R. K. DeHaan1,2, S. Sarvestani2, E. H. Huang1,2 1Cleveland Clinic Foundation,Department Of Colorectal Surgery,Cleveland, OH, USA 2Cleveland Clinic Foundation,Lerner Research Institute,Clevealnd, OH, USA
Introduction:
Colitis-associated cancer (CAC) is a feared consequence of ulcerative colitis. The inflammatory microenvironment, especially the stromal fibroblasts, is associated with increased development of neoplasia. Further, even in the absence of histological neoplasia, transcriptional inflammatory and neoplastic signatures exist. Patient-derived epithelial organoids and fibroblasts, with high-resolution single cell analysis, permit unprecedented dissection of transcriptional expression. Single cell nuclear analysis was used to discriminate cell identity and individual cell transcriptional expression. We hypothesize that co-cultures of CAC epithelial organoids with cancer associated fibroblasts (CAFs) will have a richer immune profile when compared to co-cultures of CAC epithelial organoids and proximal, histologically normal fibroblast.
Methods:
Matched patient derived epithelial organoids were co-cultured with fibroblasts from an area of CAC, or with an area of proximal colon that was histologically normal. Single-nucleus RNA-sequencing (sn-RNA-Seq) was performed (10x Genomics). Data analysis was performed in R utilizing the Seurat package for clustering and differential expression analysis. Epithelial cells were removed from the analysis through gene filtering. Dimensionality reduction was performed using Uniform Manifold Approximation and Projection. Differential expression testing was performed using a Wilcoxon rank-sum test with correction for multiple comparisons.
Results:
Integrated analysis of the two co-culture conditions generated 8 distinct cell clusters, one of which was unique to the proximal fibroblast co-culture condition. Both groups show broad transcription of IL-24, as has been previously shown in patients with ulcerative colitis, along with IL-11, a cytokine with both anti-inflammatory and pro-tumorigenic roles. The cancer fibroblast co-culture transcriptional profile contains a unique inflammatory profile including IL6, IL33, CCL3 and CCL5, which have been shown to play key roles in tumor immune evasion and trafficking of immune cell-types including tumor-associated macrophages (Figure 1).
Conclusion:
We demonstrate that in vitro modeling of colitis-associated cancer epithelia with matched normal colon and CAC stromal fibroblasts allows for the generation of a phenotype specific immune signature. Consistent with the underlying ulcerative colitis phenotype of both co-culture conditions, we see strong transcription of the anti-inflammatory IL-24 and IL-11. Despite the presence of only fibroblast and cancer epithelia, our in vitro model showed a distinct immune phenotype when CAFs were co-cultured with cancer organoids.
