Hepatocellular carcinoma (HCC) is a liver tumor that arises in patients with cirrhosis. One of the key players in the progression of cirrhosis to HCC is the hepatic stellate cell, which is activated during liver damage. Activated stellate cells play an essential role in the pathogenesis of HCC by creating a fibrotic micro-environment that sustains tumor growth and by producing growth factors and cytokines that enhance tumor cell proliferation and migration. We assessed the role of endoplasmic reticulum (ER) stress in the cross-talk between hepatic stellate cells and HCC-cells. Mice with a fibrotic HCC were treated with the IRE1A-inhibitor 4mu8C. The oncogenic protein expression was assessed using a multiplex proximity extension assay for ninety-two biomarkers in the murine exploratory panel (Olink Bioscience, Uppsala, Sweden) in liver samples from healthy mice, mice with HCC and mice with HCC treated with the IRE1A-inhibitor 4mu8C.

Mouse model

A chemically induced mouse model for hepatocellular carcinoma was used. Briefly, 5-week-oldmale sv129 mice received intraperitoneal injections once per week with 35mg/kg bodyweight diethylnitrosamine (DEN) diluted in saline. From week 10, mice were injected twice per week with 10μg/g bodyweight 4μ8C (Sigma) in saline. After 25 weeks mice were euthanized and samples were taken for analysis. The following conditions are included in the assay: healthy liver (CTL), liver cancer (DEN) and liver cancer treated with 4μ8C (DEN + 4μ8C)

Olink multiplex proximity extension assay

Liver samples were homogenized in ice-cold RIPA containing protease inhibitors (Sigma Aldrich). Homogenates were kept on ice for 20–30 min, whilst mixing vigorously to enhance disruption of the cell membranes. The homogenates were centrifuged (20 min, 13 000 rpm, 4°C) and supernatant containing protein was collected. Supernatant was stored at -20°C until protein measurement. Protein concentration was measured using the BCA kit (ThermoFisher) and all samples were diluted to 1 mg/mL protein in RIPA. Samples from 3 biological replicates per group were analyzed with a multiplex proximity extension assay for ninety-two biomarkers in the murine exploratory panel (Olink Bioscience, Uppsala, Sweden) [30]. Samples were loaded random on the assay plates.