Interestingly, these data are in contrast with a previous study using non-tumor selleck bio PtK2 cells showing progressively decreases of ��1-integrin engagement on soft substrates [3]. These observations are in accordance with other studies demonstrating that murine breast cancer cells retain high levels of active ��1-integrin after 24h of culture on a soft substrate [27]. Integrin accumulation in the cell mid-zone is also necessary to induce mitotic cell adhesion and to support cytokinesis by providing mechanical anchoring for the contractile actomyosin ring [28]. To examine the localization of ��1-integrin, immunofluorescence performed in cells in telophase revealed ��1-integrin accumulation in the mid-body, on E50 and on E20, like on glass (Figure 5D). On glass, E50 and E20 actin accumulated also in this cell mid-zone (Figure 5D).

These results suggest continuous ��1-integrin engagement during mitosis despite the soft substrates, compatible with the involvement to support cytokinesis. Figure 5 ��1-integrin engagement of mitotic SW480 cells with respect to soft substrates. 4. Mitotic spindle organization of tumor cells in response to soft substrates We next checked mitotic spindle assembly on soft matrices by immunofluorescence with anti-��-tubulin and DNA staining with Hoechst. The mitotic spindles of SW480 cells, visible on stiff substrates (glass), were preserved on E50 and even on E20 (Figure 6A and B). This result contrasts with that observed for non-cancerous mitotic PtK2 cells seeded on soft matrix since for Young modulus �� 50 kPa, the mitotic spindle could not be observed [3].

Consistent with our living cell analysis (Figure 3B and D), abnormal chromosome segregation events could be observed (Figure 6B and D), however, without evidence for multipolar or monopolar spindles on the different substrates probably due to ��1-integrin engagement maintained on the very soft substrate. Indeed, multipolar spindles were observed for cells bearing mutated ��1-integrin [23]. Figure 6 Mitotic spindle organization with respect to soft substrates. 5. Rac1 expression of tumor cells in response to soft substrates Rac1, proteins of the small GTPase Ras family, are involved in the orientation of the mitotic spindle and its activity increases at the plasma membrane of polar sides during cytokinesis [29].

We further synchronized a collection Batimastat of SW480 cells and analyzed Rac1 expression through Western blot experiments [29]. There were no differences in Rac1 expression for mitotic SW480 cells seeded either on soft substrates (E50 and E20) or on stiff substrate (glass) (Figure 7A-C). Our results showed that mitotic tumor SW480 cells maintain Rac1 expression on soft substrates. On the contrary, we previously observed that non-cancerous PtK2 cells progressively decreases this expression on soft substrates [3].