Supplementary Figure 2

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2 Supplementary Figure 2 ** ** ** * * ** ACTA2 CD34 CSPG4 NCAM TGF-b1 VEGFa Fold-change variation relative to normal tissue

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8 Piera Balzarini et al Supplementary Table 1 Pathological features of HCC biopsies Patients Gender Age Diagnosis Tumor Hcc Vascular Presence of Size (Cm) grading invasion Vascular Abnormalities* 1 F 72 HCC 2.5x1.5 G2 no yes 2 F 76 HCC 4.2x3.6 G2 yes yes 3 M 69 HCC 5.5x4.5 G2 yes yes 4 M 64 HCC 3.8x3.5 G2 yes yes 5 F 65 HCC 1.5x1.5 G2 no yes 6 M 78 HCC 3.0x2.7 G1-G2 no yes 7 M 72 HCC 5.3x4.5 G2 yes yes 8 M 68 HCC 4.5x3.5 G2 no yes 9 M 73 HCC 2.5x2.5 G2 no yes 10 M 68 HCC 4.0x3.3 G2 yes yes 11 M 64 HCC 4.2x3.0 G2 no yes 12 M 68 HCC 4.5x4.5 G2 yes yes Pathological features of 12 HCC biopsies of HCC patients. All the HCC biopsies showed a similar high grade (G2) of malignancy. Tumor size was calculated as the maximal tumor diameter of the primary lesion. In 10 out of 12 patients the nnl counterpart showed the presence of an active cirrhotic process, with the remaining two patients showing chronic liver inflammation with a diffuse steatosis. The MVD was 96±43 and 42±17 in HCC and nnl biopsies respectively. * Vascular abnormalities included: arterialization of tumor-infiltrating capillaries, sinusoid capillarization, vascular tortuosity, vascular rupture, and capillaries leakage.

9 Supplementary Table 2 Quantitative expression of MC markers on nnl and HCC specimens Biopsies liver area* % of positive cells αsma NG2 NCAM/CD56 parenchyma absent 3±2 17±7 nnl stroma absent 25±8 34±9 vessels 89±6 45±8 58±11 parenchyma 10±5 87±8** 45±7* HCC stroma 18±8 7±5 absent vessels 87±12 13±7 absent After immunohistochemical studies of HCC and nnl specimens, the same liver specimens were also investigated to determine a quantitative expression of MC markers NG2. NCAM and αsma. HCC and nnl counterpart specimens were stained with avidine-biotin peroxide staining and then we calculated the percentage of MC markers expression in three different liver specimen area: parenchyma, stroma and vessels. Note the high percentage of NG2 and NCAM positive cells mostly only in HCC parenchyma (*p< 0.05, **p<0.01 versus nnl parenchyma), while positive cells were very low, or absent in vessels ( p<0.01 versus nnl-vessels). The numbers in the table indicate the percentage of positive cells for each marker on the total cells counted in at least 10 different fields (40x magnification).and are means ± SD. Five different nnl and the corresponding HCC preparations were analyzed.. * the different liver specimen areas were define by histological examination. absent indicates no positive cells to specific antibodies staining were found in the corresponding area.

10 Piera Balzarini et al Supplementary Table 3 Isolation and expansion of nnl-stc and HCC-StCs Case Specimens Successful Growth index* weight (grs.) expansion (times of cell increment) nnl HCC nnl-stcs HCC-StCs nnl-stcs HCC-StCs yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes no 4.5 n.d yes no 2.7 n.d Successful culture expansion of HCC-StCs and their adjacent nnl-stcs counterpart was obtained in 10 out of 12 patients. *. Numbers indicate the times of cell increment for each StCs preparation after 7 days of culture. The initial cells number seeded on day 0 was 10 5 /T25 Flask. Note that most of the nnl-stcs cultures had a higher growth rate than the HCC-StCs counterpart. Indicates the case in which we could not expand HCC-StCs in culture n.d = not determined.

11 Supplementary Table 4 Expression of MC and mesenchymal markers on cultured Ad-StCs, nnl-stcs and HCC-StCs Markers % of positive cells Ad-StCs nnl-stcs HCC-StCs αsma 97±2 65±8 31±11* NG2 36±9 24±12 5±7* NCAM 43±12 26±13 13±18* TGF-b1 74±15 56±16 23±11** CD44 98±2 67±18 61±22 CD105 96±3 84±13 62±25 CD49d/VLA-4 56±14 43±13 15±9** Ki67 33±8 18±4 6±8* A quantitative evaluation of the expression of MC and mesenchymal markers was performed by counting the positive cells after avidine-biotin peroxide staining with the corresponding antibodies. The numbers in the table indicate the percentage of positive cells on the total cells counted and are the means ± SD for each marker expression in at least 10 different fields (40x magnification). Five different preparations for each kind of StCs were counted. * p<0.05, ** p<0.01 compare to marker expression on nnl-stcs;