Elevated CD163(+)/CD68(+) Ratio at Tumor Invasive Front is Closely Associated with Aggressive Phenotype and Poor Prognosis in Colorectal Cancer

Background: The interaction and crosstalk between tumor-associated macrophages (TAMs) and epithelial-mesenchymal transition (EMT) has been demonstrated to play a critical role in the progression and metastasis of multiple cancers. However, the roles of the M2-polarized TAMs in different tumor location in EMT and prognosis of colorectal cancer (CRC) have not been elucidated. Therefore, the present study was designed to set up a reliable ratio of CD163(+)/CD68(+) to assess M2-polarized TAMs infiltration in the tumor center (TC) and tumor invasive front (TF) and to further evaluate their prognostic value and biological effects on tumor cells in CRC. Methods: TAMs markers (CD68 and CD163) and EMT markers (E-cadherin and Vimentin) expression were evaluated by immunohistochemistry in 81 patients with CRC. Circulating tumor cells (CTCs) of peripheral blood from above patients was also isolated. The correlation of CD163(+)/CD68(+) ratio in different locations, EMT and CTCs counts were further analyses. Kaplan-Meier and the model analyses of univariate Cox proportional hazards were utilized to compare the survival of patients with high CD163(+)/CD68(+) ratio with those with low CD163(+)/CD68(+) ratio. Furthermore, the effects of the M2-polarized TAMs on growth, migration and invasion of CRC cells were explored in vivo and in vitro co-culture system. Results: The results showed that the level of CD163(+)/CD68(+) ratio in TF was significant higher than that in TC, and higher CD163(+)/CD68(+)(TF) ratio were closely correlated with enhanced lymphovascular invasion, tumor invasion and TNM stage. Interestingly, higher CD163(+)/CD68(+)(TF) ratio were also significantly associated with EMT program and CTCs counts. Meanwhile, Kaplan-Meier analysis showed that CD163(+)/CD68(+)(TF) was associated with both recurrence-free survival (RFS) and overall survival (OS) of patients with CRC. Multivariate Cox regression analyses demonstrated that CD163(+)/CD68(+)(TF) remained an independent prognostic factor for RFS and OS. Further receiver operating characteristic (ROC) curve analysis found that CD163(+)/CD68(+)(TF) was a better prognosticator compared with CD68(+)(TF) and CD163(+)(TF) for CRC patients. What's more, M2-polarized TAMs secreted TGF-β to facilitate the EMT, growth, proliferation and invasion of CRC cells by in vivo and in vitro experiments. Conclusions: Our studies preliminarily elucidated the prognostic value of CD163(+)/CD68(+) ratio in different tumor locations and the biological functions of M2-polarized TAMs in CRC progression via TGF-β.