Targeting non-junctional Claudin-1 with monoclonal antibodies to treat hepatocellular carcinoma [RNA-seq]

Abstract: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Despite new treatment approvals, treatment response and prognosis of patients with advanced HCC remains poor. Claudin-1 (CLDN1) is a membrane protein expressed in tight junctions but also non-junctionally at the basolateral membrane of hepatocytes. While the function of CLDN1 within tight junctions is well characterized, the role of non-junctional (NJ) CLDN1 in HCC remains unexplored. Here we show that targeting NJ-CLDN1 with a humanized monoclonal antibody (mAb) suppresses tumor growth in different pre-clinical models. Mechanistic studies including single cell RNA sequencing of multicellular patient tumorspheres suggest that non-junctional CLDN1 regulates tumor stemness, metabolism and oncogenic signaling with impact on the tumor immune microenvironment. Our results provide the rationale for targeting NJ-CLDN1 in HCC and pave the way to novel therapeutic interventions with NJ-CLDN1 mAbs aimed at improving the limited efficacy of current therapies. Fresh tumor tissues from mice bearing established primary human liver cancer (PDX models LI6280, LI6716, LI6688, LI6723, LI1055 and LI1068) were harvested and cut into small pieces (approximately 2-3 mm in diameter) and inoculated subcutaneously at the upper right dorsal flank into female BALB/c nude mice for tumor development. The randomization started when the mean tumor size reached 100 mm3. A total of 5 mice were enrolled in each model. Randomization into groups receiving CLDN1 mAb (10 mg/kg, 10 mL/kg, once weekly (QW), n=3 mice) or vehicle control (10 mL/kg, QW, n=2 mice) was performed based on "Matched distribution" method. The date of grouping was denoted as day 1 (5/12/2020). Dosing was started on day 1 and continued through day 25. Mice were sacrificed at day 28 and tumor tissue was harvested and stored snap frozen. Liver tissue was lysed in TRI-reagent (Molecular Research Center), and RNA was purified using Direct-zol RNA MiniPrep (Zymo Research) according to the manufacturer’s instructions. RNAseq was performed at Biomedical Sequencing Facility (BSF) at Ce-M-M, Vienna.