Objective To explore the possible mechanism of arotinib combined with anti-programmed death receptor-1 (anti-PD-1) in inhibiting lung adenocarcinoma by regulating Wnt/β-catenin pathway through inhibiting vascular endothelial growth factor/c-Kit (VEGF/c-Kit).

Methods A lung adenocarcinoma model was established in C57 mice, and the mice were divided into negative control group, arotinib group, anti-PD-1 monoclonal antibody group and arotinib combined with anti-PD-1 monoclonal antibody group, with 8 mice in each group. After continuous administration for 25 days, the change in tumor volume in each group was compared. Gene Set Enrichment Analysis (GSEA) was used to explore the pathways related to the enrichment of c-Kit and VEGF in the lung adenocarcinoma (LUAD) cohort of the Cancer Genome Atlas (TCGA) database; the CIBERSORTx was used to analyze the influence of c-Kit and VEGFR on the infiltration of 22 types of immune cells; the correlations of c-Kit and VEGFR with immune-related molecules were analyzed. Cell transfection was used to knock down c-Kit expression and the cells were divided into groups, the control group was normal LLC cells, the si-c-Kit group was siRNA knocked down c-Kit, and the arotinib treatment group was LLC cells processed with arotinib. Western Blot assay was used to detect expressions of c-Kit, Wnt1, β-catenin, gamma interferon (IFN-γ), tumor necrosis factor-alpha (TNF-α), PD-L1, c-Myc and c-Jun.

Results Compared with the negative control group, tumor growth was significantly inhibited in the arotinib group, PD-1 monoclonal antibody group, and the combination of arotinib and PD-1 monoclonal antibody group (P < 0.05). Compared with the arotinib group and PD-1 monoclonal antibody group, the tumor growth was significantly inhibited in the combination of arotinib and PD-1 monoclonal antibody group (P < 0.05). GSEA analysis showed that the IFN-γ pathway was enriched in c-Kit low expression samples, Wnt/β-catenin pathway was enriched in c-Kit high expression samples, while the angiogenesis and IL-6/JAK/STAT3 pathway were enriched in VEGFR high expression samples (P < 0.05). CIBERSORTx analysis found that there were fewer anti-tumor immune cells and more immunosuppressive cells in the high expression samples of c-Kit (P < 0.05). There were more anti-tumor immune cells and fewer immunosuppressive cells in the low expression samples of VEGFR (P < 0.05). The c-Kit was negatively correlated with immune promoting molecules such as granzyme A (GZMA), granzyme B (GZMB) and IFN-γ (r < 0, P < 0.05), and was positively correlated with CD274 (PD-L1), transforming growth factor-β (TGF-β), T-cell immunoglobulin and mucin domain 3 (TIM3) (r>0, P < 0.05). VEGFR was negatively correlated with immune promoting molecules in GZMA, TNF-α, GZMB and IFN-γ (r < 0, P < 0.05), and was positively correlated with immunosuppressive molecules such as CD274 (PD-L1), TIM3, PD-1, hypoxiainduciblefactor-1 alpha (HIF1A), lymphocyte activation gene 3 (LAG3), TGF-β and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) (r>0, P < 0.05). Western blot experiment had confirmed that enrotinib or knocking down c-Kit both inhibited Wnt/β-catenin pathway and downstream molecules, and regulated TNF-α expression.

Conclusion The combination of arotinib and anti PD-1 drugs can significantly inhibit tumor growth, and the possible mechanism is that arotinib inhibits Wnt/β-catenin pathway in lung adenocarcinoma by targeting c-Kit to regulate immune microenvironment and promote anti-tumor immune cell infiltration, which is helpful to improve the efficacy of anti-PD-1 monoclonal antibody in inhibiting tumor growth.