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Exploration of the mechanism of anlotinib in reversing PD-1 immunotherapy resistance: insights from single-cell sequencing

Cancer Gene Therapy volume 33, pages 248–260 (2026)Cite this article

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Abstract

Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis have revolutionized cancer therapy, yet primary and acquired resistance remain major clinical obstacles. Dysregulated angiogenesis fuels the development of an immunosuppressive tumor microenvironment, while crosstalk between immunity and angiogenesis further propels tumor immune evasion and treatment resistance. The present study aimed to establish a penpulimab-resistant model, delineate anti-PD-1 resistance traits via single-cell RNA sequencing, and unravel the precise mechanisms through which anlotinib—an anti-angiogenic agent—mitigates penpulimab resistance. These findings offer insights to guide clinical management of immune-pretreated patients. Single-cell sequencing analyses demonstrated that anlotinib reverses penpulimab resistance by reprogramming the tumor immune microenvironment, thereby boosting PD-1 blockade efficacy via modulation of immune infiltration and tumor signaling pathways. Identifying Apoe⁺ M2 macrophages, Srgn⁺ M1 macrophages, and Cxcl2⁺ T cells provides key cellular and molecular targets for developing clinically actionable immunotherapies. Taken together, this work validates the preclinical potential of anlotinib combined with immunotherapy for immunotherapy-resistant tumors.

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Fig. 1: Anti-PD-1–resistant LLC models were obtained.
Fig. 2: Cellular alternations during the acquisition of PD-1 resistance.
Fig. 3: Anlotinib reverses tumor progression and restores T cell infiltration in anti-PD-1-resistant lung cancer models.
Fig. 4: Anlotinib reverses PD-1 treatment resistance and facilitate the immunotherapy effect in PD-1 resistant tumors.
Fig. 5: Anlotinib reprograms the TME by modulating macrophages, enhancing T cell activation, and restoring immune cell communication in PD-1 resistant tumors.

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All data are presented within the article and supplementary online data.

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Acknowledgements

We thank Chia Tai Tianqing Pharmaceutical Group Co., Ltd. for providing anlotinib and penpulimab.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 82574740, 82473955, 82173842), the Fundamental Research Funds for the Central Universities (2632025TD04), and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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Author notes

  1. These authors contributed equally: Wanjin Shi, Yidong Zhang.

Authors and Affiliations

  1. School of Life Science and Technology, Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu Province, China

    Wanjin Shi, Yidong Zhang, Qiyi Yu, Huilong Li, Miaomiao Niu, Shengtao Xu, Jun Yin & Lufeng Zheng

  2. Jiangsu Key Laboratory for Pharmacology and Safety Research of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu Province, China

    Wanjin Shi & Xiaoman Li

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Contributions

Lufeng Zheng and Xiaoman Li designed the research. Wanjin Shi and Yidong Zhang analyzed the data. Wanjin Shi, Yidong Zhang, Qiyi Yu, Huilong Li, Miaomiao Niu, Shengtao Xu and Yin Jun performed the research. Wanjin Shi and Yidong Zhang wrote the paper. Lufeng Zheng, and Xiaoman Li reviewed this paper. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

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Correspondence to Xiaoman Li or Lufeng Zheng.

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Animal studies were conducted in strict compliance with guidelines and protocols submitted to and approved by the Animal Welfare and Ethics Committee (AWEC) of China Pharmaceutical University (approval number: 2024-08-003), in adherence to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

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Shi, W., Zhang, Y., Yu, Q. et al. Exploration of the mechanism of anlotinib in reversing PD-1 immunotherapy resistance: insights from single-cell sequencing. Cancer Gene Ther 33, 248–260 (2026). https://doi.org/10.1038/s41417-026-01000-3

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