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Background: Bone metastatic prostate cancer does not completely respond to androgen-targeted therapy and generally evolves into lethal castration resistant prostate cancer (CRPC). Expression of AR-V7- a constitutively active, ligand independent splice variant of AR is one of the critical resistant mechanisms regulating metastatic CRPC. TNC is an extracellular matrix glycoprotein, crucial for prostate cancer progression, and associated with prostate cancer bone metastases. In this study, we investigated the mechanisms that regulate AR-V7 expression in prostate cancer cells interacting with osteogenic microenvironment including TNC. Methods: Prostate cancer/preosteoblast heterotypical organoids were evaluated via immunofluorescence imaging and gene expression analysis using RT-qPCR to assess cellular compartmentalization, TNC localization, and to investigate regulation of AR-V7 in prostate cancer cells by preosteoblasts and hormone or antiandrogen action. Prostate cancer cells cultured on TNC were assessed using RT-qPCR, Western blotting, cycloheximide chase assay, and immunofluorescence imaging to evaluate (1) regulation of AR-V7, and (2) signaling pathways activated by TNC. Identified signaling pathway induced by TNC was targeted using siRNA and a small molecular inhibitor to investigate the role of TNC-induced signaling activation in regulation of AR-V7. Both AR-V7- and TNC-induced signaling effectors were targeted using siRNA, and TNC expression assessed to evaluate potential feedback regulation. Results: Utilizing heterotypical organoids, we show that TNC is an integral component of prostate cancer interaction with preosteoblasts. Interaction with preosteoblasts upregulated both TNC and AR-V7 expression in prostate cancer cells which was suppressed by testosterone but elevated by antiandrogen enzalutamide. Interestingly, the results demonstrate that TNC-induced Src activation regulated AR-V7 expression, post-translational stability, and nuclear localization in prostate cancer cells. Treatment with TNC neutralizing antibody, Src knockdown, and inhibition of Src kinase activity repressed AR-V7 transcript and protein. Reciprocally, both activated Src and AR-V7 were observed to upregulate autocrine TNC gene expression in prostate cancer cells. Conclusion: Overall, the findings reveal that prostate cancer cell interactions with the cellular and ECM components in the osteogenic microenvironment plays critical role in regulating AR-V7 associated with metastatic CRPC. 9BxMEZaJbqgytBg6QvviEa Video Abstract [ABSTRACT FROM AUTHOR]