SU104 - Spatial Transcriptomic Profiling of Early-Onset Colorectal Cancer

Purpose/Background: Sporadic early-onset colorectal cancer (EO CRC) is increasing at an alarming rate. Our understanding of the genetic or molecular pathogenesis of this disease is lacking as few mutational drivers or unique molecular features have been identified. Recent studies suggest that chronic inflammation is associated with EO CRC. Here, we use state-of-the-art, high resolution, spatial genomic technology to investigate the molecular profile of colorectal cancer in a tissue-dependent context. Our hypothesis is that the spatially defined immune and stromal transcriptional landscapes in EO CRC tissue will demonstrate increased fibrotic and immunosuppressive profiles compared to average-onset colorectal (AO CRC).

Methods/Interventions: Spatial transcriptomic profiling using GeoMx Human Whole Transcriptomic Atlas (Nanostring) was conducted on tumor and matched normal colonic tissue from four AO and four EO CRC patients. Fluorescently labeled antibodies for PanCK, CD45, and Vimentin were used to identify regions of interest containing epithelial, immune, and stromal cells, respectively. Biological differences were analyzed as a function of age at diagnosis of disease (EO CRC < 50 years; AO CRC ≥ 50 years), disease status, and cellular compartment comparing differential gene expression, cellular representation, and pathway enrichment. Differentially expressed gene (DEG) criteria was defined using p value < 0.05 and log fold change >1.5.

Results/Outcomes: A total of one-hundred and eleven regions of interest were transcriptionally profiled, focusing on epithelia (panCK), immune (CD45), and stroma (Vimentin) cellular compartments. In the epithelial compartment, differential expression analysis showed upregulation and downregulation of 23 and 17 genes, respectively, when comparing EO to AO CRC. TGFBI, MUC2, and MUC12 were among the top upregulated DEGs. In the immune compartment, 550 DEGs were overexpressed in EO CRC when compared to AO CRC, including genes related to TGF-beta, Wnt, NFKB, and RAS-RAF-MEK signaling pathways. Cellular representation analysis showed that EO CRC had lower abundance of CD4 and CD8 memory T cells, and higher abundance of macrophages and endothelial cells, compared to AO CRC. In the non-immune stromal compartment, EO CRC was most transcriptionally different from the matched-normal counterpart, with 339 and 17 upregulated and downregulated DEGs, respectively. Increased levels of CXCL8, a chemokine associated with chronic inflammation and colonic oncogenesis were noted in EO CRC compared to AO CRC (p < 0.0001). Gene set enrichment analysis revealed that elevated CXCL8 expression was also linked to increased expression of WNT (NES:1.47, p=2.14e-5, FDR=0.008), TGFB receptor (NES:1.82, p=1.56e-4, FDR=0.015), and MAP kinase (NES:1.28, p=2.64e-4, FDR=0.042) signaling in EO CRC compared to AO CRC. Spatial analyses further demonstrated enrichment of fibrosis-associated genes in the EO CRC stroma.

Conclusion/Discussion: Spatial profiling revealed that EO CRC transcriptional landscape is different from AO CRC and matched-normal tissue, especially in the immune and the stromal compartments. Pathways identified indicate decreased T cell infiltration of EO CRC, accompanied by a profibrotic transcriptome. Validation and mitigation of these microenvironmental protumorigenic phenotypes is the focus of our future research.