Abstract PR004: 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 183 tumors from 111 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. 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: We identified a total of 364, 504 cells in the tumor tissue using IMC. After computing a neighborhood graph using the log normalised and z-scaled signal and embedding it using UMAP, leiden clustering was performed 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 (DCs), NK cells and neutrophils in the TME of analyzed tumors. Specifically, we observed more CD8 T cells after BCG (p=0. 015), especially in females (p=0. 048). Females also had higher levels of DCs and macrophages after treatment compared to males (p=0. 024 and p=0. 048). Patients who progressed to MIBC after BCG had higher numbers of CD8 T cells and NK cells prior to BCG (p=0. 039 and p=0. 021). Prolonged exposure to interferons has been associated with immune evasion and tumor cell proliferation. In concordance, we found high pretreatment protein expression of IFNα and IFNγ in tumor regions from patients with a pronounced signature of CD8 T cell exhaustion after BCG treatment using GeoMX DSP proteomic analysis. Data analysis is currently ongoing, and additional results will be presented at the conference. 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, sex and progression. A greater understanding of the TME may help identify patients unresponsive to BCG earlier and improve the understanding of biological differences in tumor development and aggressiveness. This may ultimately improve 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 AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30 (10Suppl): Abstract nr PR004.