Exploration of the Mechanism of Anlotinib in Reversing PD-1 Immunotherapy Resistance: Insights from Single-cell Sequencing

Abstract Immune checkpoint inhibitors (ICI) targeting the PD-1/PD-L1 axis have revolutionized cancer therapy, yet primary and acquired resistance remain significant clinical challenges. Dysregulated angiogenesis fosters the formation of an immunosuppressive tumor microenvironment, and the crosstalk between immunity and angiogenesis further drives tumor immune evasion and treatment resistance. This study aims to establish a penpulimab resistance model, analyze the anti-PD-1 resistance characteristics via single-cell RNA sequencing, and explore the specific mechanisms by which anlotinib, an anti-angiogenic agent, ameliorates penpulimab resistance. The results provide evidence for clinical practice in managing immune-pretreated patients. Sequencing analyses revealed that anlotinib reversed penpulimab resistance by remodeling the tumor immune microenvironment, thereby enhancing the efficacy of PD-1 blockade through regulating immune infiltration and tumor signaling pathways. Identification of Apoe+ M2 macrophages, Srgn+ M1 macrophages, and Cxcl2+ T cells provides critical cellular and molecular targets for developing clinically relevant immunotherapies. This study offers preclinical validation for the potential use of anlotinib in combination with immunotherapy for immunotherapy-resistant tumors.