Introduction: A T cell-inflamed tumor microenvironment characterized by the presence of tumor-infiltrating T cells or expression of an immune gene signature by RNA sequencing is linked to improved prognosis and response to immunotherapy in bladder cancer. BATF-3+ dendritic cells have been observed in murine models to be critical for both priming an immune response and recruiting effector CD8+ T cells to the tumor microenvironment. However, the role of BATF-3+ dendritic cells in bladder cancers from patient specimens, especially in relation to effector CD8+ T cells, remains unknown.

Methods: We performed multiplex immunofluorescence imaging on 61 surgically resected muscle invasive bladder cancer specimens to evaluate the population of tumor infiltrating immune cells (TIICs) including CD8+, BATF-3+, PD-1+, and FOXP3+ cells. The relationship of TIICs to a previously described immune gene signature based on RNA sequencing was investigated in a subset of these samples.

Results: In the 61 muscle invasive bladder cancer samples analyzed, the average (range) numbers of each cell type per 1000 cells in tumor area were 15.73 (0-162.33) for CD8+ cells, 0.68 (0-6.32) for BATF-3+ cells, 1.42 (0-27.10) for PD-1+ cells, and 6.10 (0-27.82) for FOXP3+ cells. In the subset (n =3 4) with available RNA sequencing data, the average numbers of each cell type for the tumors with T cell-inflamed phenotype (n = 17) vs the tumors with non-T cell-inflamed phenotype (n = 17) were, 23.39 versus 8.99 (P = 0.024) for CD8+ cells, 1.26 versus 0.22 (P = 0.034) for BATF-3+ cells, 3.48 versus 0.37 (P = 0.005) for PD-1+ cells, and 11.40 versus 2.72 (P = 0.001) for FOXP3+ cells, respectively. The proportion of T cell-inflamed tumors was significantly higher in groups with above median TIICs versus below (73.3% versus 31.6% for CD8 [P = 0.016], 75.0% versus 27.8% for BATF-3 [P = 0.006], and 70.0% versus 21.4% for PD-1 [P = 0.005], and 78.9% in FOXP3high and 13.3% in FOXP3low [P < 0.001], respectively). Analyzing BATF-3 and CD8 jointly identified the group with the highest proportion of T cell-inflamed tumors (90.0% in CD8highBATF-3high versus 33.3% in others [P = 0.003]) and showed a stronger association than with CD8 alone. In a spatial analysis using G function between CD8+cells and BATF-3+ cells, the median area under the curve was 0.88, indicating that CD8+ cells clustered in proximity to BATF-3+ cells.

Conclusions: Infiltration of each immune cell type correlates with a T cell-inflamed or non-T cell-inflamed tumor microenvironment by gene expression profiling. CD8highBATF-3high tumors are nearly universally T cell-inflamed. Further analyses to assess associations between immune cell types and their spatial relations within the tumor microenvironment, and its correlation with survival outcomes and immunotherapy response are ongoing.

Citation Format: Ken Hatogai, Danny Kim, Yuanyuan Zha, Gary D. Steinberg, Alexander T. Pearson, Thomas F. Gajewski, Randy F. Sweis. Multichannel immunofluorescence imaging to assess the immune composition of tumor microenvironment in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1093.