Tumor necrosis factor promotes doublecortin-like kinase 1 expression and cellular reprogramming in intestinal epithelial cells leading to neoplastic transformation

Tumor necrosis factor (TNF) plays a crucial role in inflammatory bowel disease and colorectal cancer (CRC). However, its involvement in the regulation of cancer stem cells (CSCs) remains poorly understood. This study aims to elucidate the role of TNF in the origin of colorectal CSCs. Rat intestinal epithelial cells (IEC-6) were treated with TNF, or PBS as control, for 10 treatments and subjected to single-cell RNA sequencing (scRNA-seq). UMAP analysis identified 5 and 3 cell clusters in IEC-6 control (IEC-6Ctrl) cell line and TNF-treated (IEC-6TNF) cell line, respectively. Notably, a substantial number of stem cells emerged in IEC-6TNF cells, while goblet cells and enterocytes diminished, indicating that long-term exposure to TNF induces cellular reprogramming. Furthermore, using Dclk1 as a marker gene, pseudotime trajectory analysis revealed that transient amplifying (TA) cells represented the initial point of the developmental trajectory in both IEC-6Ctrl and IEC-6TNF cell lines. Interestingly, in the IEC-6TNF cell line, the trajectory progressed from TA cells to tuft cells and ultimately to stem cells, indicating that in TNF-treated cells, stem cells arise from Dclk1+ tuft cells. These findings suggest that prolonged TNF treatment depletes differentiated cells, such as goblet cells and enterocytes, while promoting the emergence of stem cells through dedifferentiation and reprogramming. Overall design: Rat intestinal epithelial cells (IEC-6) were treated with TNF, or PBS as control, for 10 treatments and then subjected to single-cell RNA sequencing (scRNA-seq).