Defining the microenvironment landscape of bladder cancer using highly multiplexed spatial genomic and proteomic analysis

Abstract

One Sentence Summary:A new method for capturing tumor-immune signaling in FFPE tissues explores how the PPARG signaling axis is associated with immune cell exclusion in bladder cancer. Abstract:Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. PD-1 pathway targeting immunotherapies have been approved to treat advanced bladder cancer, but most patients exhibit primary resistance, suggesting that immune evasion mechanisms exist. The PPARγ pathway has been identified as a potential therapeutic target in MIBC that is associated with reduced CD8+ T-cell infiltration and increased resistance to immunotherapies. We comprehensively profiled the tumor microenvironment (TME) in formalin-fixed, paraffin-embedded (FFPE) tissues from a cohort of PPARγ high (n=13) and PPRARγ low (n=12) MIBC, integrating bulk gene expression, targeted mutation sequencing, immunohistochemistry and multiplex spatial profiling of RNA and protein expression on the GeoMx™ Digital Spatial Profiling (DSP) platform. Molecular subtyping was consistent between traditional methods and GeoMx profiling, and, in this cohort, we observed little evidence of spatial heterogeneity in tumor subtyping. The previously characterized T-cell exclusion phenotype of PPARγ high MIBC was recapitulated on the GeoMx platform and was further extended to show that this is a general phenomenon across immune cell types, supporting potential combination of PPARγ inhibition with ICIs. Furthermore, we found that while immune cells were excluded from PPARγ high tumors, the stromal compartment from these tumors was not significantly different than those PPARγ low tumors. By preserving spatial relationships during the GeoMx analysis, we also identify a novel association between lower immune cell expression in the tumors and higher expression of β-catenin in the stroma, and differential expression of other WNT pathway members associated with PPARγ activity.Patients with the luminal I subtype rarely respond to ICIs(14,15), suggesting the existence of one or more immune escape mechanisms. We previously reported that the PPARγ/RxRa pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC (6,16). In tumors with high PPARγ pathway activity, CD8+ T-cell infiltration appears to be impaired through chemokine suppression, though additional mechanisms may also impact Tcell infiltration. These findings suggest that pharmacological inhibition of PPARγ may induce sensitivity to immunotherapies. To further our understanding of tumor-immune escape mechanisms in MIBC, analysis of both the tumor and the surrounding microenvironment is necessary. Comprehensive interrogation of the interaction between these two compartments has proved challenging thus far because most of the foundational work in MIBC has relied on commonly available assays such as single-plex immunohistochemistry (IHC) assays or bulk RNA expression and mutation profiling. With limited availability of tumor tissues during clinical trials, highly multiplexed and quant...

Digital spatial profiling of the microenvironment of muscle invasive bladder cancer

“…2 Division of Cancer Sciences, University of Manchester, Manchester, UK. 3 CRUK Manchester Centre, Manchester, UK. 4 Mount Vernon Centre for Cancer Treatment, Northwood, UK.…”

“…Our data show that tumour, stroma, and immune infiltrated ROIs of MIBC have distinct transcriptomic profiles in situ. Previous work in bladder cancer using DSP utilized focussed probe panels covering a limited number of RNA or protein targets 3,18 . Thus, we successfully generated, for the first time, a whole transcriptome spatial transcriptomics dataset using bladder cancer TMAs.…”

“…There have been only a few spatial transcriptomics studies in bladder cancer. Initial unpublished results by Reeves et al 3 showed molecular subtyping was consistent between traditional methods and DSP using an early panel profiling ~150 genes 3 . Studies of DSP in other cancers revealed novel insights into the tumour microenvironment to improve risk stratification 4 and identify interactions likely to affect biological processes 5 .…”

Systems biology approaches applied in mass spectrometry imaging

“…The last direction of the development in the near future that I would like to discuss is the integration of MSI datasets with other (imaging) technologies. More and more scientific fields, such as transcriptomics (Reeves et al, 2019;Ståhl et al, 2016) and proteomics (Doll et al, 2017), are also working towards collecting single cell-based information in a spatially resolved manner. Concurrently, online resources are being curated to match these advances to share and catalog data.…”