Abstract 3540: Effect of route of Bacillus Calmette Guerin administration on the immune microenvironment and growth of bladder tumors
Authors: Atallah Aline , Grossman Arielle , Pare Jean-Francois , Siemens Robert , Cotechini Tiziana , Graham Charles H. 
Published: June 2022

Abstract
Background: Bladder cancer is the fifth most common cancer in North America. The standard of care for high-risk non-muscle invasive bladder cancer (NMIBC) involves intravesical immunotherapy with Bacillus Calmette Guérin (BCG). However, it is possible that the efficacy of this modality of BCG administration is suboptimal, as most patients do not respond fully to this immunotherapy. Furthermore, our current understanding of the immunotherapeutic effect of BCG is incomplete. Using a mouse model of NMIBC, we compared the tumor immune microenvironment (TiME) following intravesical versus intravenous administration of BCG, as well as changes in tumor volume and mice survival.
Methods: Female C57Bl/6 mice (6-8 weeks old) were catheterized and instilled with 2.5x105 MB49 bladder cancer cells after poly-L-lysine treatment. Beginning on day 7, mice were treated with intravenous (IV) or intravesical BCG (8 mg/ml) or saline once weekly for three weeks. Ultrasound was performed weekly to monitor tumor growth. Similarly, a cohort of non-tumor bearing mice was treated with poly-L-lysine on day one followed by three weekly intravesical instillations of BCG. In both cohorts, mice were sacrificed on day 23 and bladders were harvested, enzymatically dispersed to generate single-cell suspensions for analysis by flow cytometry, or snap frozen for transcriptomic analysis using NanoString nCounter Platform.
Results: Mice receiving IV BCG had better survival and their bladder tumors were significantly smaller compared with mice receiving intravesical BCG. This reduction in tumor size was associated with a significantly increased proportion of CD8+ T cells and a significantly reduced proportion of inflammatory monocytes in bladder tumors from mice treated with IV BCG compared with intravesical BCG. Whole tumor transcriptome analysis revealed alterations in various signalling pathways associated with route of BCG administration. Our results demonstrate similar trends in the distribution of immune populations in the TiME following intravesical saline and IV BCG treatment, as well as following IV saline and intravesical BCG treatment. We also found that the TiME after intravesical BCG treatment has more immune cells which are predominantly immature myeloid cells. Moreover, our results indicate that intravesical BCG treatment leads to a significant population of immature myeloid cells in the bladders of non-tumor bearing mice.
Conclusion/Significance: These results provide evidence that the route of BCG administration is an important determinant of the TiME composition and may influence anti-tumor responses. Understanding the link between the TiME and BCG therapy may facilitate the development of new approaches to improve outcomes and reduce recurrence rates in patients with NMIBC.
Citation Format: Aline Atallah, Arielle Grossman, Jean-Francois Pare, Robert Siemens, Tiziana Cotechini, Charles H. Graham. Effect of route of Bacillus Calmette Guerin administration on the immune microenvironment and growth of bladder tumors [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 3540.