Immortalized Fibroblasts with Decreased Rho GTPase Activity Suppress Tumor Cell Growth in Vitro and in Vivo

Cancer-associated fibroblasts (CAFs) are important factors for the growth and spread of tumors. Fibroblasts that initially inhibit tumor growth may lose their inhibitory capacity and even become stimulatory during tumor development.
Using high-throughput imaging system and field live cell microscopy of tumor-fibroblast co-cultures, we showed, that the inhibitory capacity of human hTERT-immortalized fibroblasts BjhTERT correlates with morphology and of their growing layer. The organized ‘‘whirly’’ growth pattern is linked to strong inhibition of tumor cell proliferation and motility. Compared to disorganized “crossy” sub-clone, “whirly” fibroblasts had lower activity of RhoA, one of the major governor of mechanical properties. To check, if RhoA activity in fibroblasts correlates with tumor inhibitory capacity, we knocked out RhoA in BjhTERT. RhoA knocked out (KO) fibroblasts had “whirly” phenotype with significantly down-regulated expression of alpha-SMA and actin stress fibers, decreased in width. They were less spread and elongated and showed pronounced peripheral actin protrusions and diffuse, feather-like, adhesions. Moreover the RhoA(KO)-BjhTERT demonstrated increased tumor inhibitory capacity in vitro and in vivo. After 80 days, only 13% of SCID mice injected with PC3 cells and RhoA(KO)-BjhTERT had formed a tumor. In contrast, 88% of animals injected with a mixture of prostate cancer cells PC3 and BjhTERT with active RhoA had developed tumor.

Our findings confirm, that mechanical properties of fibroblasts play important role in tumor growth. Activation of RhoA leads to changes of the cytoskeleton and cell-matrix adhesion that reduces the ability of fibroblasts to inhibit tumor growth.

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