Intrahepatic inflammatory IgA+PD-L1high monocytes in hepatocellular carcinoma development and immunotherapy.

BACKGROUND: IgA neutralizes pathogens to prevent infection at mucosal sites. However, emerging evidence shows that IgA contributes to aggravating inflammation or dismantling antitumor immunity in human diseased liver. The aim of this study was to elucidate the roles of inflammation-induced intrahepatic inflammatory IgA+ monocytes in the development of hepatocellular carcinoma (HCC). METHODS: Patient cohorts including steatohepatitis cohort (n=61) and HCC cohort (n=271) were established. Patients' surgical and biopsy specimens were analyzed using immunohistochemistry. Multicolor flow cytometry was performed with a subset of patient samples. Single-cell RNA-Seq analysis was performed using Gene Expression Omnibus (GEO) datasets. Additionally, we performed in vitro differentiation of macrophages, stimulation with coated IgA, and RNA sequencing. Hepa1-6 cells and C57BL/6N mice were used to obtain HCC syngeneic mouse models. RESULTS: Serum IgA levels were associated (p<0.001) with fibrosis progression and HCC development in patients with chronic liver diseases. Additionally, immunohistochemical staining of inflamed livers or HCC revealed IgA positivity in monocytes, with a correlation between IgA+ cell frequency and IgA serum levels. Compared with IgA- monocytes, intrahepatic IgA+ monocytes expressed higher levels of programmed death-ligand 1 (PD-L1) in inflamed livers and in HCC tumor microenvironment. Single-cell RNA sequencing using NCBI GEO database indicated an upregulation in inflammation-associated genes in the monocytes of patients whose plasma cell IGHA1 expression was greater than or equal to the median value. Bulk RNA sequencing demonstrated that in vitro stimulation of M2-polarized macrophages using coated IgA complex induced PD-L1 upregulation via YAP-mediated signaling. In vivo blockade of IgA signaling decreased the number of tumor-infiltrating IgA+PD-L1high macrophages and increased the number of CD69+CD8+ T cells to enhance antitumor effects in HCC mice models. CONCLUSIONS: Overall, the findings of this study showed that serum IgA levels was correlated with intrahepatic and intratumoral infiltration of inflammatory IgA+PD-L1high monocytes in chronic liver diseases and HCC, providing potential therapeutic targets.

Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma.

A predictive biomarker of immune checkpoint inhibitor (ICI)-based treatments in hepatocellular carcinoma (HCC) has not been clearly demonstrated. In this study, we focused on the infiltration and programmed death ligand 1 (PD-L1) expression of tumor-associated macrophages (TAMs) in the tumor microenvironment of HCC. Immunohistochemistry demonstrated that PD-L1 was preferentially expressed on CD68+ macrophages in the tumor microenvironment of HCC, suggestive of its expression in TAMs rather than in T cells or tumor cells (P < 0.05). A co-culture experiment using activated T cells and M2 macrophages confirmed a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8+ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4+ and CD8+ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8+ T cells in HCC.

Nardostachys jatamansi Root Extract Attenuates Tumor Progression in Hepatocellular Carcinoma via Inhibition of ERK/STAT3 Pathways.

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) is a highly prevalent disease and treatment is limited. Therefore, development of new therapeutic agents is urgent. The aim of this study was to investigate the in vitro and in vivo anti-cancer effects of Nardostachys jatamansi root extract (NJRE) against HCC and underlying mechanisms involved in such effects. MATERIALS AND METHODS: Effects of NJRE on viability of HCC cell lines were determined by MTT analysis and annexin/PI apoptosis assays. Expression levels of proteins in MAPK and STAT3 pathways and caspase-3 and PARP after treatment with NJRE in HCC cell lines were determined by western blotting. In a syngeneic model using mouse HCC cells Hepa1-6, inhibition of tumor formation after oral administration of NJRE was determined and expression levels of phospho-ERK and phospho-STAT3 in liver tissues were analyzed by immunohistochemical staining. RESULTS: NJRE reduced the activation of STAT3 by inhibiting the expression of ERK and finally attenuated the proliferation of HCC. CONCLUSION: NJRE has anti-cancer effects against HCC. It has potential to be used in the treatment of human HCC.

EpCAM-high liver cancer stem cells resist natural killer cell-mediated cytotoxicity by upregulating CEACAM1.

BACKGROUND: Natural killer (NK) cells can recognize and kill cancer cells directly, but their activity can be attenuated by various inhibitory molecules expressed on the surface. The expression of epithelial cell adhesion molecule (EpCAM), a potential marker for cancer stem cells (CSCs), is known to be strongly associated with poor clinical outcomes in hepatocellular carcinoma (HCC). NK cells targeting CSCs may be a promising strategy for anti-tumor therapy, but little is known about how they respond to EpCAMhigh CSCs in HCC. METHODS: EpCAM expression was assessed by immunohistochemistry in 280 human HCC tissues obtained from curative surgery. To investigate the functional activity of NK cells against liver CSCs, EpCAMhigh and EpCAMlow Huh-7 cells were sorted by flow cytometry. The functional role of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is related to NK cells, was determined by in vitro co-culture of NK cells and hepatoma cells using Hepa1-6 mouse hepatoma cells, as well as in vivo experiments using C57/BL6 mice. RESULTS: The frequency of recurrence after curative surgery was higher in patients with positive EpCAM expression than in those with negative EpCAM expression. In subsequent analysis based on the anatomical location of EpCAM expression, patients with peritumoral EpCAM expression showed worse prognosis than those with pantumoral EpCAM expression. Co-culture experiments demonstrated that CEACAM1 was upregulated on the surface of EpCAMhigh HCC cells, resulting in resistance to NK cell-mediated cytotoxicity. Inversely, silencing CEACAM1 restored cytotoxicity of NK cells against EpCAMhigh Huh-7 cells. Moreover, neutralizing CEACAM1 on the NK cell surface enhanced killing of Huh-7 cells, suggesting that homophilic interaction of CEACAM1 is responsible for attenuated NK cell-mediated killing of CEACAM1high cells. In mouse experiments with Hepa1-6 cells, EpCAMhigh Hepa1-6 cells formed larger tumors and showed higher CEACAM1 expression after NK cell depletion. NK-mediated cytotoxicity was enhanced after blocking CEACAM1 expression using the anti-CEACAM1 antibody, thereby facilitating tumor regression. Moreover, CEACAM1 expression positively correlated with EpCAM expression in human HCC tissues, and serum CEACAM1 levels were also significantly higher in patients with EpCAM+ HCC. CONCLUSION: Our data demonstrated that EpCAMhigh liver CSCs resist NK cell-mediated cytotoxicity by upregulation of CEACAM1 expression.