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

J Transl Med. 2025 Apr 25;23(1):478. doi: 10.1186/s12967-025-06485-4.

Abstract

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.

Keywords: Anlotinib; NSCLC; Neoadjuvant immunotherapy; PD-1 blockade; PLA2G4A; Single-cell; Tumor microenvironment; VEGFR.

MeSH terms

  • Carcinoma, Non-Small-Cell Lung* / drug therapy
  • Carcinoma, Non-Small-Cell Lung* / genetics
  • Carcinoma, Non-Small-Cell Lung* / pathology
  • Female
  • Gene Expression Regulation, Neoplastic / drug effects
  • Humans
  • Immune Checkpoint Inhibitors* / pharmacology
  • Immune Checkpoint Inhibitors* / therapeutic use
  • Indoles* / pharmacology
  • Indoles* / therapeutic use
  • Lung Neoplasms* / drug therapy
  • Lung Neoplasms* / genetics
  • Lung Neoplasms* / pathology
  • Male
  • Middle Aged
  • Neoadjuvant Therapy*
  • Programmed Cell Death 1 Receptor* / antagonists & inhibitors
  • Programmed Cell Death 1 Receptor* / metabolism
  • Quinolines* / pharmacology
  • Quinolines* / therapeutic use
  • RNA-Seq*
  • Signal Transduction* / drug effects
  • Signal Transduction* / genetics
  • Single-Cell Analysis*
  • Single-Cell Gene Expression Analysis
  • Tumor-Associated Macrophages
  • Vascular Endothelial Growth Factor A* / metabolism

Substances

  • Indoles
  • anlotinib
  • Quinolines
  • Vascular Endothelial Growth Factor A
  • Programmed Cell Death 1 Receptor
  • Immune Checkpoint Inhibitors