Abstract 3517: Targeting pH regulators to modulate human hepatocellular carcinoma microenvironment
Authors: Tuccitto Alessandra , Kuchuk Olga , Citterio Davide , Huber Veronica , Camisaschi Chiara , Milione Massimo , Vergani Barbara , Villa Antonello , Rivoltini Licia , Castelli Chiara , Mazzaferro Vincenzo 
Published: August 2018

Abstract
Hepatocellular carcinoma (HCC) arises in a hypoxic/acidic microenvironment in which the pO2 is about 0.8%. This condition favors tumor progression and fosters immunosuppression. Tumor cells survive this obstile environment by overexpressing pH regulators such as carbonic anhydrase (CA) IX, XII and V-ATPase complex. We investigated the expression and functional properties of these molecules in HCC cell lines and patients, to evaluate their potential role as novel therapeutic agents against HCC. The expression of CAs and V-ATPase subunits was assayed by qRT-PCR, western blot and confocal microscopy in HCC cell lines exposed to normoxia (21% O2) or hypoxia (1% O2). The effect of pH regulatory inhibitors on HCC cell viability was evaluated by MTT assay. Paired tumoral and non-tumoral liver tissues (n=57) from HCC patients were analyzed for the expression of pH regulatory molecules by qRT-PCR and by immunohistochemistry. Multi-paramentric flow cytometry analysis was performed on patient-derived HCC cell suspensions exposed to V-ATPase inhibitor or vehicle for 24h (n=6). Human HCC tissue explants (n=8) were cultured in presence of V-ATPase inhibitor for 24h and gene expression was analysed by qRT-PCR. CAs and V-ATPase are expressed by HCC cell lines grown under normoxia (21% O2) and a selective increment of CAIX and CAXII was observed after hypoxia (1% O2) exposure. These molecules were involved in cancer cell survival, as demonstrated by the significant reduction of tumor viability by CAIX and V-ATPase inhibitors. Ex vivo analyses showed a focal and intense plasma membrane immunoreactivity for CAIX in discrete nests of hepatocytes inside HCC lesions and in cholangiocytes within normal and non-tumoral tissues. Tumor cells selectively expressed CAXII in the cytoplasm. In vivo and ex vivo we observed that in the hypoxic microenvironment of HCC CAXII was retained in the endoplasmic reticulum of hepatocytes. Significant, albeit heterogeneous, positivity for V-ATPase was detected in most of HCC lesions analyzed. Importantly, infiltrating immune cells compatible with M2 macrophage phenotype expressed the V-ATPase. The inhibiton of V-ATPase by omeprazole in cell suspensions obtained from clinical HCC tissues showed a decreased production of CCL22 paralleled by an increment of IFNγ by M2 macrophages. Short-term treatment of HCC tissue explants with omeprazole exerted important effects on tumor microenvironment reducing the immunosuppressive gene CCL22 together incrementing IFNG, but also affected the aggressiveness of tumoral cells inducing a down-modulation of epithelial to mesenchymal transition associated genes VIM and MYC and increasing the transcription of CDH1. In conclusion our data indicate that pH regulators, overexpressed in tumor and immune cells composing the HCC microenvironment, play a role in maintaining an aggressive and immunosuppressive milieu in HCC and likely they represent a promising target.
Citation Format: Alessandra Tuccitto, Olga Kuchuk, Davide Citterio, Veronica Huber, Chiara Camisaschi, Massimo Milione, Barbara Vergani, Antonello Villa, Licia Rivoltini, Chiara Castelli, Vincenzo Mazzaferro. Targeting pH regulators to modulate human hepatocellular carcinoma microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3517.