Vascular abnormalization confers tumor resistance to antiangiogenic therapy via downregulating angiostatic factors

BACKGROUND: Angiogenesis is a hallmark of cancer. However, the efficacy of antiangiogenic therapy is hindered by drug resistance. The underlying mechanisms are complex and multifactorial, and the practical strategies to overcome resistance remain limited. METHODS: By establishing single cell clones (SCCs) from EO771 breast tumor cell line, we classified SCCs as resistant and sensitive to Anlotinib, a new antiangiogenic agent. Resistant and sensitive SCCs were then mixed in different ways to uncover novel mechanisms of antiangiogenic resistance by integrating orthotopic tumor models, RNA sequencing (RNA-seq), immunohistochemistry, blocking antibodies, and recombinant proteins. RESULTS: Increasing the diversity and quantity of sensitive SCCs overcame the influences of resistant SCCs and improved Anlotinib efficacy. Sensitive SCC tumors exhibited a more normalized vasculature and increased intratumoral CD8+ T cells compared to resistant SCC tumors. Moreover, elevated vessel perfusion was significantly correlated with better Anlotinib efficacy. RNA-seq data revealed upregulation of dual-function genes (e.g., Ifng, Cxcl9, and Vcam1) linking angiostasis and immune activation. Strikingly, the addition of an angiostatic factor (e.g., recombinant murine IFN-γ or Endostar) converted resistant breast tumors to sensitive to Anlotinib treatment and induced vascular normalization, whereas neutralizing IFNγ, a key angiostatic factor in sensitive SCC tumors, impeded their responses to Anlotinib. CONCLUSIONS: The downregulation of angiostatic factors promotes breast tumor vascular abnormalization and resistance to antiangiogenic therapy. Therefore, integrating angiostatic factors with pro-angiogenic blockers may overcome resistance, offering a translatable approach to improve antiangiogenic outcomes in breast cancer.