Elevated soluble TNFα levels and upregulated TNFα mRNA expression in purified peripheral blood monocyte subsets associated with high-grade hepatocellular carcinoma.

BACKGROUND: Chronic inflammation is involved in the initiation and progression of various cancers, including liver cancer. The current study focuses on the characterization of the peripheral immune response in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) patients, before and after surgical procedure, in order to assess the effect of tumor resection in the immune system homeostasis and to determine possible prognostic factors associated with high-grade tumors. We developed a whole-blood assay to monitor immune alterations and functional competence of peripheral monocytes in a group of 10 healthy individuals (HG), in 20 HCC patients and 8 CCA patients, by multi-color flow cytometry, qRT-PCR, and ELISA techniques. RESULTS: The qRT-PCR analysis showed an upregulation of TNFα expression by classical and intermediate monocytes purified from HCC patients presenting tumors in grade G3-G4 as compared to G1-G2 HCC patients. Moreover, ELISA assay confirmed elevated serum levels of TNFα in G3-G4 compared to G1-G2 HCC patients. A significant decrease of circulating non-classical monocytes was detected in both CCA and HCC patients before and after surgical procedure. In addition, a functional defect in circulating classical and intermediate monocytes was observed in both groups of cancer patients when compared to the HG, with partial recovery after the surgical intervention. CONCLUSIONS: This integrated analysis permitted the identification of altered functional competence of monocyte subsets in CCA and HCC patients. In addition, our results point to a potential role of TNFα as a prognostic peripheral biomarker in HCC patients, indicating the presence of high-grade tumors that should be further validated.

Functional and Phenotypic Characterization of Tumor-Infiltrating Leukocyte Subsets and Their Contribution to the Pathogenesis of Hepatocellular Carcinoma and Cholangiocarcinoma.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represent the most common primary liver malignancies whose outcome is influenced by the immune response. In the present study, we evaluated the tumor-infiltrating leukocyte (TIL) populations in 21 HCC patients and 8 CCA patients by flow cytometry immediately after the surgical procedure. Moreover, CD4+ T cells, CD8+ T cells, monocytes, and macrophages were purified by cell sorting for further analysis of gene expression by quantitative reverse-transcription polymerase chain reaction. Regarding tumor-infiltrating macrophages, we observed a significantly higher expression of markers associated with M2 phenotype and a higher expression of PD-L1 in patients with HCC in comparison to CCA. In addition, for HCC patients, we found a significant increase in the expression of CD200R in macrophages from tumors that were in grade G3-G4 as compared to tumors in grade G1-G2. Besides, a significantly higher frequency of tumor-infiltrating lymphocytes, CD8+CD56+ T cells, and natural killer cells was detected in HCC biopsies in comparison to CCA. In summary, this study has revealed functional and phenotypic differences in TIL cell subpopulations between CCA and HCC, as well as among different histopathological grades and tumor aggressiveness degrees, and it has provided evidence to better understand the tumor immune microenvironment of CCA and HCC.