[Cinobufagin inhibits CD244+ macrophages to enhance T-cell activity and chemosensitivity in colorectal cancer].

Objective To investigate the effects of Cinobufotalin (Cino) on the tumor microenvironment (TME) of colorectal cancer (CRC) and the chemosensitivity to Oxaliplatin (OX). Methods We explored the clinical expression differences of CD244 in the TCGA-COAD cohort. Through UMAP dimensionality reduction analysis of single-cell sequencing data, we clarified the cellular localization of CD244 and analyzed its interaction characteristics with immune cells. In vivo experiments, a CRC tumor-bearing mouse model was established and randomly divided into 4 groups: Tu group, Cino group, OX group, and Cino combined with OX group. Intraperitoneal injection was performed every other day for treatment. Experimental techniques such as tumor volume measurement, fluorescence signal/mass changes and tissue morphology changes, real-time quantitative PCR, Western blot, and immunofluorescence staining were used to detect tumor proliferation, metastasis, expression distribution of CD244+ macrophages and T cell activity. In vitro experiments, RAW264.7 cells were stimulated with interleukin 4 (IL-4) to construct a M2-type macrophage model. The effects of Cino, OX or Cino combined with OX on the expression of CD244+ macrophages were observed, and the conditioned medium (CM) of the corresponding treated macrophages was used to culture T cells. The expression of T cell exhaustion and activation markers was detected, and the killing function of T cells was tested. Results In vivo experiments, Cino treatment had no significant effect on tumor growth, proliferation and metastasis in mice. OX treatment and its combination with Cino inhibited tumor proliferation and metastasis, with the Cino-OX combination exhibiting an enhanced inhibitory effect. Both Cino and OX inhibited the expression of CD244+ macrophages in tumor tissues, and their combination showed a synergistic inhibitory effect. Neither Cino nor OX alone had significant effect on T cell activity, their combination could inhibit T cell exhaustion and increase CD8+ T cell infiltration. In vitro experiments, Cino and OX treatments inhibited the expression of CD244+ macrophages, with their combination exhibiting an enhanced inhibitory effect; after IL-4 treatment, the supernatant was collected to culture T cells, and the tumor-killing ability of T cells decreased. The CM treated with Cino or OX could enhance the killing ability of T cells, and the combination showed a stronger killing ability. Conclusion Cino enhances the tumor-killing capacity of T cells and reshapes the tumor microenvironment by specifically targeting and inhibiting CD244+ macrophages, thereby increasing the chemosensitivity of CRC to OX.