Tumor-associated macrophages (TAMs), the most abundant infiltrating immune cells in tumor microenvironment, have distinct functions in hepatocellular carcinoma (HCC) progression. CD68 + TAMs represent multiple polarized immune cells mainly containing CD86 + antitumoral M1 macrophages and CD206 + protumoral M2 macrophages. TAMs expression and density were assessed by immunohistochemical staining of CD68, CD86, and CD206 in tissue microarrays from 253 HCC patients. Clinicopathologic features and prognostic value of these markers were evaluated. We found that CD68 + TAMs were not associated with clinicopathologic characteristics and prognosis in HCC. Low presence of CD86 + TAMs and high presence of CD206 + TAMs were markedly correlated with aggressive tumor phenotypes, such as multiple tumor number and advanced tumor-node-metastasis (TNM) stage; and were associated with poor overall survival (OS) (p = 0.027 and p = 0.024, respectively) and increased time to recurrence (TTR) (p = 0.037 and p = 0.031, respectively). In addition, combined analysis of CD86 and CD206 provided a better indicator for OS (p = 0.011) and TTR (p = 0.024) in HCC than individual analysis of CD86 and CD206. Moreover, CD86 + /CD206 + TAMs predictive model also had significant prognosis value in α-fetoprotein (AFP)-negative patients (OS: p = 0.002, TTR: p = 0.005). Thus, these results suggest that combined analysis of immune biomarkers CD86 and CD206 could be a promising HCC prognostic biomarker.

Chemokines play a key role in orchestrating the recruitment and positioning of myeloid cells within tumor microenvironment. However, the tropism regulation and functions of these cells in HCC are not completely understood. Herein, by scrutinizing the expression of all chemokines in HCC cell lines and tissues, we found that CCL15 was the most abundantly expressed chemokine in human HCC. Further analyses showed that CCL15 expression was regulated by genetic, epigenetic and microenvironmental factors, and negatively correlated with patient clinical outcome. In addition to promoting tumor invasion in an autocrine manner, CCL15 specifically recruited CCR1 cells toward HCC invasive margin, approximately 80% of which were CD14 monocytes. Clinically, high-density of marginal CCR1 CD14 monocytes positively correlated with CCL15 expression and was an independent index for dismal survival. Functionally, these tumor-educated monocytes directly accelerated tumor invasion and metastasis through bursting various pro-tumor factors and activating STAT1/3, ERK1/2 and AKT signaling in HCC cells. Meanwhile, tumor-derived CCR1 CD14 monocytes expressed significantly higher levels of PD-L1, B7-H3, and TIM-3 that may lead to immune suppression. Transcriptome sequencing confirmed that tumor-infiltrating CCR1 CD14 monocytes were reprogrammed to upregulate immune checkpoints, immune tolerogenic metabolic enzymes (IDO and ARG), inflammatory/pro-angiogenic cytokines, matrix remodeling proteases, and inflammatory chemokines. Orthotopic animal models confirmed that CCL15-CCR1 axis forested an inflammatory microenvironment enriched with CCR1 monocytes and led to increased metastatic potential of HCC cells CONCLUSION: A complex tumor-promoting inflammatory microenvironment was shaped by CCL15-CCR1 axis in human HCC. Blockade of CCL15-CCR1 axis in HCC could be an effective anti-cancer therapy. This article is protected by copyright. All rights reserved.