Fig. 1
Sulfarotene selectively targets HCC TRCs. a Inhibition of colony spheroid formation in TRCs. Upper panel, the representative images of HCC TRC colony spheroids on day 5 (treatment day 4). Middle panel, quantitative analysis of time-dependent changes of spheroid sizes. Down panel, change of spheroid size on day 5 (n = 3). b Apoptotic effects of sulfarotene at 1.0, 5.0 and 10 µM on HCC cell derived TRCs were determined by flow-cytometry with Annexin V and propidium iodide (PI) double staining, compared with DMSO (0.1 % DMSO-containing medium), 10 µM ACR, and 10 µM sorafenib on treatment day 2 (left) (n = 3). Right, bar graph presentation for the percentage of apoptotic cells based on flow-cytometry analysis. c The IC50 values of sulfarotene, ACR and sorafenib for HCC TRCs were determined in the CCK8 assay after treatment for 48 h (n = 3). d Representative immunofluorescence images for the changes of Ki-67 (red) and Caspase-3 (green) in the HCC TRCs in response to treatment with 10 µM each of sulfarotene, acyclic retinoid and sorafenib for 48 h as compared to parental cancer cells. e, f Inhibitory effects of sulfarotene on growth and xenograft tumor node formation of Hep3B-TRCs subcutaneously transplanted to the flanks of BALB/c nude mice (n = 6). Five days after transplantation of TRCs, mice were treated with sulfarotene at 0.22 and 2.2 mg/kg compared to ACR at 1.8 mg/kg and the DMSO carrier. g, h Inhibition of lung metastasis in mice produced by sulfarotene compared to ACR. Hep3B-TRCs were injected through the tail vein into mice. After 5 days, 0.22 and 2.2 mg/kg sulfarotene and 1.8 mg/kg ACR in 0.1 % DMSO (vehicle control) in PBS were injected every 2 days for 25 days. Left, representative bioluminescence images of lung metastasis after treatment for 25 days. Right, HE staining of lung sections with TRC-derived metastasis foci (inset). Tukey’s post hoc test. SFT, sulfarotene; sora, sorafenib; ACR, acyclic retinoid. Data are presented as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant