A biomimetic liver model recapitulating bio-physical properties and tumour stroma interactions in hepatocellular carcinoma

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 micro-environment 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 mimicking the bio-physical properties of the tumor ECM. In our model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted insert membrane of a Transwell system. 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 determined by rheology. Cell viability was determined by the AlamarBlue(R) assay over 21-days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both a 2D co-culture and our 3D model for a period of 72h. We show that this model is viable for 25-days and gives rise to metastatic tumor nodules after 17 days in culture. 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. This model could in future provide a valuable new platform to study multifocal HCC or to identify mechanisms that contribute to early stages of metastasis. SUMMARYA protocol for a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature, to study endocrine and paracrine signaling in liver cancer. The model uses physiological relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, which is an active mediator of cellular interactions, tumor growth and metastasis.