Figure 1: The tumour-initiating properties of α2δ1⁺ HCC cells.

(a) Phase contrast micrographs demonstrate that FACS-sorted α2δ1⁺ Huh7 cells are able to form more primary (1°) and serially passaged (2°) spheroids as compared with α2δ1⁻ counterparts. Scale bar, 250 μm. (b) Histograms show the primary (1°) and serially passaged (2°) spheroid formation efficiency of the α2δ1⁺ cells as compared with α2δ1⁻ cells sorted from indicated sources. Bars are the mean±s.d. of three independent experiments (n=6). * Student’s t-test. (c) Representative photograph showing tumour formation in NOD/SCID mice was restricted to the α2δ1⁺ cell population (red arrow). No tumour growth was usually observed on injection of α2δ1⁻ cells (black arrow) on the opposite flanks of the same animals. (d) Representative images showing the dissected tumours formed by sorted α2δ1⁺ Huh7 cells. A total of three mice per group were transplanted. Scale bar, 2 cm. (e) The histology of tumours formed by α2δ1⁺ cells purified from a primary HCC tissue (Case-2) and Huh7 cell line was compared with that of original patient tumour and parent Huh7 tumour, respectively, by HE staining. Scale bar, 100 μm. (f) qRT–PCR analysis of α2δ1 mRNA levels in HCC tissues and paired normal tissues adjacent to tumours. Horizon lines indicate the median values of each group. (g,h) Kaplan–Meier curves for the disease-free survival (DFS) and overall survival (OS) of 85 HCC patients were compared between groups with high and low levels of α2δ1 mRNA in HCC tissues, which were divided according to the cutoff of 1.26, the median value of α2δ1 relative to GAPDH mRNA.