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

Liver cancer is the second most common cause of cancer-related death worldwide, with a steady increasing incidence and mortality [1] and, therefore, a major public health challenge. It is considered an immunogenic cancer because 90% of cases develop under conditions of chronic inflammation [1, 2]. This inflammation conducts to the development of tumors and it is associated with higher tumor immunogenicity. For this reason, the most suitable therapeutic strategies to be applied in these types of carcinomas would be immunotherapeutic approaches [3]. Hepatocellular carcinoma (HCC) is the most frequent liver cancer and is associated to high morbidity and mortality rates [1, 4]. It presents poor prognosis, generally due to its late presentation and thus, late diagnostic. Cholangiocarcinoma (CCA), a malignancy that originates from biliary epithelia, is an aggressive cancer with a high mortality rate [5] and, along with HCC, represents a major primary liver cancer [2]. CCA is difficult to diagnose due to its silent and nonspecific clinical features and, in most cases, the symptoms occur when the tumor has reached an advanced stage [6]. For patients with advanced disease, there are only limited therapeutic treatment options that provide limited benefits for a subset of patients. Therefore, novel therapeutic options are needed [7]. Partial surgical resection and liver transplantation are potentially curative treatments in selected patients with HCC and CCA [8, 9]. Unfortunately, a high postoperative tumor recurrence rate significantly decreases long-term survival outcomes [9].

Prognosis of cancer patients is based on tumor-related factors as well as on host-related factors, including systemic immune cell activation [10]. Given the difficulties in acquiring liver tissues, circulating biomarkers as well as comprehensive studies monitoring the peripheral immune system homeostasis, are needed. In fact, it has been demonstrated that immune monitoring of peripheral blood (PB) cells might lead to the identification of biomarkers, which could serve to predict prognosis and/or therapy response [11]. Due to its immunogenic origin, further knowledge of the changes on immune cell populations may help define the potential of the combination of therapies, such as adoptive immunotherapy and/or checkpoint inhibition [12]. Moreover, monitoring of the peripheral immune response after surgical resection will provide information to advance our understanding of the mechanisms underlying the clinical response to surgical resection.

In the present study, we have characterized, by multi-color flow cytometry, the frequency, composition and activity of circulating monocyte subsets in PB samples from HCC and CCA patients, in order to understand the immune status of these patients, and to obtain evidence about the changes in the proportion or functions of these populations, for application in the clinical diagnosis and future development of treatments to HCC and CCA.

The current study has identified a functional defect on some circulating monocyte subsets in HCC and CCA patients, regarding their capability to produce TNFα under stimulation, displaying a partial recovery after surgical procedure. Moreover, a peripheral pro-inflammatory state in HCC and CCA patients was observed, and, a potential role for TNFα circulating levels, as well as TNFα mRNA expression by classical and intermediate monocytes, as prognostic peripheral biomarkers in HCC patients, indicating the presence of high-grade (G3 or G4) tumors, was revealed.

Inflammatory factors affect nearly all the stages of tumor development and the effectiveness of the applied therapies. TNFα is considered one of the most important inflammatory mediators of the cancer-associated inflammatory networks. In this regard, preclinical studies in breast cancer have suggested that TNFα promotes tumor growth in vivo and could be considered a therapeutic target [14]. In the present study, we pointed out an upregulation of TNFα mRNA expression in peripheral classical, intermediate and non-classical monocytes purified from HCC patients presenting tumors classified as grade G3-G4, as compared to G1-G2 HCC patients. Moreover, in line with these results, we detected a significant increase of TNFα serum levels in HCC patients with tumors in grade G3-G4 as compared to grade G1-G2 HCC. In this regard, further studies will be needed to validate the potential use of TNFα serum levels as a prognostic factor or indicator of histopathological grading. Supporting this idea, it has been reported that aberrant elevated TNFα levels might promote not only tumor growth, but also invasion and poor prognosis [15]. Additionally, a previous work in a rat liver cancer model demonstrated that TNFα inhibition and deletion could decrease tumor incidence and showed that clinically TNFα expression was correlated to hepatic progenitor cells activation and HCC recurrences [16].

Regarding the characterization of circulating monocytes, we observed a significant decrease in the frequency and absolute counts of PB monocytes, both in CCA and HCC patients, at T0, as compared to the HG. Interestingly, values continued to be diminished comparatively to the HG after surgical procedure (at T1). The observed decrease in the frequency of circulating monocytes could be associated to an increase in the frequencies of circulating neutrophils since previous works have identified elevated levels of circulating neutrophils that were significantly and independently associated with poorer prognosis in HCC [17‐19]. Peripheral blood monocytes constantly enter the liver to replenish hepatic macrophages and dendritic cells, playing important roles in the pathogenesis of inflammatory disorders [20]. Our previous findings had already shown altered frequencies and altered absolute numbers of circulating FcεRI⁺ monocytes and myeloid dendritic cells in CCA and HCC patients, as well as functional defects in both cell subsets [21]. Thus, we wondered whether other monocyte subsets might be affected in these patients. The results of the present study extend our previous findings, showing alterations on monocytes function, as well as a significant decrease in the relative frequency and absolute numbers of circulating non-classical monocytes both in CCA and HCC patients. These values remained diminished as compared to the HG at T1, excepting the frequency of non-classical monocytes in CCA patients, that was partially restored after tumor resection. Non-classical monocytes “patrol” the vasculature via a mechanism that requires the fractalkine receptor CX3CR1, covering apoptotic endothelial cells and sensing danger signals coming from the tissue [22]. A previous work has shown that this population of monocytes reduces tumor metastasis by recruiting NK cells [23]. Therefore, we hypothesized that the observed decrease in this monocyte subset may be having a role in cancer progression. Additionally, it has been suggested that non-classical monocytes are crucial regulators in the pathogenesis of chronic liver disease in humans, by the secretion of abundant cytokines, perpetuating chronic inflammatory processes within the liver and by directly activating hepatic stellate cells that, in turn, can secrete multiple chemokines for monocyte recruitment into the liver [24]. Therefore, indicating that the modulation of monocyte-subset recruitment into the liver may represent possible novel approaches for interventions targeting pro-inflammatory actions of monocyte subsets in liver cancer.

When evaluating monocyte function, we observed, at T0, a significant decline in TNFα production by classical and HLADR⁺⁺ intermediate monocyte subsets, under LPS plus IFNγ stimulation, in both CCA and HCC patients as compared to the HG, whereas no significant differences were detected for non-classical monocytes. TNFα production by HLADR⁺ intermediate monocyte was also affected in CCA patients before tumor resection, but not in HCC patients. The frequencies of TNFα-producing cells seem to be restored after tumor resection, excepting classical monocyte subsets that displayed a significant decrease in TNFα production in HCC patients at T1. These data indicate a functional defect in classical and intermediate monocytes in both, CCA and HCC patients that it is partially restored for intermediate monocytes at T1. In line with these results, previous studies in other carcinomas have reported a diminished percentage of TNFα-producing CD14⁺ cells in PB of lung adenocarcinoma patients, showing that malignant cells inhibited the capability of monocytes to produce TNFα [25]. Thus, the decreased TNFα expression, observed in classical and intermediate monocytes, may be caused by tumor cells, but also, may influence tumor progression and relapse, since an altered response of innate immune cells might underscore a reduced capacity to mount an efficient antitumor immune response [26]. Monocytes can differentiate into a variety of macrophage or dendritic cell subtypes that can either activate or inhibit the immune response [27], and it has been shown that CX3CR1 is rapidly downregulated during monocytes differentiation [28]. In relation with this process, classical and non-classical monocytes purified from CCA and HCC patients presented a downregulation of the mRNA expression of the fractalkine receptor, CX3CR1, at T0, when compared to the HG, with partial recovery at T1, indicating a possible differentiation process in these monocyte subsets occurring before surgical procedure. Additionally, CX3CR1 expression could participate in promoting cell-to-cell interactions with an inflamed endothelium, as well as increasing monocytes survival [28]. We also detected, in intermediate monocytes from CCA patients, an upregulation of TNFα mRNA expression at T0 that was restored after surgical procedure, which is further supported by the results obtained from the measurement of serum TNFα levels, in which we detected significantly elevated TNFα serum levels in CCA patients at T0, that were restored at T1. Indicating an important role of peripheral TNFα in CCA associated inflammatory response.

In addition, we detected elevated circulating levels of CXCL10 in HCC patients, before surgery, that were only partially restored at T1. CXCR3 is the receptor for CXCL10, a member of the CXC chemokine family with pro-inflammatory and anti-angiogenic properties [29] that has been associated with a variety of human diseases, including tumor development, metastasis, and dissemination. More importantly, CXCL10 has been identified as a major biological marker mediating disease severity and may be used as a prognostic indicator for various diseases [30], thus, its possible role as disease severity biomarker for liver cancers should be further evaluated. Moreover, circulating levels of CCL20 in HCC patients before and after tumor resection were significantly increased, in comparison to the HG, as well as in CCA patients at T0, which presented a partial recovery after tumor resection. CCL20, alternatively called liver activation regulated chemokine (LARC), is a strong chemoattractant for leukocytes expressing its sole receptor CCR6 [31]. Many studies have shown that the CCL20/CCR6 axis contribute to the initiation and progression of HCC by mediating the migration of circulating T cells into the tumor microenvironment [32]. Thus, the observed increase in the circulating levels of CCL20 indicates a probable migration of T cells to the tumor microenvironment of these carcinomas.

The detected increase in the circulating levels of CCL20, CXCL10, and TNFα suggests a peripheral pro-inflammatory state in HCC and CCA patients and an activate state for monocyte subsets although classical and intermediate monocyte subsets displayed a functional defect at T0 and, therefore, a limited capacity to respond under further stimulation processes.

This integrated analysis permitted the identification of different immune background in CCA and HCC patients, as well as altered functional competence of circulating monocytes in these carcinomas. Intriguingly, most of these alterations appear to be restored in CCA patients after tumor resection, however, most of them remained in HCC patients after surgical procedure. Moreover, this work has demonstrated that flow cytometry serves as a powerful analytical platform for the rapid characterization of individual cells within heterogenic cell populations and it may help in monitoring functional competence of immune cell populations to better evaluate immune dysfunctions in cancer patients. This approach could be applied in clinical routine to evaluate innate and adaptive immunity and to monitor responses to different treatments. In addition, the identified immune alterations should be further studied to consider clinically relevant therapeutic targets. A better understanding of the potential of inflammation and innate immunity to inhibit tumor progression should lead to the development of new and improved immunomodulatory approaches for the treatment of liver carcinomas. Notably, our results point out a potential role for TNFα as a prognostic factor in HCC patients indicating the presence of high-grade tumors. However, further studies should be conducted with larger sample sizes to validate the findings we have presented and correlate these findings with post-surgery outcomes after long-term follow-up.

The aim of the present study was to characterize, by multi-color flow cytometry, the frequency, composition and activity of circulating monocyte subsets in PB samples from HCC and CCA patients, in order to understand the immune status of these patients, and to obtain evidence about the changes in the proportion or functions of these populations, for application in the clinical diagnosis and future development of treatments to HCC and CCA.