Introduction

Chronic inflammation in patients with inflammatory bowel disease (IBD) increases the risk of colorectal cancer (CRC). Both sporadic CRC (SCRC) and colitis-associated CRC (CAC) bear similar mutational patterns, including APC, KRAS, and TP53 mutations. However, the dysplasia to carcinoma sequence in CAC is different in that adenomatous transformation often starts with TP53 and not with APC mutations (characteristic of SCRC). This illustrates that to understand epithelial- and immune cell changes involved in CAC etiology, it is critical to study pre-cancerous tissues instead of end stage disease.

Aims & Methods

We aimed to provide a detailed characterization of the epithelial and immune cell transcriptome in cancers, dysplastic lesions and adjacent normal tissue of both CAC and SCRC patients, using GeoMx Digital Spatial Profiling, to identify chronic inflammation-induced transcriptional changes that predispose for dysplasia and cancer development in IBD. We collected FFPE tissue of 13 IBD patients with either dysplasia (colitis-associated dysplasia (CAD)), CAC, or both. 6 patients without IBD with sporadic dysplasia (SD) or SCRC were used as a control. An expert IBD pathologist scored the tissues to define 3 regions: histologically non-inflamed non-dysplastic mucosa (adjacent normal mucosa: ANM), dysplasia (SD/CAD) or cancer SCRC/CAC). GeoMx Digital Spatial Profiling was used for spatial transcriptomic analysis of both the epithelial and immune cell compartment. Immunohistochemistry was performed to study immune cell composition. Data analyses were performed using R. Multivariate linear modelling was used for gene and pathway differential expression analyses (significance p<0.01).

Results

Differential gene expression analysis in the epithelial compartment showed that the region with most differentially expressed genes (n=408) was the ANM. Interestingly, transcriptional pathways related to T cell immune responses were downregulated (“Cytotoxicity” and “Lymphocyte Trafficking”) in ANM of CAC patients, as compared to SCRC patients. Furthermore, we observed decreased intraepithelial T lymphocytes (IELs) abundance in CAC by deconvolution of transcriptional data, which was confirmed by immunohistochemistry (83.71 vs 50.79 IELs cell density/mm²,p<0.05). Reduction in IELs in ANM of CAC patients was accompanied by decreased IL15 expression in epithelial cells, a cytokine critical for IEL recruitment. In the immune compartment, most transcriptional differences were observed in dysplastic tissue (415 differentially expressed genes), with the “T cells” pathway being the most differentially expressed in CAD regions versus SD. Genes encoding for T cell activation were upregulated in CAD, including CD69, ICOS and GZMB, while at the same time, increased gene expression of T-cell checkpoint molecules such as LAG3, CTLA4 and TIGIT was observed.

Conclusion

In conclusion, we demonstrated altered cytokine gene expression patterns in ANM of CAC patients versus SCRC patients, including decreased IL15 expression, a cytokine critical for IEL attraction. This was accompanied by decreased IELs numbers, potentially compromising immunosurveillance and clearance of pre-malignant cells. In contrast, dysplastic tissues in IBD patients were more infiltrated with immune cells, amongst which T cells, as compared to SD patients. Increased expression of T cell checkpoints in the CAD compartment however suggests an immunosuppressed state in CAD. Together, this study may add a novel concept to how chronic inflammation-induced cell adaptations may favour dysplasia development and progression to cancer.