pH Regulatory Molecules In The Tumour Microenvironment: Modulators Of Aggressiveness And Immune Profile Of Human Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) arises in an inflammatory, hypoxic/acidic microenvironment that favours tumour progression and fosters immunosuppression. Tumour cells survive this hostile environment by over-expressing pH regulatory molecules such as carbonic anhydrase (CA) IX, XII and V-ATPase complex, but the relevance of these molecules in HCC is poorly defined. Aim: The aim of this study was to dissect the relationships between pH regulatory molecules and the aggressive behaviour of malignant hepatocytes, and to evaluate how pH regulatory molecules influence the immune microenvironment of HCC. Methods: HCC, non-tumour and normal liver tissue samples were analysed by qRT-PCR for the expression of genes encoding the pH regulatory molecules (CAIX, CAXII and V-ATPase), of genes associated to epithelial-to-mesenchymal-transition (EMT) (TWIST, CDH1, VIM) and those encoding for HCC stem cell-associated markers (CD13, CD24, CD44, CD90, EpCAM, CD133, KRT19, OCT4, NANOG and SOX2). Selected HCC, non-tumour and normal liver tissue samples were evaluated by immunohistochemistry (IHC) to detect the presence and localization of CAIX, CAXII and VATPase and to assess the distribution of macrophages and T cells. Confocal microscopy and flow cytometry were implemented to assess the co-expression of selected markers. HCC cell lines, characterised for the expression of pH regulators, were tested for the sensitivity to the CAIX, CAXII, and V-ATPase specific inhibitors. The effects of V-ATPase specific drug were also studied ex vivo in primary human HCC tumour explants by qRT-PCR and by flow cytometry in HCC single cell suspensions obtained by the enzymatic digestion of HCC specimens. Results: Our mRNA analysis showed that the expression of CA9 was significantly correlated with the expression of the hypoxia-inducible factor 1α-related gene (HIF1A) and of the stem cell-associated markers CD24, CD133, EpCAM and KRT19. Moreover, mRNA for CA9 and for the different CA12 isoforms were associated with tumour grading, thus indicating their possible role in tumour malignancy. Applying a machine learning tool known as the ‘Adaptive Index Model’ the combined expression of different CA12 isoforms, CD209 and CDH1 defined a ‘signature’ classifying HCC patients in groups at different risk of recurrence, thus indicating a link between pH regulators, myeloid and EMT markers likely influencing HCC prognosis. IHC analysis indicated that HCC displays a complex expression pattern for the pH regulatory proteins. Both CAIX and CAXII were detected in transformed, but not in normal hepatocytes. CAIX protein had a focal distribution in the tumour, thus supporting its possible association with hypoxic and the most aggressive tumour area. Conversely, CAXII was homogeneously expressed by all tumour hepatocytes, but mainly retained in the endoplasmic reticulum (ER). The majority of HCC expressed V-ATPase which, importantly, was also present in immune infiltrating cells. This expression pattern qualified the CAIX, CAXII and V-ATPase as possible targetable molecules. Our in vitro data indicated that blockage of their enzymatic activities by specific drugs affected the viability of HCC cell lines in a dose dependent fashion, although with the CAXII specific inhibitor showing low efficacy, likely related to the preferential ER localization of CAXII molecules inside the HCC cells. Ex vivo experiments with HCC tissue explants and HCC cellular suspensions showed that inhibition of VATPase modulated the epithelial/mesenchymal features of HCC cells and the levels of pro- and anti-tumour cytokines expressed by M2 macrophages and T cells infiltrating HCC. Conclusions: Herein, our data demonstrated that the pH regulatory molecules, CAIX, CAXII and V-ATPase are over expressed in the HCC microenvironment and interfering with their pathways exerted anti-tumour activities, although these data also lead to the conclusion that more effective CAXII specific drugs should be designed. The results of this thesis also suggest that pH regulatory molecules might have a role in HCC aggressiveness and prognosis. Importantly, one of these pH regulators, namely V-ATPase complex, influences the mesenchymal features of tumour cells and the immunosuppressive tumour microenvironment (TME). Interfering with tumour metabolism is an emerging strategy for treating cancers that are resistant to standard therapies. Thus, targeting the unique crosstalk between tumour cells and the microenvironment, played by the pH regulatory molecules, can be considered as a new option for HCC treatment and the blockage of the V-ATPase complex might represent a multi-task strategy for the treatment of HCC patients

Cancer acidity: An ultimate frontier of tumor immune escape and a novel target of immunomodulation

The link between cancer metabolism and immunosuppression, inflammation and immune escape has generated major interest in investigating the effects of low pH on tumor immunity. Indeed, microenvironmental acidity may differentially impact on diverse components of tumor immune surveillance, eventually contributing to immune escape and cancer progression. Although the molecular pathways underlying acidity-related immune dysfunctions are just emerging, initial evidence indicates that antitumor effectors such as T and NK cells tend to lose their function and undergo a state of mostly reversible anergy followed by apoptosis, when exposed to low pH environment. At opposite, immunosuppressive components such as myeloid cells and regulatory T cells are engaged by tumor acidity to sustain tumor growth while blocking antitumor immune responses. Local acidity could also profoundly influence bioactivity and distribution of antibodies, thus potentially interfering with the clinical efficacy of therapeutic antibodies including immune checkpoint inhibitors. Hence tumor acidity is a central regulator of cancer immunity that orchestrates both local and systemic immunosuppression and that may offer a broad panel of therapeutic targets. This review outlines the fundamental pathways of acidity-driven immune dysfunctions and sheds light on the potential strategies that could be envisaged to potentiate immune-mediated tumor control in cancer patients

Soft-landed protein voltammetry: A tool for redox protein characterization

Microperoxidase-11 (MP-11) was first soft landed onto the gold surface of a screen-printed electrode. Intact protein deposition was verified by time-of-flight secondary ion mass spectrometry. The coupling of soft landing with electrochemical techniques allowed unique information to be obtained about the deposition features. A full characterization of the direct electron-transfer properties was performed by modeling data obtained from cyclic voltammetry experiments; calculated values of kinetic electron-transfer constant, formal redox potential, and reorganization energy allow us to hypothesize the mechanism involved in soft landing immobilization and demonstrate the different conformation of the enzyme deposited from two different charged species. The strong interaction between MP-11 and the gold surface and long-term stability of the functionalized electrode characterizes the peculiar features of this procedure, which enhance its power with respect to the existing immobilization procedure and ensure its suitability for those practical applications that could benefit from an unmediated bridgeless bioeletrochemical electron transfer (e.g., biosensor transducers or electrode elements in biofuel cells)

Astrocyte-derived lipoxins A4 and B4 promote neuroprotection from acute and chronic injury

Astrocytes perform critical non-cell autonomous roles following CNS injury that involve either neurotoxic or neuroprotective effects. Yet the nature of potential prosurvival cues has remained unclear. In the current study, we utilized the close interaction between astrocytes and retinal ganglion cells (RGCs) in the eye to characterize a secreted neuroprotective signal present in retinal astrocyte conditioned medium (ACM). Rather than a conventional peptide neurotrophic factor, we identified a prominent lipid component of the neuroprotective signal through metabolomics screening. The lipoxins LXA4 and LXB4 are small lipid mediators that act locally to dampen inflammation, but they have not been linked directly to neuronal actions. Here, we determined that LXA4 and LXB4 are synthesized in the inner retina, but their levels are reduced following injury. Injection of either lipoxin was sufficient for neuroprotection following acute injury, while inhibition of key lipoxin pathway components exacerbated injury-induced damage. Although LXA4 signaling has been extensively investigated, LXB4, the less studied lipoxin, emerged to be more potent in protection. Moreover, LXB4 neuroprotection was different from that of established LXA4 signaling, and therapeutic LXB4 treatment was efficacious in a chronic model of the common neurodegenerative disease glaucoma. Together, these results identify a potential paracrine mechanism that coordinates neuronal homeostasis and inflammation in the CNS

Targeting Immune Regulatory Networks to Counteract Immune Suppression in Cancer

The onset of cancer is unavoidably accompanied by suppression of antitumor immunity. This occurs through mechanisms ranging from the progressive accumulation of regulatory immune cells associated with chronic immune stimulation and inflammation, to the expression of immunosuppressive molecules. Some of them are being successfully exploited as therapeutic targets, with impressive clinical results achieved in patients, as in the case of immune checkpoint inhibitors. To limit immune attack, tumor cells exploit specific pathways to render the tumor microenvironment hostile for antitumor effector cells. Local acidification might, in fact, anergize activated T cells and facilitate the accumulation of immune suppressive cells. Moreover, the release of extracellular vesicles by tumor cells can condition distant immune sites contributing to the onset of systemic immune suppression. Understanding which mechanisms may be prevalent in specific cancers or disease stages, and identifying possible strategies to counterbalance would majorly contribute to improving clinical efficacy of cancer immunotherapy. Here, we intend to highlight these mechanisms, how they could be targeted and the tools that might be available in the near future to achieve this goal

Immune landscape and in vivo immunogenicity of NY-ESO-1 tumor antigen in advanced neuroblastoma patients

Abstract Background Indirect evidence suggesting the immunosensitivity/immunogenicity of neuroblastoma is accumulating. The aims of this study were to investigate the immune landscape of neuroblastoma and to evaluate the in vivo immunogenicity of the NY-ESO-1 tumor antigen in advanced neuroblastoma patients. Methods The immune infiltrating cells of the NY-ESO-1+ tumors from three HLA*A201 patients with metastatic neuroblastoma who relapsed after conventional treatments were evaluated by immunohistochemistry. The patients were vaccinated with the HLA-A*0201-restricted peptide NY-ESO-1157-165(V). The peptide was emulsified in Montanide ISA51 and given subcutaneously in a phase I pilot study. The immunogenicity of NY-ESO-1 antigen was evaluated by monitoring mononuclear cells in patient peripheral blood, pre- and post-vaccine, by short-term in vitro sensitization, HLA-multimer staining and IFN-γ ELISpot analysis. Results Both CD3 T cells and CD163 myeloid cells were present in pre-vaccine tumors and PD-1 and PD-L1 expression was mainly found in the immune infiltrate. Despite the advanced stage of the disease, the vaccination induced systemic NY-ESO-1 specific CD8 T cells releasing IFN-γ in response to activation with the NY-ESO-1 peptide and an HLA-A2 positive neuroblastoma cell line. Conclusions Our results indicate that vaccination with a tumor-associated peptide is able to boost NY-ESO-1-specific, functionally active T cells in advanced neuroblastoma patients with lymphocyte infiltration in their pre-vaccine tumors. Trial registration EudraCT #2006–002859-33

Complex Immune Contextures Characterise Malignant Peritoneal Mesothelioma: Loss of Adaptive Immunological Signature in the More Aggressive Histological Types

Malignant peritoneal mesothelioma (MpM), arising in the setting of local inflammation, is a rare aggressive tumour with a poor prognosis and limited therapeutic options. The three major MpM histological variants, epithelioid (E-MpMs), biphasic, and sarcomatoid MpMs (S-MpMs), are characterised by an increased aggressiveness and enhanced levels of EZH2 expression. To investigate the MpM immune contexture along the spectrum of MpM histotypes, an extended in situ analysis was performed on a series of 14 cases. Tumour-infiltrating immune cells and their functionality were assessed by immunohistochemistry, immunofluorescence, qRT-PCR, and flow cytometry analysis. MpMs are featured by a complex immune landscape modulated along the spectrum of MpM variants. Tumour-infiltrating T cells and evidence for pre-existing antitumour immunity are mainly confined to E-MpMs. However, Th1-related immunological features are progressively impaired in the more aggressive forms of E-MpMs and completely lost in S-MpM. Concomitantly, E-MpMs show also signs of active immune suppression, such as the occurrence of Tregs and Bregs and the expression of the immune checkpoint inhibitory molecules PD1 and PDL1. This study enriches the rising rationale for immunotherapy in MpM and points to the E-MpMs as the most immune-sensitive MpM histotypes, but it also suggests that synergistic interventions aimed at modifying the tumour microenvironment (TME) should be considered to make immunotherapy beneficial for these patients

Hepatocellular Carcinoma is Associated with Gut Microbiota Profile and Inflammation in Non-Alcoholic Fatty Liver Disease

The gut-liver axis plays a pivotal role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), which is the third cause of hepatocellular carcinoma (HCC) worldwide. However, the link between gut microbiota and hepatocarcinogenesis remains to be clarified. The aim of this study is to explore what features of the gut microbiota are associated with HCC in cirrhotic patients with NAFLD. A consecutive series of patients with NAFLD-related cirrhosis and HCC (group 1: 21 patients), NAFLD-related cirrhosis without HCC (group 2: 20 patients) and healthy controls (group 3: 20 patients) was studied for gut microbiota profile, intestinal permeability, inflammatory status and circulating mononuclear cells. We finally constructed a model depicting the most relevant correlations among these features, possibly involved in hepatocarcinogenesis. Patients with HCC showed increased levels of fecal calprotectin, whilst intestinal permeability was similar to cirrhotic patients without HCC. Plasma levels of interleukin (IL) 8, IL13, C-C motif chemokine ligand (CCL) 3, CCL4 and CCL5 were higher in the HCC group and were associated with an activated status of circulating monocytes. The fecal microbiota of the whole group of cirrhotic patients showed higher abundance of Enterobacteriaceae and Streptococcus, and a reduction in Akkermansia. Bacteroides and Ruminococcaceae were increased in the HCC group, while Bifidobacterium was reduced. Akkermansia and Bifidobacterium were inversely correlated with calprotectin concentration, which in turn was associated with humoral and cellular inflammatory markers. A similar behavior was also observed for Bacteroides