Abstract
The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroidmodel which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 µL media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.
Highlights
The global incidence and mortality from liver cancer have continued to increase, despite advances in treatments for liver disease and most other types of cancer
Chronic liver diseases, which progress to cirrhosis, and hepatocellular carcinoma (HCC), include Hepatitis B virus (HBV), Hepatitis C virus (HCV), alcohol-related liver disease (ARLD), non-alcoholic fatty liver diseases (NAFLD) - the latter attributed to obesity and type 2 diabetes mellitus (T2DM)[2, 3]
The same optimized protocol was applied to the COS7 primate kidney fibroblast cell line (Figure 1, middle row)
Summary
The global incidence and mortality from liver cancer have continued to increase, despite advances in treatments for liver disease and most other types of cancer. In 2020, there were more than 9,00,000 new diagnoses, accounting for 4.7% of total cancer cases worldwide[1 ] This is disappointing, given that the significant risk factors for the development of HCC, the most common form of liver cancer, are well characterised[2 ]. The in vitro cell line models remain a good option for scientists working with limited resources, with a lesser need to have a constant supply of fresh human tissues[10 ]. These models provide a tool that can be used as a first screen to help with target validation of drug selection before proceeding to more complex in vivo models. The recent modification of the traditional 2D monolayer cultures into 3D cultures has improved the efficacy of these in vitro models ,
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