Patient-derived multicellular tumor spheroids towards optimized treatment for patients with hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and has poor prognosis. Specially, patients with HCC usually have poor tolerance of systemic chemotherapy, because HCCs develop from chronically damaged tissue that contains considerable inflammation, fibrosis, and cirrhosis. Since HCC exhibits highly heterogeneous molecular characteristics, a proper in vitro system is required for the study of HCC pathogenesis. To this end, we have established two new hepatitis B virus (HBV) DNA-secreting HCC cell lines from infected patients.

Methods: Based on these two new HCC cell lines, we have developed chemosensitivity assays for patient-derived multicellular tumor spheroids (MCTSs) in order to select optimized anti-cancer drugs to provide more informative data for clinical drug application. To monitor the effect of the interaction of cancer cells and stromal cells in MCTS, we used a 3D co-culture model with patient-derived HCC cells and stromal cells from human hepatic stellate cells, human fibroblasts, and human umbilical vein endothelial cells to facilitate screening for optimized cancer therapy.

Results: To validate our system, we performed a comparison of chemosensitivity of the three culture systems, which are monolayer culture system, tumor spheroids, and MCTSs of patient-derived cells, to sorafenib, 5-fluorouracil, and cisplatin, as these compounds are typically standard therapy for advanced HCC in South Korea.

Conclusion: In summary, these findings suggest that the MCTS culture system is the best methodology for screening for optimized treatment for each patients with HCC, because tumor spheroids not only mirror the 3D cellular context of the tumors but also exhibit therapeutically relevant pathophysiological gradients and heterogeneity of in vivo tumors.

Keywords: Hepatocellular carcinoma (HCC); MCTS-based chemosensitivity assays; Multicellular tumor spheroids (MCTS); Optimized treatment.

Fig. 1

Characterization of primary HCC cells. a The morphology of AMC-H1 and AMC-H2 HCC cells in monolayer culture condition. b AFP, albumin, and HepPar-1 immunostaining of AMC-H1 and AMC-H2 cells to examine the cellular origin of primary HCC cells. c AFP and albumin mRNA expression levels in AMC-H1 and AMC-H2 HCC cells. d Expression levels of EGFR, β-catenin, N-cadherin, p53, PTEN, ERK and Akt in AMC-H1 and AMC-H2 cells were measured by western blot analysis. Activation of signaling pathways was examined by measuring phosphorylation of ERK and Akt. e Spheroid cultures of AMC-H1 and AMC-H2 cells in stem cell permissive medium for 5 day. The expression level of each molecule was quantitated by densitometric analysis. f CD133 immunostaining of AMC-H1 and AMC-H2 cells. g Dose response curve of AMC-H1 and AMC-H2 cells treated with 5-FU, cisplatin, and sorafenib at the indicated concentrations for 48 h under monolayer culture conditions. Bright field images were obtained using a Zeiss microscope. The fluorescence images were obtained and analyzed using the Operetta® High Content Screening (HCS) system. Data are shown as the mean ± SD of triplicate experiments. Scale bar = 200 μm

Fig. 2

Kinetics and drug sensitivity of tumor spheroids from primary HCC cells. a Kinetics of tumor spheroids from AMC-H1 and AMC-H2 cells at the indicated times. Cells were cultured in 96-well ultra-low attachment (ULA) plates for 96 h. b Drug sensitivities in tumor spheroids of AMC-H1 and AMC-H2 HCC cells. Cells were cultured in 96-well ULA plates for 3 day and then treated with 5-FU, cisplatin, and sorafenib at the indicated concentrations for 7 day. Then, tumor spheroids were stained with ethidium homodimer-1 (EthD-1; left panel), and the staining intensity was analyzed (right panel). All images were obtained and analyzed using the HCS system. Data are shown as the mean ± SD of triplicate experiments. Scale bar = 200 μm. *p < 0.05, **p < 0.005

Fig. 3

MCTS formation and drug sensitivity of primary HCC cells. a Schematic of MCTS model representing the in vivo microenvironment. b Morphology of tumor spheroids and MCTS of AMC-H1 and AMC-H2 HCC cells. c Drug sensitivities in MCTS of AMC-H1 and AMC-H2 HCC cells grown with hepatic stellate cells (HSC; LX2), fibroblasts (WI38), and endothelial cells (HUVECs). Cells were cultured in 96-well ULA plates for 3 day and then treated with 5-FU, cisplatin, and sorafenib at the indicated concentrations for 7 day. Then, MCTS were stained with EthD-1 (upper panel), and the staining intensity was analyzed (lower panel). d EthD-1 intensity of tumor spheroids and MCTS composed with AMC-H1 or AMC-H2 after treatment with the indicated drugs. All images were obtained and analyzed using the HCS system. Data are shown as the mean ± SD of triplicate experiments. Scale bar = 200 μm. *p < 0.05, **p < 0.005