Cancer-associated fibroblasts (CAFs), which occupy a major portion of the TME, play an important role in tumor growth, immunosuppression, treatment resistance. Our studies found that fibroblast activation protein α (FAPα) based whole cell tumor vaccine (WCTV) can induce a cross-immune response to produce specific neutralizing antibodies and CTL effects to kill tumor cells and CAFs. However, the therapeutic efficacy of vaccination is limited. Recent studies have revealed that stereotactic ablation radiotherapy (SABR) also elicit systemic antitumor immunity, which makes it an ideal partner for immunotherapy. The goal of this study was to examine synergistic effect of SABR and FAP-based WCTV.We produced FAPα-based WCTV by infecting tumor cells with lentiviral vector pCDH encoding FAPα, and then inactivating them with radiation (100Gy) for immunotherapy in vivo. To investigate the optimal protocol, we compared different SABR schemes and sequence of the combination of irradiation and FAPα- based WCTV in mouse LLC Lewis lung cancer model. Subsequently, we tested whether the combination protocol would result in enhanced anti-tumor activity and antifibrotic effect in mouse 4T1 breast cancer and CT26 colon cancer models. Furthermore, we analyzed the impact of treatment on cellular immunophenotypic characterization in TME via immunohistochemical staining and flow cytometry.We demonstrated that the therapeutic effect of SABR combined with FAPα-based WCTV is better than radiotherapy or vaccination alone. We identified the optimal dose scheme of SABR synergistically with FAPα- based WCTV is a single dose of 8Gy × 3 times, and the best treatment sequence is radiotherapy within one week after the first vaccination (three times in total). The optimal combination protocol also resulted in enhanced anti-tumor activity and antifibrotic effect against 4T1 breast cancer and CT26 colon cancer, and inhibited lung metastasis in breast cancer. No adverse effects were found based on gross measures of health. Importantly, collagen type I and FAPα expression in tumor tissue was significantly lower in SABR+ Vaccine treated mice than in control mice. SABR combined with FAPα-based WCTV reduced microvascular and lymphatic invasion, allowed polarization of tumor-associated macrophages to the M1-like phenotype. The relative percentage of M2 macrophages, MDSCs, and Tregs significantly decreased in the peripheral blood and lymph nodes. By contrast, CD8+ T lymphocytes especially activated cells increased.Our findings indicate that combined FAPα-based WCTV and SABR synergize to suppress tumor growth and metastasis via activating the systemic anti-tumor immune response, attenuating CAFs-mediated immunosuppression, interstitial fibrosis and treatment resistance. This novel combination of radio-immunotherapy may add a new perspective to cancer treatments.Copyright © 2021. Published by Elsevier Inc.