OP24 SPP1 in colitis-associated colon cancer

Abstract

Abstract Background Patients with ulcerative colitis (UC) and Crohn’s disease (CD) face a lifelong risk of developing colitis-associated carcinoma (CAC). Current insights into CAC development originate from murine CAC models, while human CAC studies primarily focus on mutational analyses. Although the mutational landscape reveals distinct patterns and frequencies compared to colorectal cancer, it falls short in elucidating the inflammatory mechanisms of CAC development. Consequently, we adopted a multi-omics approach to unravel CAC carcinogenesis, utilizing human samples from patients with CD, CD-associated CAC (CD-CAC), UC, and UC-associated CAC (UC-CAC). Findings were further validated in vitro using human colon organoids. Methods A Nanostring Immunology v2 panel was employed on RNA (archival FFPE sections) from 40 patients suffering from either IBD or CAC, alongside inflammation-free healthy controls (n=10 per group). Our data revealed a robust upregulation of the SPP1 (Secreted Phosphoprotein 1) gene encoding osteopontin (OPN) in CAC patients. Prospectively, we extended our methodology to an unbiased imaging-based spatial RNA in situ sequencing (ISS) approach (477 genes, Xenium, 10X Genomics) using FFPE sections from CAC and UC patients (n=3 per group). This provided insights into the spatial expression of SPP1 and its target receptors at a subcellular resolution. Findings were validated using whole FFPE section confocal microscopy. In addition, in vitro models, including human UC organoids, were analyzed by bulk RNA-Seq, RT-qPCR, and downstream functional assays. Results Nanostring Immunology v2 gene expression data revealed upregulation of the SPP1 gene encoding OPN (Fig 1A) and EMT transcription factors FN1 and ZEB1 in CAC patients. Xenium RNA in situ sequencing (ISS) unveiled the spatial heterogeneity of SPP1 expression in CAC (Fig 1B). Unsupervised graph-based clustering identified an SPP1+CD68+ macrophage cluster exclusively in CAC (Fig 1C), validated through OPN/CD68 immunostainings (Fig 1D). In vitro OPN stimulation across multiple human and mice colon organoid models excluded OPN as the trigger of EMT. Interestingly, Xenium ISS and immunostainings revealed a mutually exclusive spatial location of SPP1/OPN+ macrophages and CD8+ T cells (Fig 1E, F and G). Conclusion SPP1 is significantly upregulated in CAC (RNA and protein), expressed by CD68+ macrophages. In vitro OPN stimulation demonstrated no direct effect on intestinal epithelial organoids. The mutually exclusive spatial locations of SPP1/OPN+ macrophages and CD8+ T cells suggests a crucial role of SPP1/OPN in modulating the immunosurveillance of CAC cells by CD8+ tumour lymphocytes.