Indeed, on these substrates only 60% (E50) and 10% (E20) of cells are able to progress in mitosis Figure 7 Rac1 expression of mitotic SW480 cells with respect to soft substrates. selleck chemical 6. DNA replication activity of tumor cells in response to soft substrates To determine whether SW480 cells that were able to progress through mitosis were capable of reenter cell cycle, we investigated their capability to undergo DNA replication 4h after being seeded on different substrates. SW480 cells seeded on E50 and E20 show site of replication uniformly distributed in the nucleus (Figure 8A) with respectively 60% and 23% of cells with nuclear BrdU signal (Figure 8B).
It is noteworthy that the percentages of SW480 cells incorporating BrdU correlated with the percentage of cells achieving mitosis on E50 and E20 (Figures 3C and and8B),8B), suggesting that SW480 cells able to complete chromosome segregation on soft substrates are further able to undergo a new cycle of DNA replication. Figure 8 DNA Replication with respect to soft substrates. Conclusion In conclusion, we report that despite massive cell death on extremely soft substrates (E0), tumor cells like the SW480 colon cancer cells, even when bearing abnormal chromosome segregation, resist to the very soft substrates (E20 and E50) and achieve mitosis. These findings might be highly relevant for the pathophysiology of cancer and the dissemination of colon tumor cells. Indeed, their cancerous nature at least some of the tumor cells might help them to overcome chromosomal segregation abnormalities linked to the change in substrate stiffness and therefore escape the soft substrates to pursue their journey up to the site of metastasis formation.
Moreover, this ability to overcome segregation abnormalities could result in more chromosomal rearrangements, which may contribute to increasing tumor cell aggressiveness. Further investigating the response of tumor cells to physical environmental changes may help to identify new potential targets for anticancer therapy. Materials and Methods 1. Materials and fabrication of PEM PLL (MW = 5.7 x 104 Da, Sigma, St. Quentin Fallavier, France) and HA (MW = 4.0 x 105 Da, BioIberica, Barcelona) were used for buildup (PLL/HA)24 films, and PSS (MW = 7.0 x 104 Da, Sigma, St. Quentin Fallavier) and PAH (MW = 7.0 x 104 Da, Sigma) for (PSS/PAH)n capping films (n corresponds to the number of layer pairs), which were deposited on top of (PLL/HA)24 strata.
PLL, HA, PSS, and PAH were dissolved at 1 mg/mL in a buffer solution containing 150 mM NaCl and 20 Dacomitinib mM of tris(hydroxymethyl)-aminomethan (TRIS, Merck) at pH 7.4, and all rinsing steps were performed in the same buffer. (PLL/HA)24 strata and (PSS/PAH)n capping films were prepared using a dipping machine (Dipping Robot DR3, Riegler & Kirstein GmbH, Berlin, Germany), on glass slides (VWR Scientific, Fontenay sous Bois, France).