Introduction: In non-muscle invasive bladder cancer (NMIBC), immunosuppressive factors responsible for post-BCG therapy recurrence remains unclear. In this study, we performed comprehensive genetic analysis and functional analysis in untreated NMIBC to explore key players of immunosuppression associated with recurrence after BCG therapy. Methods: 13 patients (73.5±8.9 years old) with untreated NMIBC who were treated over 6 months BCG and could be followed up for 5 years were included. They were divided into two groups; recurrence (n=6) and non-recurrence groups (n=7). Genes that are upregulated in bladder cancer tissues compared to normal bladder tissues and associated with recurrence and immunosuppression were extracted from the microarray of untreated bladder cancer tissue. In bladder cancer cells (RT4 and UMUC5), the expression of the genes was evaluated by qPCR and Western blot (WB). Cell proliferation (WST assay), adhesion (CytoSelectTM), invasion (scratch assay) and secretion of immunosuppressive cytokine TGF-ß1 (ELISA and immunostaining) were also assessed after knockdown of the genes to evaluate the function of the gene in bladder cancer cells. Results: Microarray analysis revealed integrin beta 8 (ITGB8) was significantly upregulated in the recurrence group (p < 0.0001). The microarray results were validated with ITGB8 immunostaining of the same patient's tissue and showed similar results to the microarray. qPCR and WB results showed that ITGB8 was highly expressed in UMUC5 compared to RT4. In UMUC5, ITGB8 knockdown significantly decreased cell proliferation, adhesion, invasion (p < 0.01) and TGF-ß1 secretion (p < 0.01). It also significantly reduced staining for TGF-ß1 and TGF-ß1 LAP-D, which indicates TGF-ß1 activation (p < 0.01). Conclusions: In untreated NMIBC, ITGB8 may contribute to recurrence after BCG by suppressing tumor immunity through the activation of TGF-ß1. Thus, inhibition of ITGB8 was considered to prevent the recurrence of NMIBC via inducing an immune response by BCG therapy. SOURCE OF Funding: This study was supported by a Grant-in-Aid for Young Investigators from the Japan Society for Promotion of Science (JSPS KAKENHI 19K18590, S. Hoshi).
