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Introduction: Cancer-associated fibroblasts (CAFs), the main cellular component of the tumor microenvironment (TME), contribute to tumor metastasis. Prostate cancer (PCa) is the most common malignancy among the elderly male population, and it is typically fatal when the disease progresses to metastasis and castration-resistant stage (CRPC). We have previously demonstrated that ADAM9 increased during PCa progression, and ROS is the critical mediator regulating ADAM9 expression in CRPC cells to promote tumor growth. In addition to membrane-anchored ADAM9, a secreted variant of ADAM9 (sADAM9) was recently identified in activated liver stromal cells that facilitated colon cancer liver metastasis. However, it is not clear whether sADAM9 is expressed by prostatic CAFs and, if so, whether it contributes to aggressive tumor phenotypes through tumor-stromal interactions. Objective: To investigate the sADAM9 variants in CAFs and to elucidate the effects of CAF-derived sADAM9 on the aggressiveness of PCa cells. Methods: Immunohistochemistry staining and ELISA of clinical samples were used to assess the expression of ADAM9 during PCa tumor progression. Laser microdissected tissues, primary cell lines, and experimental cell model of paired normal fibroblasts and CAF cells were analyzed. The effects of sADAM9 on CRPC progression were conducted in PC3 cells by WST-1 cell proliferation assay, transwell migration assay, and μ-slide chemotaxis assay. Immunoblot analyses of integrin β1 and migration-related kinases were used to verify the possible downstream signaling pathway of sADAM9. Results: Increased overall intensity of ADAM9 protein expression in PCa patients with metastasis compared. Elevated ADAM9 expression was observed in both cancerous epithelial cells and the tumor stroma. The average serum ADAM9 concentration measured by ELISA was 6.253 ± 0.448 ng/mL in BPH patient samples versus 10.89 ± 0.953 ng/mL in PCa patient samples. A smaller molecular weight (~50-kDa) of ADAM9 was detected in CM from cultured CAFs but not normal fibroblasts or PCa cell lines except for DU145 cells. Treatment of PC3 cells with sADAM9 induced a significant chemotactic migration while did not affect cell proliferation. Furthermore, a stark increase in activated integrin β1, phosphorylated focal adhesion kinase (p-FAK), and p-AKT, but not p-mTOR in PCa cells by sADAM9 suggested the induction of cell migration occurs through activation of the integrin β1/FAK signaling pathways. Conclusion: Herein, we have provided in vitro evidence to demonstrate the pro-metastatic function of sADAM9 from CAFs that facilitate the migration of CRPC cells through integrin receptor signaling, which could be partly responsible for the promotion of CRPC metastasis. Further molecular identification of controlling spliced ADAM9 expression in CAFs remains to investigate. Citation Format: Weichieh Chen, Le Thi Huynh Trang, Chia-Ling Hsieh, Katsumi Shigemura, Masato Fujisawa, Leland W.K. Chung, Shian-Ying Sung. ADAM9 secreted by cancer-associated stromal cells promotes metastasis of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6125.