A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Abstract

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma microenvironment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the in vivo situation compared to traditional 2D cultures. Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.

In Vitro Cell Toxicity and Intracellular Uptake of Doxorubicin Exposed as a Solution or Liposomes: Implications for Treatment of Hepatocellular Carcinoma

“…Cell viability was evaluated after drug treatment for 24, 48 or 72 h by a resazurin reduction assay, as previously described [ 31 ]. A 1% solution of resazurin sodium salt (Sigma-Aldrich) was prepared according to manufacturer’s recommendation and diluted 1/80 in CCM SM .…”

Inhibiting IRE1α-endonuclease activity decreases tumor burden in a mouse model for hepatocellular carcinoma

“…The HCC-cell lines (HepG2, ATCC HB-8065 and Huh7, kind gift from Dilruba Ahmed, Karolinska Institute, Sweden) and the human hepatic stellate cell-line LX2 (SCC064, Sigma-Aldrich, Darmstadt, Germany) were cultured at 37°C with 5% CO 2 in high glucose Dulbecco modified eagle medium, GlutaMAX supplemented (DMEM) (31066047, ThermoFisher Scientific, Stockholm, Sweden) supplemented with 1% antibiotic antimycotic solution (A5955-100ML, Sigma-Aldrich, Darmstadt, Germany) followed by 10% and 2% fetal bovine serum (FBS) (10270106, ThermoFisher Scientific, Stockholm, Sweden) for the HCC cell lines and LX2 cell line, respectively ( Calitz et al, 2020 ). No FBS was used during starvation and stimulation with growth factors.…”

“…For transwell co-culturing experiments, cells were grown on 12-well Corning Costar Transwell plates (3460, Sigma-Aldrich, Darmstadt, Germany) with 0.4 μm-pore polyester membrane, allowing the exchange of soluble factors, but preventing direct cell contact ( Calitz et al, 2020 ). Cells were detached using 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific, Stockholm, Sweden), re-suspended in growth medium and seeded at a density of 1.0 × 10 5 cells per well and 4.0 × 10 4 cells per insert.…”

Limitations and Possibilities of Transarterial Chemotherapeutic Treatment of Hepatocellular Carcinoma

“…The cirrhotic liver is characterized by a severe inflammatory reaction and an excessive deposition of extracellular matrix, which actively contributes to the progression of HCC [ 45 , 46 ]. Several studies have suggested that therapeutic response relies partly on extrinsic mechanisms provided by the crosstalk between tumour cells and components of the tumour microenvironment ( Figure 3 ).…”