Establishment of multicellular tumor spheroids (MCTS) as an in vitro model for studying sorafenib resistance in hepatocellular carcinoma (HCC)

PALMA, NF; CHARES, LG; LIVORRE, VI; FERRETTI, AC; LUCCI, A; COMANZO, CG; VERA, MC; ALVAREZ ML; MOTTINO, AD; CARRILLO, MC; QUIROGA, AD; CEBALLOS, MP

Congreso; Reunión Anual de la Sociedad Argentina de Fisiología (SAFIS); 2021

Multidrug resistance counteracts the efficiency of sorafenib (Sfb), an important first-line therapy for HCC, and effectivechemotherapy strategies are still missing and under research. Spheroids provide more reliable results than standard 2D in vitrocell cultures since they mimic features of in vivo tumors. We have shown that EX-527 (EX), a sirtuin 1/2 inhibitor, enhances theinhibitory effect of Sfb in spheroids of HCC cells (HCC-S). Stromal cells in solid tumors contribute to cancer progression andchemoresistance. Aim: to generate MCTS composed of HCC and stroma cells. Methods: MCTS of HCC (HepG2 or Huh7),endothelial (EA.hy926) and hepatic stellate (LX-2) cells were obtained at different ratios (A)1:1:1, B)1:0.6:0.4, C)1:0.3:0.3,D)1:0.1:0.1) by liquid overlay technique and, once generated, morphology (microscopy) and volume (diameter) were evaluated.EA.hy926 and LX-2 were labeled with the CFSE and DiI probes, respectively, to examine their distribution inside MCTS (fluorescentmicroscopy). Proliferation rate was calculated at 72h (volume 72/0h). Viability (APH assay) and proliferation were determined inHuh7 treated with Sfb 8μM or EX 40μM for 72h. HCC-S were used as control. Results: While HepG2 cells formed loose aggregates,HepG2-MCTS formed highly compact and more spherical cell aggregates (50%* less volume than HepG2-S). Huh7-MCTS weresimilar in shape and compactness to Huh7-S, but presented with higher volumes (A)+66%*, B)+47%*, C)+49%*), except for D).Cell tracker dyes confirmed the presence of cancer and stromal cells inside the MCTS. Proliferation rate was similar betweenMCTS and HCC-S from both cell lines, except for A) that grew more slowly (-30%*). Compared with Huh7-S, Huh7-MCTS exhibitedstronger resistance to Sfb (viability: A)+68%*, B)+38%*, C)+42%*); similar volume and EX (viability: A)+218%*, B)+150%*,C)+127%*; volume: (A)+145%*, B)+112%*, C)+91%*), except for D). *p<0.05 vs. HCC-S. Conclusion: MCTS seem to be a better 3Dmodel for studying drug response in the context of cancer-stroma interactions