Aberrant NOTUM+ Program Induced in LGR5+ Crypt Base Columnar Cells Maintains an Immunosuppressive Niche in Colorectal Cancer

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, with treatment failure largely driven by cancer stem-like cells that resist conventional chemoradiation and subsequently initiate tumor recurrence. While immune checkpoint blockade is effective in microsatellite instability-high (MSI-H) CRCs, the majority of CRCs are microsatellite stable (MSS) and exhibit immune exclusion, rendering them refractory to immunotherapy. Here, we identify a previously uncharacterized cancer cell subtype, which we term cancerous Crypt Base Columnar (canCBC) cells. These cells transcriptionally resemble normal LGR5+ CBC cells but activate an aberrant WNT/ß-catenin signalling inhibitory program, marked by NOTUM expression. We show that canCBC cells are specifically enriched in MSS tumors, where their presence correlates with reduced CD8? T cell infiltration, broader immune exclusion, and a propensity for regional lymphatic dissemination. Consistently, targeted ablation of canCBCs enhances the tumor-clearing potential of CD8? T cells. This study identifies a novel therapeutic target for overcoming immune exclusion and improving immunotherapy responses in MSS CRCs. Overall design: Local colorectal cancer biopsies were extracted and converted into patient derived colorectal cancer speroids organoids. Organoids were then singled and scRNA sequencing was conducted.