Prostate Tumor Cell Metastatic Potential Drive Responsiveness to Matrix Composition and Stiffness

Introduction: Solid tumors are characterized by a progressive increase in stromal stiffness that correlates with progression to metastatic stage. This increasing stiffness is primarily attributed to the deposition of the extracellular matrix (ECM) components such as type I collagen (COL) and fibronectin (FN). These changes in ECM structure influence various cellular functions, including cell migration, invasion, and contractility. Both the mechanical properties and composition of the ECM impact cellular behaviors that contribute to tumor progression. The complex interplay between ECM stiffness and composition is essential for understanding the cellular response and its impact on tumor progression. This research explores how prostate cancer cells (PCa) respond to varying ECM composition and stiffness level using highly and lowly metastatic cell lines. By examining how different ECM environments influence cancer cell functions and behaviors, the study may offer insights into tumor progression and metastasis.

Materials and

Methods: Highly metastatic PC-3 cells, lowly metastatic 22Rv1 cells and murine non-invasive MyC-CaP cells were seeded on either COL- or FN-coated polyacrylamide (PA) gels of 5 or 20 kPa. Mechanotransduction was assessed by monitoring YAP nuclear translocation in immunofluorescence (nucleus/cytoplasm intensity ratio) and cell spreading with actin staining, using confocal microscopy. It was also measured with cells seeded on 20kPa gels coated with different FN/COL ratio (100% FN, 25% COL/FN, 50% COL/FN, 75% COL/FN and 100% COL). To establish the cell’s preference to COL or FN, cells were plated for 1 hour on gels coated with perpendicular COL and FN gradients generated using a PRIMO UV-patterning system. For the measurement of durotaxis, stiffness gradient from 1 to 50 kPa were engineered, by the polymerization of 2 polyacrylamide gels with different stiffness on the same coverslip. The gels were coated with COL, FN or both. Cells were plated and incubated for 72 hours to allowed them to migrate on the gel. The whole gel was then imaged with microscopy. Using prostate tissue from a High-Myc or PTEN -/-homozygote murine model, COL and FN ratio at protein level was quantified with immunofluorescence.

Results, Conclusions, and Discussions: The analysis of the ECM components in PCa murine model revealed an increase in COL expression after 12 weeks in the PTEN tumor progression model, while the increase of FN expression was minimal (Figure 1). In the High-Myc model, COL increase was observed in 55 weeks old mice, indicating a slower change in ECM composition (data not shown). Analysis of data from PCa patient showed a decrease in the FN/COL ratio with the tumor progression, which correlates with our findings (data not shown). To investigate the consequence of the altered ECM composition on cell behavior, we examined how PCa cells behaved on substrates coated with either COL or FN. For instance, PC-3 cells preferred spreading on COL, MyC-CaP cells spread more readily on FN, while 22Rv1 cells did not show a particular preference (data not shown). We then investigated the impact of these preferences on mechanotransduction by monitoring YAP nuclear translocation and cell spreading. Our results showed that PC-3 cells responded more strongly to the stiffness of COL-coated substrates compared to FN-coated ones, whereas MyC-CaP cells displayed an opposite pattern, and 22Rv1 did not shown any difference in response (Figure 2). Additionally, through stiffness gradient, we examined if the change in ECM composition could influence durotaxis. Our results indicated that PC-3 cells migrate towards areas of lower stiffness when exposed to COL compared to FN (Figure 3). In contrast, MyC-CaP cells exhibited a preference for lower stiffness areas when exposed to FN compared to COL, whereas 22rv1 cells had the same response on COL and FN (data not shown). To determine the invasiveness of the different cell lines depending on the composition of the matrix, 3D invasion tests are currently performed in collagen gel supplemented, or not, with FN. All together, these results show that metastatic cells are more responsive on COL, compared to non-metastatic cells that are more responsive to FN. Overall, our study highlights the crucial roles of ECM composition and stiffness in guiding PCa cell behavior. These findings could provide insights into tumor progression and metastasis.

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