Yanyang Liu, Li Tu, Li Wang, Jianlin Long, Jiantao Wang, Yuyi Wang, Feng Luo, Dan Cao

Although anti-angiogenic therapy is widely applied clinically, its efficacy has been less than expected. Screening for regulatory factors and sensitive indicators to define the effectiveness of these drugs is required. Through a retrospective study of clinical data, we found that patients with a higher peripheral monocyte-to-lymphocyte ratio (MLR) obtained less benefit from recombinant human endostatin (rhES, Endostar(®)), an anti-angiogenic drug, in lung cancer. Because MLR is positively correlated with macrophage count in tumors, this result suggests that macrophages may influence the effectiveness of rhES therapy in lung cancer. Clinical data from 72 lung cancer patients treated with rhES were collected. Animal study, flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay, Western blot analysis, and transwell migration assays were carried on Lewis lung carcinoma (LLC) cells, bone marrow-derived macrophages, macrophage cell line RAW264.7, and ANA-1 cells. Clinical data showed that compared with the baseline MLR before rhES treatment, patients with progressive disease had higher MLRs than those of patients with partial response. Experimental results showed that more macrophages were recruited in the LLC tumors after rhES treatment and the majority of them displayed an M2-like phenotype. rhES aggravated hypoxia and the inflammatory response in the tumor microenvironment. Hypoxia promoted the expression of CCL2 by endothelial and fibroblast cells, which could induce macrophages recruitment, and increased levels of inflammatory cytokines (interleukin-4 [IL-4], IL-6, and IL-10) skewed macrophage polarization toward the M2-like phenotype. Hypoxia or inflammation cytokine-treated macrophages enhanced the progression of LLC in vitro and in vivo. We found rhES could aggravate hypoxia and the inflammatory response in the tumor microenvironment. These changes were favorable for macrophage accumulation, and skewed their polarization toward the M2-like phenotype which could help LLC to escape from the anti-angiogenic therapy. Thus, these data indicate the accumulation of macrophages in the tumor microenvironment may adversely affect the efficacy of rhES on lung cancer.