I20 - Sp1 mechanotransduction regulates breast cancer cell invasion in engineered viscoelastic extracellular matrices

Introduction: : Invasive ductal carcinomas are associated with changes in both composition and mechanical properties of the extracellular matrix such as increased stiffness, altered viscoelasticity (stress relaxation rate), and increased production of collagen I. The increased collagen density and stiffness serve as important diagnostic and prognostic markers of disease, which can be assessed through manual palpation and radiographic imaging. Furthermore, differences in viscoelasticity have been shown to discriminate between malignant and benign breast tumors. While it is well established that individual tumor-mimicking ECM cues can enhance malignant traits in cancer cells, how cells generate an integrated response to multiple cues in complex microenvironments remains poorly understood, primarily because decoupling these cues remains a challenge.

Materials and

Methods: : To address this knowledge gap, we have developed an alginate-based 3D hydrogel cell culture platform with independently tunable stiffness (100 Pa vs. 2000-4000 Pa), stress relaxation (half-time in order of ≈ 100 s vs. ≈ 1000 s), and collagen I concentration (0.5 mg/ml vs. 2 mg/ml) to map a range of cellular microenvironments. Hydrogel mechanical properties were validated using shear rheology. We cultured MDA-MB-231 and MCF10A cells, as well as spheroids made from these cell lines in these matrices to analyze invasion and tracked their migration. RNA-seq was performed to study changes in gene expression in response to presence of individual as well multiple tumor-mimicking ECM cues. Transcription factor and pathway enrichment were performed using TRRUST and GSEA analysis respectively.

Results, Conclusions, and Discussions:: Our results show that these combined tumor-mimicking ECM cues reinforced invasive morphologies and promoted spheroid invasion in both breast cancer and mammary epithelial cells. Specifically, high stiffness and low collagen density within slow relaxing matrices led to the greatest cell migration speed and displacement. RNA-seq revealed Sp1 target gene enrichment across all matrix conditions that promoted invasion, with a greater enrichment observed when multiple ECM cues were present. Notably, high expression of Sp1 target genes upregulated by fast stress relaxation correlated with poor patient survival. Mechanistically, we found that phosphorylated Sp1 (T453) exhibited increased nuclear localization in both stiff and fast relaxing matrices, which was regulated by PI3K and ERK1/2 signaling, as well as actomyosin contractility. Our study emphasizes that multiple ECM cues within complex viscoelastic microenvironments collectively reinforce malignant traits, with Sp1 acting as a mechanoresponsive transcription factor that transduces these signals.

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