The water channel protein aquaporin-4 (AQP4) and the gap junction forming proteins connexin-43 (Cx43) and connexin-30 (Cx30) are astrocytic proteins critically involved in brain water and ion homeostasis. While AQP4 is mainly involved in water flux across the astrocytic endfeet membranes, astrocytic gap junctions provide syncytial coupling allowing intercellular exchange of water, ions, and other molecules. We have previously shown that mice with targeted deletion of Aqp4 display enhanced gap junctional coupling between astrocytes. Here, we investigate whether uncoupling of the astrocytic syncytium by deletion of the astrocytic connexins Cx43 and Cx30 affects AQP4 membrane localization and expression. By using quantitative immunogold cytochemistry, we show that deletion of astrocytic connexins leads to a substantial reduction of perivascular AQP4, concomitant with a down-regulation of total AQP4 protein and mRNA. Isoform expression analysis shows that while the level of the predominant AQP4 M23 isoform is reduced in Cx43/Cx30 double deficient hippocampal astrocytes, the levels of M1, and the alternative translation AQP4ex isoform protein levels are increased. These findings reveal a complex interdependence between AQP4 and connexins, which are both significantly involved in homeostatic functions and astrogliopathologies.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and was listed as the third cause of death by cancer in 2020 (WHO). Hepatocarcinogenesis is a multistep process and to date it is still poorly understood. HCC occurs almost entirely in patients with underlying chronic liver disease and cirrhosis. Pre-clinical animal models play an important role in unveiling cancer biology which can facilitate development and testing of new antineoplastic drugs. The aim of this study was to establish an orthotopic model of HCC utilizing two cell lines Huh-7 and HepG2, which could be effectively used to study efficacy of new potential treatments. In a follow up study, the correlation of tumor marker alpha-fetoprotein (AFP) and tumor growth was investigated. AFP was measured using Quantikine® ELISA kit. For the intrahepatic (ih) inoculation cancer cells were injected into the left lobe of the liver. For the intrasplenic (is) inoculations of HCC cells, the spleen was exposed, and cells were injected into spleen parenchyma along the long axis of the spleen. Afterwards spleen was removed to avoid intrasplenic tumor growth. The animals were weighed two times a week throughout the study and appearance of any clinical signs was closely monitored. The animals were terminated 70 days after cancer cell implantation. The mice did not present with any acute symptoms before the end stage disease. However, prior sacrifice some swelling and darkening of the abdominal area especially in the ih-model was observed. To visualize liver ExiTron Nano 6000 nanoparticle contrast agent was used. The contrast agent was administered intravenously once during the study, prior the first imaging. Disease progression was monitored using Bruker SkyScan 1276 High-Resolution Micro-CT Scanner. The in vivo measurements were performed using one field of view (FOV) and an image pixel size of 18 μm. Each in vivo scanning was completed within 12 minutes and the estimated radiation dose was kept under 500 mGy at each imaging session. The first tumours were detected by µCT between 2-3 weeks depending more on the cancer cell implantation route than the cell line used. The take rate in the liver was 85% in the ih-model, and less than 50% in the is-model. The intrahepatic inoculation led to more uniform and measurable tumor nodules observed in majority of operated animals, whereas the intrasplenic tumors were dispersed throughout with multiple nodules. In addition, a tight correlation between tumor volume and amount of alpha-fetoprotein detected from serum samples was found. In conclusion, an orthotopic model using intrahepatic approach of cancer cells implantation was established. Application of µCT enabled following tumor growth and recognition of cancer spread in the abdomen. Altogether this provides a promising tool contributing to the development of novel therapies targeting HCC in vivo. Citation Format: Justyna Zdrojewska, Mari Suominen, Shirin Katoozi, Katja Fagerlund, Jukka Rissanen, Roger Malerbakken-Bjerke, Jenny Karlsson. Orthotopic model of hepatocellular carcinoma (HCC) using Huh-7 and HepG2: Application of micro-CT in detection of cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1662.