In summary, these studies provide new insights into the mechanism whereby ovarian tumor spheroids induce mesothelial cell clearance. Clearance-competent tumor spheroids were found to adhere to the dorsal surface of the mesothelial cells and initiate spreading. Protrusions from the spreading cells penetrated underneath the mesothelial cells causing localized breakdown of the mesothelial cell matrix adhesions, and provoked migration of the mesothelial cells. In tumor spheroids, α5β1 integrin, talin I and myosin II were found to be required for spheroid –induced mesothelial clearance. These experiments suggest that ovarian cancer spheroids use actomyosin contractility to exert force via matrix adhesion to the fibronectin organized on the mesothelial monolayer, ultimately leading to mesothelial clearance (see model, ). The mesothelial clearance we observe in vitro may be relevant in human tumors since it has been shown that mesothelial cells are not present under ovarian tumor masses found attached to the peritoneal tissues.
FIGURE 7 Model depicting the events associated with ovarian tumor cell intercalation into a mesothelial monolayer. Cancer spheroids attach to the mesothelial monolayer using various cell adhesion molecules including CD44,α5β1, αvβ1 (more ...)
In contrast to other epithelial tumors that employ hematogenous or lymphatogenous routes to metastasize, ovarian cancer cells predominantly move within the ascites fluid to metastasize to new sites within the peritoneal cavity(8
). The mesothelial monolayer surface provides a variety of ligands to support the attachment of ovarian cancer cells (1
). These ligands include hyaluronic acid, mesothelin and extracellular matrix molecules that are able to engage integrins (19
). Both CD44 and β1-containing integrin dimers have been implicated as receptors that can mediate ovarian cancer cells adherence to the mesothelium. However, function-blocking antibodies directed against β1 integrin or CD44 only partially block ovarian cancer cells adherence to the mesothelial monolayer in short term, in vitro adhesion assays(9
). This suggests that multiple ligands and receptors can support ovarian tumor cell adhesion to the mesothelial monolayer and targeting a single molecule will not abrogate cancer cell interaction with mesothelial cells. Consistent with this, we found that blocking CD44 or selected β1 integrin containing integrin heterodimers expressed by OVCA433 spheroids (α2
) did not significantly block OVCA433 spheroid attachment to the mesothelium after 10 hours of co-culture. Interestingly, however, our data indicated that interfering with the function of α5β1 integrin alone can significantly decrease OVCA433, DOV13 and SKOV3 spheroid-induced mesothelial clearance over a period of 10 hours (Supplementary Figure 3A,C
). As α5β1 integrin is a fibronectin receptor, these results suggest that cancer spheroids can utilize α5β1 to bind to the fibronectin surrounding the mesothelial cells to mediate mesothelial clearance. Supporting this, we found that as a spheroid clears a space in a mesothelial monolayer, the fibronectin fibrils organized on the top of the mesothelial cells are redistributed away from the mesothelial cells and under the spheroid. This process was dependent on functional α5β1 integrin expressed by the cancer spheroids. In addition, we also observed that the expression level of α5β1 integrin in various ovarian cancer cell lines correlated with the ability of these cells to clear the mesothelium (data not shown). However, it is likely that other, α5
integrin-independent mechanisms can mediate clearance as well.
Integrins are the major molecules that can transmit traction forces to the outside environment (23
). While α2
integrin binding to collagen I can induce fibril reorganization and transmit traction forces to the ECM in certain contexts(24
), OVCAR5 ovarian tumor spheroids that express high levels of α2
, but not α5
integrin, were unable to clear the mesothelium in our experiments (Supplementary Figure 3C
). In addition, blocking α2
integrin in OVCA433 cells that express both α2
integrin did not prevent mesothelial clearance (Supplementary figure 3C
). It is possible that α2 integrin does not transmit sufficient traction force under conditions of adherence to mesothelial cells to induce clearance of the mesothelial cells. The generation of traction force on fibronectin has been shown to involve two steps: First, clustering of α5
integrins promotes strong adhesiveness to matrix and second, recruitment of talin I stabilizes and reinforces formed α5
) promoting the exertion of traction force on the matrix force. Our study indicated that the interaction between fibronectin receptor α5
integrin expressed by tumor cells and mesothelial-associated fibronectin is a molecular event that contributes to the clearance process. In addition, we show that expression of talin I by tumor spheroids is required for α5
-mediated formation of traction force and mesothelial clearance. We found that interfering with the function of another fibronectin receptor, αv
integrin, did not affect spheroid–induced mesothelial clearance (Suplementary ), suggesting that these receptors do not contribute to development of myosin contractility by OVCA433 spheroids that adhered to the mesothelial monolayer. This is consistent with previous experiments implicating α5
but not αv
integrins, in the development of contractility (26
). Our data as well as earlier findings(3
) show that the mesothelial cells retract in response to cancer cluster attachment. This raises an interesting question: how does the tumor induce retraction in the mesothelial cells? One possibility is that retraction is induced by the physical force of the spreading tumor cells pulling on the mesothelial cell’s associated ECM and provoking mesothelial cells migration away from the spheroid. Alternatively, the retraction of mesothelial cells could be provoked by a repulsive ligand presented on the tumor cells. In this study, we have shown that force produced by a spreading ovarian cancer cell cluster, via α5
integrin, myosin II and talin I, is important for mesothelial clearance. The evidence that ovarian cancer spheroids deficient in non-muscle myosin II were unable to sustain mesothelial clearance suggests that mere contact between tumor cells and mesothelial cells is not sufficient to induce retraction and migration of mesothelial cells and that a repulsive ligand presented by spheroid does not trigger retraction of the mesothelial cells. However, it is possible that interfering with myosin function also perturbs expression or activity of repulsive ligands present on tumor cell plasma membrane. Spheroid-induced mesothelial clearance was accompanied by disassembly of mesothelial cell matrix adhesion sites, indicating that force induced on the mesothelium by cancer spheroids initiates migratory response in individual mesothelial cells. In spheroid-induced matrix adhesion turnover experiments mesothelial cells that originated from peritoneal wall (LP9) exhibited much more dynamic integrin adhesion when compared to mesothelium isolated from lungs (MET5A) (compare movie 4A and 4B). This suggests that mesothelial cells covering different organs might elicit distinct migratory responses when contacting tumor spheroids.
Earlier studies implicated mesothelial apoptosis as a mechanism of clearance as result of tumor cluster attachment (29
). In our assays clearance of the mesothelium started about 30 minutes after spheroid attachment and was accompanied by migration of individual mesothelial cells from underneath of tumor spheroid. This observation argues that in our assay mesothelial cells respond to contacting tumor cells by activating migratory, but not apoptotic pathways. However it is possible that mesothelial cells that are “stuck” underneath the spheroid and cannot escape, undergo apoptosis.
In patients with advanced disease ovarian tumor clusters predominantly implant into mesothelial lining of peritoneal cavity-associated organs. Invasive tumor implants are able to cross mesothelial layer and gain access to stroma beneath mesothelium (30
). These observations suggest that the mesothelium presents a functional barrier to the spread and progression of ovarian tumors. Hence, one would expect that progression toward invasive disease would be associated with alterations that enable the tumor cells to adhere to the mesothelium and brake mesothelial barrier by provoking mesothelial clearance. Our studies suggest that integrin-dependent activation of myosin contractility in tumor cells is required to perturb the mesothelial barrier. Therefore, our results suggest that acquisition of contractile phenotypes in ovarian tumor cells represents a step towards malignant progression.