Cancer is among the leading causes of death worldwide. Early diagnosis and prognosis are vital to improve patients’ outcomes. The gold standard of tumor characterization leading to tumor diagnosis and prognosis is tissue biopsy. Amongst the constraints of tissue biopsy collection is the sampling frequency and the incomplete representation of the entire tumor bulk. Liquid biopsy approaches, including the analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating miRNAs, and tumor-derived extracellular vesicles (EVs), as well as certain protein signatures that are released in the circulation from primary tumors and their metastatic sites, present a promising and more potent candidate for patient diagnosis and follow up monitoring. The minimally invasive nature of liquid biopsies, allowing frequent collection, can be used in the monitoring of therapy response in real time, allowing the development of novel approaches in the therapeutic management of cancer patients. In this review we will describe recent advances in the field of liquid biopsy markers focusing on their advantages and disadvantages.

According to the WHO’s recently released worldwide cancer data for 2020, liver cancer ranks sixth in morbidity and third in mortality among all malignancies. Hepatocellular carcinoma (HCC), the most common kind of liver cancer, accounts approximately for 80% of all primary liver malignancies and is one of the leading causes of death globally. The intractable tumor microenvironment plays an important role in the development and progression of HCC and is one of three major unresolved issues in clinical practice (cancer recurrence, fatal metastasis, and the refractory tumor microenvironment). Despite significant advances, improved molecular and cellular characterization of the tumor microenvironment is still required since it plays an important role in the genesis and progression of HCC. The purpose of this review is to present an overview of the HCC immune microenvironment, distinct cellular constituents, current therapies, and potential immunotherapy methods.

Considering the increasing importance of immune checkpoints in tumor immunity we investigated the clinical relevance of serum T-cell immunoglobulin and mucin domain-3 (TIM-3) in patients with hepatocellular carcinoma (HCC). Serum TIM-3 levels were measured and their association with HCC stage and the detection of serum programmed death ligand-1 (PD-L1) were assessed. In patients submitted to transarterial chemoembolization (TACE), pre- and 1-week post-treatment TIM-3 levels were also evaluated. We studied 53 HCC patients with BCLC stages: 0 (5.7%), A (34%), B (32.1%), C (22.6%), and D (5.7%). The patients with advanced HCC (BCLC C) had significantly higher TIM-3 levels than patients with BCLC A (p = 0.009) and BCLC B (p = 0.019). TIM-3 levels were not associated with HCC etiology (p = 0.183). PD-L1 detection (9/53 patients) correlated with TIM-3 levels (univariate analysis, p = 0.047). In 33 patients who underwent TACE, post-treatment TIM-3 levels (231 pg/mL, 132–452) were significantly higher than pre-TACE levels (176 pg/mL, 110–379), (p = 0.036). Complete responders had higher post-TACE TIM-3 levels (534 pg/mL, 370–677) than partial responders (222 pg/mL, 131–368), (p = 0.028). Collectively, TIM-3 may have a role in anti-tumor immunity following TACE, setting a basis for combining immunotherapy and chemoembolization.

L-dopa decarboxylase (DDC) that catalyzes the biosynthesis of bioactive amines, such as dopamine and serotonin, is expressed in the nervous system and peripheral tissues, including the liver, where its physiological role remains unknown. Recently, we reported a physical and functional interaction of DDC with the major signaling regulator phosphoinosite-3-kinase (PI3K). Here, we provide compelling evidence for the involvement of DDC in viral infections. Studying dengue (DENV) and hepatitis C (HCV) virus infection in hepatocytes and HCV replication in liver samples of infected patients, we observed a negative association between DDC and viral replication. Specifically, replication of both viruses reduced the levels of DDC mRNA and the ~120 kDa SDS-resistant DDC immunoreactive functional complex, concomitant with a PI3K-dependent accumulation of the ~50 kDa DDC monomer. Moreover, viral infection inhibited PI3K-DDC association, while DDC did not colocalize with viral replication sites. DDC overexpression suppressed DENV and HCV RNA replication, while DDC enzymatic inhibition enhanced viral replication and infectivity and affected DENV-induced cell death. Consistently, we observed an inverse correlation between DDC mRNA and HCV RNA levels in liver biopsies from chronically infected patients. These data reveal a novel relationship between DDC and Flaviviridae replication cycle and the role of PI3K in this process.

Cell senescence constitutes a physiological process that serves as protection from malignant transformation of cells. However, recent scientific discoveries also identify cell senescence as pivotal in hepatocellular cancer (HCC) biology. The review herein aimed to accumulate evidence on senescence as a mediator of HCC occurrence in hepatitis B (HBV), C (HCV) virus infections, and non-alcoholic fatty liver disease (NAFLD). In HBV infection, the carcinogenic HBV X protein frequently mutates during chronic infection, and subsequently exhibits different effects on senescence. In HCV infection, senescent non-functional T-cells do not effectively clear pre-malignant hepatocytes. Furthermore, the HCV Core protein inhibits the occurrence of normal stress-induced hepatocyte senescence, allowing damaged cells to maintain their proliferative potential. In NAFLD-mediated HCC, current data point towards the gut microbiome and hepatic stellate cell senescence. Additionally, senescence contributes in the development of resistance in targeted therapies, such as sorafenib. Finally, the promising role of senotherapeutics in HCC was also explored. Overall, although we may still be at a primitive stage in fully unraveling the role of senescence in cancer, it seems that understanding and harnessing senescence may have the potential to revolutionize the way we treat hepatocellular cancer.

Background: Many studies have suggested that the immune response may play a crucial role in the progression of hepatocellular carcinoma (HCC). Therefore, our aim was to establish a (i) functional culture of primary human tumor hepatocytes and non-tumor from patients with hepatocellular carcinoma (HCC) and (ii) a co-culture system of HCC and non-HCC hepatocytes with autologous peripheral blood mononuclear cells (PBMCs) in order to study in vitro cell-to-cell interactions.Methods: Tumor (HCC) and non-tumor (non-HCC) hepatocytes were isolated from the liver resection specimens of 11 patients operated for HCC, while PBMCs were retrieved immediately prior to surgery. Four biopsies were obtained from patients with no liver disease who had surgery for non malignant tumor (normal hepatocytes). Hepatocytes were either cultured alone (monoculture) or co-cultured with PBMCs. Flow cytometry measurements for MHC class II expression, apoptosis, necrosis and viability (7AAD) were performed 24 h, 48 h and 72 h in co-culture and monocultures.Results: HCC and non-HCC hepatocytes exhibited increased MHC-II expression at 48h and 72h in co-culture with PBMCs as compared to monoculture, with MHC II-expressing HCC hepatocytes showing increased viability at 72 h. PBMCs showed increased MHC-II expression (activation) in co-culture with HCC as compared to non-HCC hepatocytes at all time points. Moreover, CD8+ T cells had significantly increased apoptosis and necrosis at 48h in co-culture with HCC hepatocytes as compared to monocultures.Interestingly, MHC-II expression on both HCC and non-HCC hepatocytes in co-culture was positively correlated with the respective activated CD8+ T cells.Conclusions: We have established an in vitro co-culture model to study interactions between autologous PBMCs and primary HCC and non-HCC hepatocytes. This direct interaction leads to increased antigen presenting ability of HCC hepatocytes, activation of PBMCs with a concomitant apoptosis of activated CD8+ T cells. Although, a partially effective immune response against HCC exists, still tumor hepatocytes manage to escape.