Abstract 1633: Comprehensive cell- and gene-based tumor profiling using flow cytometry and Nanostring in a murine bladder cancer model
Authors: Lapinski Philip E. , Draper David W. , Wise Scott 
Published: June 2022

Abstract
Background: Five checkpoint immunotherapies that target the PD-1/PD-L1 axis are currently FDA approved. Novel approaches are helping to identify new combination treatment strategies for therapeutic intervention of bladder cancer, which is the thirteenth leading cause of cancer-related deaths. Using anti-mPD-1 treatment in a murine bladder cancer model MB49, we show that non-targeted immune gene expression profiling combined with flow cytometry provides a gene and cell-specific signature for the tumor microenvironment, which aids in the identification of targets for novel treatment approaches.
Methods: C57BL/6 mice with established MB49 tumors were treated with anti-mPD-1 or isotype control antibodies. Tumors were collected 3 days after the last treatment. Treatment-induced immunophenotypic changes were examined in tumor-infiltrating immune subsets using T cell- and myeloid-focused flow cytometry panels. We used the mouse PanCancer IO 360™ Nanostring panel for transcriptomic analysis of 770 genes and the ROSALIND™ platform (OnRamp BioInformatics) to identify differentially regulated genes between treatment groups.
Results: Treatment of tumors with anti-mPD-1 showed moderate anti-tumor activity, with a 58% tumor growth inhibition at day 18 post-implant. Immunophenotyping by flow cytometry revealed that anti-mPD-1 triggered an increase in tumor-infiltrating CD8+ T cells compared to control animals. Additionally, the CD8+ T cell phenotype was altered by treatment, with increased frequency of ICOS and LAG-3 in CD8+ T cells in tumors from treated animals. Changes in the T cell compartment also included reduction in the proportion of central memory CD8+ and CD4+ T cells compared to controls. In the myeloid compartment, iNOS expression increased in tumor-associated macrophages from treated animals. NanoString analysis revealed 62 genes were differentially regulated in tumors from treated animals compared to controls. ROSALIND analysis classified 30 of the genes as regulators of interferon, cytotoxicity, antigen presentation, and cytokine signaling. Among the genes upregulated by anti-mPD-1 were IDO, TIM-3, and CSFR1, which can promote tumor growth and are clinical targets being investigated for new immunotherapies.
Conclusions: NanoString analysis complemented immunophenotyping to provide a comprehensive profile of the MB49 tumor model. Together, these data demonstrate that anti-mPD1 increases T cell recruitment into the tumor and upregulates the expression of genes known to enhance T cell recruitment and anti-tumor activity. iNOS protein upregulation suggests that anti-mPD-1 treatment may also exert effects by driving M2 macrophages towards an M1 phenotype. Further investigation may elucidate clinical implications for inhibitors of these gene products as treatment options in combination with anti-mPD-1.
Citation Format: Philip E. Lapinski, David W. Draper, Scott Wise. Comprehensive cell- and gene-based tumor profiling using flow cytometry and Nanostring in a murine bladder cancer model [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 1633.