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Abstract Breast cancer progression is marked by extracellular matrix (ECM) remodeling, including increased stiffness, faster stress relaxation, and elevated collagen levels. In vitro experiments have revealed a role for each of these factors to individually promote malignant behavior, but their combined effects remain unclear. To address this, we developed alginate-collagen hydrogels with independently tunable stiffness, stress relaxation, and collagen density. We show that these combined tumor-mimicking ECM cues reinforced invasive morphologies and promoted spheroid invasion in breast cancer and mammary epithelial cells. High stiffness and low collagen density in slow-relaxing matrices led to the greatest cell migration speed and displacement. RNA-seq revealed Sp1 target gene enrichment in response to both individual and combined ECM cues, with a greater enrichment observed under multiple cues. Notably, high expression of Sp1 target genes upregulated by fast stress relaxation correlated with poor patient survival. Mechanistically, we found that phosphorylated-Sp1 (T453) was increasingly located in the nucleus in stiff and/or fast relaxing matrices, which was regulated by PI3K and ERK1/2 signaling, as well as actomyosin contractility. This study emphasizes how multiple ECM cues in complex microenvironments reinforce malignant traits and supports an emerging role for Sp1 as a mechanoresponsive transcription factor.
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Three-dimensional matrix stiffness modulates mechanosensitive and phenotypic alterations in oral squamous cell carcinoma spheroids
Extracellular biophysical cues such as matrix stiffness are key stimuli tuning cell fate and affecting tumor progression in vivo. However, it remains unclear how cancer spheroids in a 3D microenvironment perceive matrix mechanical stiffness stimuli and translate them into intracellular signals driving progression. Mechanosensitive Piezo1 and TRPV4 ion channels, upregulated in many malignancies, are major transducers of such physical stimuli into biochemical responses. Most mechanotransduction studies probing the reception of changing stiffness cues by cells are, however, still limited to 2D culture systems or cell-extracellular matrix models, which lack the major cell–cell interactions prevalent in 3D cancer tumors. Here, we engineered a 3D spheroid culture environment with varying mechanobiological properties to study the effect of static matrix stiffness stimuli on mechanosensitive and malignant phenotypes in oral squamous cell carcinoma spheroids. We find that spheroid growth is enhanced when cultured in stiff extracellular matrix. We show that the protein expression of mechanoreceptor Piezo1 and stemness marker CD44 is upregulated in stiff matrix. We also report the upregulation of a selection of genes with associations to mechanoreception, ion channel transport, extracellular matrix organization, and tumorigenic phenotypes in stiff matrix spheroids. Together, our results indicate that cancer cells in 3D spheroids utilize mechanosensitive ion channels Piezo1 and TRPV4 as means to sense changes in static extracellular matrix stiffness, and that stiffness drives pro-tumorigenic phenotypes in oral squamous cell carcinoma.
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- Award ID(s):
- 2046037
- PAR ID:
- 10584251
- Publisher / Repository:
- American Institute of Physics
- Date Published:
- Journal Name:
- APL Bioengineering
- Volume:
- 8
- Issue:
- 3
- ISSN:
- 2473-2877
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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