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

Abstract

<b>Background:</b> 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. <b>Methods:</b> 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 <i>in vivo</i> and <i>in vitro</i> co-culture system. <b>Results</b>: 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 <i>in vivo</i> and <i>in vitro</i> experiments. <b>Conclusions:</b> 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.