scRNA-seq reveals that VEGF signaling mediates the response to neoadjuvant anlotinib combined with PD-1 blockade therapy in non-small cell lung cancer.

BACKGROUND: Clinical trials have shown that neoadjuvant anlotinib combined with PD-1 blockade therapy can prolong the survival of patients with driver gene negative non-small cell lung cancer (NSCLC), but some patients fail to benefit from the combination therapy. METHODS: To explore the potential drug resistance mechanism and predict the efficacy of neoadjuvant therapy in NSCLC patients, we used scRNA-seq to observe and analyze the dynamic changes of immune cells, stromal cells and cancer cells in NSCLC patients who received neoadjuvant combination therapy. We analyzed transcriptome data of ~ 47,000 single cells from 9 NSCLC patients, including 3 treatment naïve patients, 3 post-treatment patients with major pathological response (MPR), and 3 Non-MPR patients. Subsequently, the infiltration of immune cells was detected by immunohistochemistry and multiplex immunofluorescence in NSCLC. RESULTS: In MPR patients, we found that neoadjuvant therapy reduced the expression of the T cell exhausted signature, reduced the transition of T_THEMIS cells to Tregs, and enhanced the positive feedback between CD4(+) T cells and PAX5(+) memory B cells. In Non-MPR patients, tumor-associated macrophages (TAMs) dampen therapeutic efficiency by being the hub of cell communication. TAMs and fibroblasts stimulate endothelial cells via VEGF, endothelial ZEB1 may up-regulate FLT1 (VEGFR) expression in response to anlotinib, and VEGFR(+) endothelial cell signature can predict survival of NSCLC cohort in TCGA. In addition, PLA2G4A, the key enzyme in the VEGF pathway, was highly expressed in the tumor cells of Non-MPR patients after anlotinib treatment. In 135 NSCLC patients, we confirmed by immunohistochemistry that PLA2G4A was positively correlated with poor prognosis and Tregs infiltration. CONCLUSION: In conclusion, VEGF signaling dependent dynamic changes in endothelial and epithelial cells are deeply involved in the formation of anlotinib resistance and immunosuppression phenotypes in NSCLC patients.