Bevacizumab and anlotinib combination therapy acts via HIF-1α suppression to exert synergistic anti-angiogenic and anti-tumor effects in non-small cell lung cancer

Nafeisha Simayi¹Jiaying Li¹Junkai Hu²Ning Jia³Rongxue Wan¹Jinbiao Xie⁴Wenhua Huang^5*

• ¹Southern Medical University, Guangzhou, Guangdong, China
• ²Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong Province, China
• ³Yan 'an University, Yan 'an, China
• ⁴Affiliated Hospital of Putian University, Putian, Fujian Province, China
• ⁵School of Basic Medical Science, Southern Medical University, Guangzhou, China

Introduction: Tumor angiogenesis is required for the progression of non-small cell lung cancer (NSCLC) and the anti-VEGF antibody, bevacizumab, and multitarget tyrosine kinase inhibitor, anlotinib, are anti-cancer treatment options the combined effect of which in NSCLC remains unclear. Methods: A vascularized microfluidic chip was applied to model angiogenesis, together with in vitro assays, molecular analyses, and an in vivo xenograft mouse model to evaluate therapeutic effects. Immunological changes were examined by assessing T cell infiltration and cytokine levels, and the role of HIF-1α was validated using an inhibitor and activator. Results: Bevacizumab plus anlotinib (B+A) inhibited angiogenesis, reducing vessel density to 10% of control values, also reducing diameter and GFP area ratio. B+A inhibited cell viability by 78 %, colony formation by 90 % and invasion by 75% in the NSCLC cell-lines, A549 and H1299, downregulating N-cadherin 5.34-fold, vimentin 6.46-fold and α-SMA 4.35-fold, and upregulating E-cadherin 3.75-fold. Rates of apoptosis of A549 and H1299 cells were increased 3.85-fold. Phosphorylation of VEGFR2, PDGFRβ and FGFR1 were also reduced. B+A reduced tumor volume 7.23-fold and weight 7.08-fold, decreased tumor cell density and lowered Ki-67 expression an in vivo mouse xenograft model. HIF-1α was inhibited in a time-and dose-dependent manner. Importantly, combination therapy enhanced CD4⁺and CD8⁺T cell infiltration, increased the production of pro-inflammatory cytokines such as IL-2, and reduced the expression of immunosuppressive factors such as IL-6, indicating an immunomodulatory effect that improved anti-tumor immunity. Conclusion: The HIF1α inhibitor, PX478, did not enhance anti-tumor effects of B+A but the HIF1α activator, DMOG, reversed them. In addition, combination therapy enhanced CD4⁺ and CD8⁺T cell infiltration, increased pro-inflammatory cytokines. These findings highlight the therapeutic potential of combining anlotinib and bevacizumab for NSCLC treatment and identify HIF-1α as a key target.