Abstract 769: Spatial proteomics and transcriptomics reveal an altered immune cell landscape in bladder cancer patients unresponsive to BCG treatment

Abstract Introduction: Patients with high-risk non-muscle invasive bladder cancer (NMIBC) are recommended treatment with Bacillus Calmette-Guérin (BCG). The therapeutic effect of BCG is highly dependent on the host immune system and the tumor microenvironment (TME). The cellular composition and the functional status of the TME have been shown to play crucial roles for treatment efficacy. However, much is still unknown regarding the cellular interactions and mechanisms of BCG. Using spatial proteomics and transcriptomics we investigated the immune cell landscape in a cohort of BCG treated patients. Materials and methods: A total of 150 tumors from 105 patients with NMIBC treated with BCG were included on a tissue microarray (TMA). The TMA included paired pre- and post-BCG tumors. We analyzed the tumor tissue using Imaging Mass Cytometry (IMC; StandardBioTools) for simultaneous detection of 35 immune and tumor-related proteins at single cell resolution. Ilastik was used to generate probability maps and DeepCell Mesmer was used for cell segmentation. Markers were quantified for the mean intensity per cell. The GeoMX Digital Spatial Profiling (DSP) was used for spatial proteomics and transcriptomics profiling of tumor and stromal areas, targeting 63 protein targets and whole transcriptome amplification, respectively. Results: After computing a neighborhood graph using the log normalised and z-scaled signal and embedding it using UMAP, leiden clustering was performed on 360, 673 cells to detect communities of unique cell types by interrogating the presence of phenotypic markers. We identified major lineages such as macrophages, CD8 T cells, dendritic cells, natural killer (NK) cells and neutrophils in the TME of analyzed tumors. We observed more macrophages, CD4 T cells and CD8 T cells after BCG (p=0.02, p<0.001 and p=0.004, respectively) and similarly, we found enrichment of cellular neighborhoods composed of immune cells after treatment. Patients who progressed to muscle-invasive bladder cancer after BCG had higher numbers of CD4 T cells, CD8 T cells and NK cells prior to BCG (p=0.034, p=0.023 and p<0.001, respectively). Prolonged exposure to interferons has been associated with immune evasion and tumor cell proliferation. In concordance, we found high pretreatment expression of interferon signaling pathways in tumor regions from patients with a signature of CD8 T cell exhaustion after BCG using GeoMx DSP transcriptomic analysis. Conclusion: The composition and functional status of the TME is associated with clinical and biological features such as immune cell abundance, CD8 T cell exhaustion, and progression. A greater understanding of the TME may help identify patients unresponsive to BCG and improve the understanding of biological differences in tumor development and aggressiveness ultimately improving patient outcomes. Citation Format: Trine Strandgaard, Tine Ginnerup Andreasen, Tessa Jane Divita, Liina Salminen, Sean Houghton, Kasper Thorsen, Iver Nordentoft, Iyinyeoluwa Okulate, Alexander Schmitz, Søren Riis Paludan, John Sfakianos, Amir Horowitz, Jørgen Bjerggaard Jensen, Lars Dyrskjøt. Spatial proteomics and transcriptomics reveal an altered immune cell landscape in bladder cancer patients unresponsive to BCG treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 769.