissue-based early detection of breast

Materials and

Methods: : As shown in Figure 1 (A), Normal human dermal fibroblasts (NHDFs) and MDA-MB-231 breast cancer cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C with 5% CO₂. Conditioned medium (CM) was collected after 3 days of culturing MDA-MB-231 cells at 60% confluence and filtered through a 0.2 μm filter. Polyacrylamide (PA) gels were fabricated using a standard protocol and activated with SulfoSANPAH under UV light. Type I rat tail collagen was coated onto the gels and incubated overnight at 4°C. NHDFs were seeded on the collagen-coated gels (7,000 cells/gel) and cultured for 4 days. Analyzes included mRNA expression, FACS, morphology, and traction force microscopy.

Results, Conclusions, and Discussions:: Exposing normal human dermal fibroblasts (NHDFs) to conditioned medium (CM) from MDAMB-231 cells led to significant upregulation of cancer-associated fibroblast (CAF) markers, including PDGFRβ, FAP, ACTA2, PODPN, and FSP1, as confirmed by qPCR analysis [Fig. 1 (B)]. Notably, stiffer substrates (40 kPa) induced more pronounced CAF marker expression compared to softer gels, indicating that mechanical cues interact with tumor-derived factors to enhance fibroblast reprogramming [Fig. 1 (C)]. Morphological analysis of CAFs on stiffer substrates (40 kPa) demonstrated enhanced focal adhesion formation and increased expression of paxillin and F-actin compared to cells on softer gels (2 kPa) [Fig. 1 (D–E)], suggesting increased cytoskeletal tension and adhesion dynamics in response to ECM stiffness. However, other geometric features, such as cell perimeter and aspect ratio, were unaffected by substrate stiffness, implying that mechanical cues selectively influence specific aspects of CAF morphology. Overall, TNBC-derived CM reprograms fibroblasts, with ECM stiffness further amplifying this process. The synergistic interaction between biochemical and biomechanical signals in CAF activation may contribute to tumor invasiveness, offering potential targets for therapeutic intervention.
Our results demonstrate that conditioned media derived from triple-negative breast cancer (TNBC) cells reprograms normal fibroblasts into activated fibroblasts, and that extracellular matrix (ECM) stiffness further promotes this activation. In particular, stiffer matrices promote increased expression of CAF markers and cytoskeletal reorganization, suggesting that biochemical and mechanical cues exert a synergistic effect to promote fibroblast activation. These insights suggest the potential of targeting ECM remodeling and CAF activation as therapeutic strategies to mitigate TNBC cutaneous metastasis.

Acknowledgements and/or References (Optional):: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2021R1A2C3008408), and the Korea-US Collaborative Research Fund (KUCRF) funded by the Ministry of Science and ICT and the Ministry of Health & Welfare, Republic of Korea (No. RS-2024-00468873).