Lipopolysaccharide Induces Resistance to CAR-T Cell Therapy of Colorectal Cancer Cells through TGF-β-Mediated Stemness Enhancement.

Chimeric antigen receptor-T (CAR-T) cell therapy is a cellular immunotherapy that has emerged in recent years, and increasing studies showed that therapeutic resistance to CAR-T cell therapy presents in colorectal cancer patients. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is known to preserve a high concentration in the colon. Whether LPS is a contributing factor to the development of resistance in colorectal cancer cells against CAR-T cell therapy remains unclear. For in vivo experiments, colorectal cancer cells COLO205 were pretreated with LPS for 24 h and then were injected into nude mice through the tail vein, followed by CAR-T cells transplantation one day later. Later, the number of tumors in the lung tissues of the mice was observed. The in vitro experiments were performed on COLO205 cells, which were treated with LPS for 24 h. The effect of LPS on the stemness of COLO205 and SW620 cells was observed by using the colony formation assay and spheroidization experiments. The effect of LPS on the expression of stemness-related genes, including CD44, SOX2, and NANOG, was observed by qRT-PCR assay, Western blotting assay, and immunofluorescence staining. Inhibitors of TGF-β and the MYD88 inhibitor were used to study the mechanisms by which LPS induces the stemness enhancement and resistance to CAR-T cell therapy of COLO205 cells. The correlation between MYD88 and TGFB1, as well as the correlation between TGFB1 and stemness-related genes was analyzed using the TCGA database. Both the in vivo assay of nude mice and the in vitro assay showed that LPS pretreatment could induce resistance to CAR-T cell therapy of colorectal cancer cells. LPS could enhance COLO205 and SW620 cells stemness presented by upregulation of CD44, SOX2, and NANOG. The reverse interfering assay using the TGF-β inhibitor indicated that the autosecretion of TGF-β induced by LPS played a critical role in the stemness enhancement of colorectal cancer cells. The TCGA database analysis revealed a strong positive correlation between MYD88 and TGFB1. Additionally, TGFB1 has been found to upregulate the expression of genes associated with stemness. Further mechanism studies showed that the TLR4/MYD88 pathway medicates LPS-induced TGF-β expression. Our results suggested that LPS-induced resistance to CAR-T cell therapy of colorectal cancer cells through stemness enhancement. TLR4/MYD88 signal pathway-dependent TGF-β expression was involved in stemness enhancement and CAR-T cell therapy resistance. In conclusion, our findings help us to understand the underlying mechanisms of CAR-T cell therapy resistance and indicate that inhibitors of TGF-β and MYD88 are promising targeting candidates to promote a therapeutic effect of CAR-T cell therapy in colorectal cancer in the clinic.