Loss of CD151 can promote invasion and growth in a 3D matrix. (A-H) Parental, CD151, α3, and α6 integrin-silenced cell spheroids were embedded in 3D collagen and photographed immediately (Day 0) and 2 d later (Day 2). (I-L) Parental and CD151-silenced cell spheroids were embedded in 3D collagen and grown 2 d in the presence of 1 µg/ml cell permeable C3 transferase to inhibit Rho activity. (M) Quantification of spheroid size on Day 2, plotted as fold change from size on Day 0. Bars indicate mean ± SEM for 6–12 spheroids per cell type. CD151sh3 spheroids were significantly larger and α3sh4 spheroids were significantly smaller than parental spheroids, *P < 0 .0001, and CD151sh3 spheroids treated with C3 transferase were significantly smaller than untreated CD151sh3 spheroids, #P < 0 .0001. ANOVA with Tukey post-tests. (N&O) Parental and CD151-silenced spheroids were embedded in 3D collagen and cultured for 12 d (P) Spheroids cultured for 12 d in 3D collagen were excised and relative cell number was determined by WST assay after digestion with collagenase type II. Bars indicate mean ± SEM for 12 spheroids per cell type. CD151sh3 spheroids had grown significantly larger, *P < 0.0001, unpaired t test.