Intercellular Mechanisms of Therapeutic Resistance at the Tumor-Stromal Interface Using Ultra High-Plex Single-Cell Spatial Transcriptomics and Genetically-Engineered Tumoroids

Abstract

There is a major gap in knowledge regarding how intercellular interactions in the tumor microenvironment (TME) mediate therapeutic resistance. Achievement of this goal has been limited by a lack of (1) spatial context in dissociated single-cell methods; (2) single-cell resolution in spatial profiling approaches; (3) high quality data and yield with FFPE patient specimens; and (4) computational methods for ligand-receptor analyses that consider both gene expression and spatial coordinates. We developed an innovative spatial biology paradigm that combines cutting-edge experimental and computational methods to enable high-resolution, spatially-guided discovery of critical mediators of therapeutic resistance. We applied this approach to dissect the single-cell spatial transcriptomic landscape of untreated vs. chemoradiotherapy-treated primary human pancreatic ductal adenocarcinoma (PDAC; n = 21) using ultra-high plex spatial molecular imaging (SMI) optimized for high-sensitivity, subcellular detection of up to 6000 gene transcripts in FFPE sections-an order of magnitude greater than contemporary methods. We recovered over 1,000,000 high-quality single cells in situ representing more than 20 distinct cell types, including epithelial, immune, endothelial, endocrine, and diverse stromal cells. We developed an optimal transport-based computational method to infer cell-cell communication at the cancer-stromal interface. Treatment with chemoradiotherapy was associated with the largest increase in fibroblast-malignant interactions. Comparing the SMI data with orthogonal single-nucleus RNA-sequencing and digital spatial profiling data, we identified CLCF1-CNTFR as the fibroblast-malignant interaction most associated with resistance to chemoradiotherapy in PDAC. CLCF1 is a gp130-family cytokine that activates Jak-STAT signaling and acts as a potent neurotrophic factor. Notably, the CLCF1-CNTRF (fibroblast-malignant) interaction has prominent pro-oncogenic effects in lung adenocarcinoma and an engineered CNTFR decoy receptor with therapeutic potential has been developed. To functionally validate the role of the CLCF1-CNTFR (fibroblast-malignant) interaction in mediating resistance to cytotoxic therapy, we created CRISPR-engineered cancer-fibroblast tumoroids and modulated expression of this ligand-receptor pair. Pancreatic cancer cell viability in the presence of 5-fluorouracil was better maintained with increased CLCF1-CNTFR signaling. In this study, we integrated ultra high-plex single-cell spatial transcriptomics, optimal transport ligand-receptor predictions, and genetically-engineered stromal tumoroids to identify and validate CLCF1-CNTFR as an important intercellular mechanism of resistance to chemoradiotherapy in PDAC-pioneering a paradigm for translating single-cell spatial biology to clinical oncology.