(A) Ovarian cancer spheroids (OVCA433) labeled with CMTCX cell tracker dye (red) were pipetted on top of a confluent monolayer of GFP-labeled primary TERT immortalized human lung mesothelial cells (green) and incubated for 60 min. The dynamics of these two cell populations were followed in parallel for 10 hours. Images show a time course of mesothelial clearance at 0, 7 and 10 hours. Scale bar = 100μm (B) Quantification of mesothelial clearance from experiment shown in A. (C) Representative images from a mesothelial clearance assay utilizing a primary tumor cluster isolated from the ascites fluid of an ovarian cancer patient. The sample was labeled and assayed as in (A) Scale bar = 50μm. (D) Time lapse images of multiple Z sections (side view) of OVCA433 CMTCX-labeled spheroids inducing clearance of GFP- labeled lung mesothelial cells.

(A) Temporal analysis of adhesion dynamics of Paxillin-GFP labeled LP9 human peritoneal mesothelial cells incubated with or without RFP-actin expressing OVCA433 cancer spheroids. Events of adhesion assembly and disassembly were scored. Scale bar = 5μm (B) The bar graph shows the ratio of adhesion assembly and disassembly events within the same area. A total of 150 assembly and disassembly events were analyzed per condition. (C) GFP-labeled lung mesothelial cells were mixed at 1:500 ratio with non -labeled lung mesothelial cells. Images show temporal behavior of single mesothelial cells in the presence or absence of cancer spheroids. Scale bar =100μm (D) The migration distance of single mesothelial cells was measured in the presence and absence of OVCA433 cancer spheroids. 50 GFP-labeled mesothelial cells were analyzed per condition.

(A) Western blot of non-muscle myosin heavy chain A and B (NM IIA or NM IIB) expression levels in control or NM IIA/NM IIB shRNA treated OVCA433 cells. (B) Images show the temporal behavior of mesothelial cells contacting control or myosin shRNA expressing OVCA433 ovarian cancer cells. Scale bar = 50μm (C) Quantification of mesothelial clearance from B. 20 spheroid attachment sites were analyzed per condition. (D) Western blot of talin I expression levels in control or talin 1 shRNA expressing OVCA433 cells. (E) Images show temporal behavior of mesothelial cells contacting control or talin 1 shRNA expressing OVCA433 ovarian cancer cells. Scale bar = 50μm (F) Quantification of mesothelial clearance from E. 15 randomly chosen regions were analyzed per condition.

(A) Western blot analysis of α5 integrin expression levels in OVCAR5 and OVCA433 ovarian cancer cells. Scale Bar = 10μm (B) Images depict mesothelial clearance induced by OVCA433 cancer spheroids in the presence of control or α5β1 integrin blocking antibody at 0 and 10 hour time points. Scale bar = 50μm (C) Quantification of mesothelial clearance from E. 12 independent regions were analyzed per condition..
(D) Western blot analysis of α5 integrin, phospho-MLC and MLC expression levels in control or α5 overexpressing OVCAR5 ovarian cancer cells plated on fibronectin-coated glass. Images show organization of the actin cytoskeleton in control and α5 integrin overexpressing OVCAR5 ovarian cancer cells plated on fibronectin-coated glass. (E) Images represent mesothelial clearance induced by control or α5 integrin overexpressing OVCAR5 cells at 0 and 10 hour time points. Scale bar = 50μm (F) Quantification of mesothelial clearance from B. 20 randomly chosen regions were analyzed per condition.

(A) The upper image shows a laser scanning confocal image shows the top view of an OVCA433 cancer spheroid (blue) inserted into a mesothelial monolayer (green) stained with an antibody directed against human fibronectin (red). Scale bar = 10μm. The lower image shows a side view reconstruction of multiple Z planes of the same image. (B) Images show temporal acquisition of mesothelium (green) associated fibronectin (red) by OVCA433 cancer spheroids (blue). Scale bar = 10μm (C) Dissociation of fibronectin (red) from mesothelial monolayer (green) after 7 hours in response to spheroids (blue) pretreated with either control or α5β1 blocking antibody. Scale bar = 10μm. (D) Quantification of fibronectin dissociation from a mesothelial monolayer in response to an attached cancer spheroid pretreated with either control or α5β1 antibody. 8 randomly chosen regions were analyzed in the control group and 20 randomly chosen regions were analyzed in the ITGA5 group.

OVCAR5 ovarian cancer cells were plated on a polyacrylamide (PAA) gel substrate coated with fibronectin (10ug/ml). Cells were allowed to adhere to and spread on the PAA substrate and the displacement of fluorescently labeled beads embedded in the substrate was monitored. The strain energy was calculated from measured bead displacement data and used to reconstruct traction stresses. The strain energy is the energy invested by the cells to deform the substrate and a measure for the overall contractility of a cell (37). (A) OVCAR5 cells have low endogenous α5-integrin (ITGA5) expression levels, and are significantly less contractile than OVCAR5 cells overexpressing ITGA5 (p<0.001). The high contractility of ITGA5-overexpressing OVCAR5 cells is attenuated by treatment with 1uM blebbistatin (p<0.001). (B) The contractility of ITGA5-overexpressing OVCAR5 cells can also be significantly attenuated by siRNA-mediated knockdown of Talin 1, but only slightly attenuated by knockdown of Talin II. Bar = 20μm.

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 and α2β1 integrins. Interaction of α5β1 integrin, expressed on cancer spheroids with fibronectin presented on the mesothelium promotes activation of myosin in the tumor spheroids and transmission of the force from the spheroid to the fibronectin on the monolayer. This promotes dissociation of mesothelial cells adhesions and their migration away form intruding tumor cells, leading to mesothelial cells exclusion from the base of cancer spheroid.