Anlotinib Combined with PD-1 Inhibitor Suppresses MSS Colorectal Cancer via Vascular Normalization and Immune Reprogramming
核心洞察
Anlotinib combined with a PD-1 (搜索)/PD-L1 inhibitor significantly suppressed tumor growth and prolonged survival in a murine microsatellite-stable (MSS) colorectal cancer (搜索) model.
The combination therapy reduced immunosuppressive cells including MDSCs, M2-type macrophages, and Tregs while increasing CD8+ T cell infiltration within the tumor microenvironment.
Combined treatment alleviated tumor hypoxia, inhibited angiogenesis, and normalized tumor vasculature by downregulating VEGF (搜索), IL-17A, and HIF-1α expression.
Anlotinib in combination with a PD-1 (搜索)/PD-L1 inhibitor demonstrates potent antitumor activity in microsatellite-stable (MSS) colorectal cancer (搜索), according to a preclinical study published in ImmunoTargets and Therapy. The research provides experimental evidence that dual targeting of angiogenesis and immune checkpoint pathways can remodel the immunosuppressive tumor microenvironment (TME) and overcome immunotherapy resistance in MSS colorectal tumors.
Investigators at Zhongyan Zichuang (Beijing) Biotechnology Co., Ltd. employed a CT26 syngeneic subcutaneous tumor model in BALB/c mice to evaluate the combination of anlotinib—a multi-target tyrosine kinase inhibitor targeting VEGFR, FGFR, PDGFR, and c-kit—with PD-1/PD-L1-IN-9 hydrochloride (搜索), a small-molecule inhibitor that selectively blocks the PD-1 (搜索)/PD-L1 interaction.
Tumor Growth Suppression and Survival Benefit
The combination therapy significantly reduced tumor size and volume compared to vehicle control and either monotherapy arm. Tumor growth curves measured every three days during the 10-day treatment period showed marked inhibition in the combination group. More notably, survival analysis extending to day 100 demonstrated significantly prolonged survival in mice receiving the dual regimen. At the molecular level, Western blot analysis revealed downregulated Bcl-2 expression and increased cleaved caspase-3 and Bax, while TUNEL staining confirmed elevated apoptosis in tumors from the combination group.
Vascular Normalization and Hypoxia Alleviation
Immunohistochemical analysis showed that combined treatment significantly decreased expression of IL-17A and VEGF (搜索), two key drivers of tumor angiogenesis. CD31 and CD34 staining, used to quantify microvessel density (MVD), indicated that the combination therapy reduced aberrant vascularization. Western blot further confirmed that HIF-1α and VEGF levels were suppressed, suggesting that anlotinib plus PD-1 (搜索) blockade alleviates tissue hypoxia and induces normalization of the remaining tumor vasculature.
Reprogramming of the Immunosuppressive Microenvironment
Flow cytometric analysis of tumor-infiltrating immune cells revealed that combination therapy significantly reduced the proportions of myeloid-derived suppressor cells (MDSCs), M2-type tumor-associated macrophages (TAMs), and regulatory T cells (Tregs). Immunofluorescence staining corroborated the reduction in MDSC fluorescence intensity. Concurrently, the combination enhanced infiltration of CD8+ T cells at the tumor site and reduced PD-1 (搜索) expression on both CD4+ and CD8+ T cells, suggesting partial reversal of T-cell exhaustion.
Cytokine Remodeling
ELISA analysis of both peripheral blood and tumor tissue homogenates demonstrated consistent immune reprogramming. In the combination group, immunostimulatory factors IFN-γ and TNF-α were significantly upregulated, while immunosuppressive mediators—including IL-10, ARG1, IL-17, VEGF (搜索), and iNOS—were downregulated. These findings indicate that the combination therapy improves the immune microenvironment at both systemic and intratumoral levels.
The authors propose that anlotinib transiently normalizes abnormal tumor vasculature, alleviating hypoxia and improving perfusion, which facilitates CD8+ T cell infiltration and function. In turn, activated CD8+ T cells may contribute to sustaining neovascularization through cytokine regulation, creating a positive feedback loop that explains the observed synergistic effect.
Limitations and Future Directions
The researchers acknowledge several limitations, including the relatively small sample size, the use of a subcutaneous rather than orthotopic colorectal cancer (搜索) model, and the analysis of only selected immune cell subsets. Additionally, the precise molecular pathways underlying the observed immune microenvironment changes remain to be elucidated. Despite these constraints, the study provides robust preclinical evidence supporting further clinical evaluation of anlotinib combined with PD-1 (搜索) inhibition as a therapeutic strategy for MSS colorectal cancer patients who currently derive minimal benefit from immune checkpoint blockade alone.