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HEP_26257_sm_SuppFig1.tif4524KSupporting Information Figure 1. Characteristics and differentiation potential of LN-MSCs derived from adjacent non-tumor tissues. (A) Representative morphology of LN-MSCs. Magnification: x 40 and x 100. After osteogenic-specific induction, the LN-MSCs showed strong ALP positive staining, and lots of calcium deposition in the extracellular matrix, which was verified by von Kossa staining, x 100. After the adipogenic differentiation induction, they showed many small lipid vacuoles via Oil red O staining, x 100. (B) Flow cytometric analysis showed cells were positive for mesenchymal lineage markers (CD29, CD73, CD166, CD90 and CD105), negative for hematopoietic and endothelial markers (CD45, CD14, CD144 and CD31), and negative for HLA-DR.
HEP_26257_sm_SuppFig2.tif5562KSupporting Information Figure 2. LC-MSCs promoted HepG2 and SMMC-7721 proliferation in vivo. (A) Subcutaneous grafting assays demonstrated that tumors formed by HepG2 (3×106) mixed with LC-MSCs (1×107) were significantly heavier than HepG2 alone, the tumor weight of HepG2 alone was 0.282±0.211g, and HepG2 with LC-MSCs was 0.584±0.232g, *P<0.05. (B) Similar tumor-promoting results were obtained from SMMC-7721 cells co-injected with LC-MSCs. The number of SMMC-7721 cells and LC-MSCs used was 5×106. The tumor weight of SMMC-7721 alone was 0.231±0.077g, and SMMC-7721 co-injected with LC-MSCs was 0.513±0.146g, **P<0.01.
HEP_26257_sm_SuppFig3.tif3958KSupporting Information Figure 3. Immunohistochemistry staining of HCC tissues and adjacent non-tumor tissues with rabbit polyclonal S100A4 antibody. (A) Representative results of immunohistochemistry staining of S100A4 on 95 HCC tissues and 41 adjacent cancer-free tissues in tissue array were shown. (B) The difference of S100A4 expression between the HCC tissues and adjacent cancer-free tissues was highly significant (***P<0.001, Fisher's exact test). Among 41 adjacent cancer-free tissues, 26.8% (11 of 41) were positive for S100A4 but only in stromal cells, and 73.2% (30 of 41) were negative for S100A4 expression. In contrast, 84.2%, (80 of 95) were positive for S100A4 staining in HCC tissues, and 15.8% (15 of 95) were negative for S100A4 expression in HCC tissues. (C) In 95 HCC samples, 70.5% (67 of 95) were positive for S100A4 expression in stromal cells, 8.4% (8 of 95) were both positive for S100A4 in stromal and HCC cells, 5.3% (5 of 95) were only expressed in HCC cells, 15.8% (15 of 95) were negative in stromal cells and HCC cells.
HEP_26257_sm_SuppFig4.tif1044KSupporting Information Figure 4. S100A4 promoted HCC cell proliferation. (A) S100A4 overexpression significantly promoted SMMC-7721 tumor formation in vivo. (B) Western blotting analysis showed that RNAi suppressed 70%-90% expression of S100A4 in HCC cells. MTT assays indicated that knockdown of S100A4 suppressed Huh7 cell proliferation in vitro.
HEP_26257_sm_SuppFig5.tif3210KSupporting Information Figure 5. miRNAs expression was altered in HCC cells after co-culture with LC-MSCs. Quantitative RT-PCR was used to detect 18 different miRNAs in GFP-positive MHCC97L and Huh7 after co-cultured with LC-MSCs. The results showed many miRNAs were altered by LC-MSCs co-culture. Among them, miR-155 expression was dramatically enhanced in HCC cells after co-culture with LC-MSCs.
HEP_26257_sm_SuppFig6.tif2677KSupporting Information Figure 6. Knockdown of MMP9 expression inhibited the effect of S100A4 on cancer cell invasion. Matrigel assays for invasion of MHCC97H cells (endogenous high levels expression of S100A4) and 7721-S100A4 cells (exogenous overexpression of S100A4), these cells were transfected with MMP9 shRNA or scrambled shRNA, respectively. The results showed that knockdown of MMP9 expression suppressed the invasive-promoting effect of S100A4 in HCC cells, x 200.
HEP_26257_sm_SuppFig7.tif1011KSupporting Information Figure 7. A schematic diagram illustrating how LC-MSCs can modulate HCC progression through S100A4-miR-155-SOCS1-STAT3 -MMP9 axis.
HEP_26257_sm_SuppTab1.doc55KSupporting Information Table 1. The expression of different trophic factors from LC-MSCs and LN-MSCs of patient 8 by cDNA array analysis.
HEP_26257_sm_SuppTab2.doc131KSupporting Information Table 2. S100A4 expression in 95 clinical HCC samples.
HEP_26257_sm_SuppTab3.doc70KSupporting Information Table 3. S100A4 expression in 41 adjacent cancer-free liver tissues.
HEP_26257_sm_SuppTab4.doc40KSupporting Information Table 4. The primer sequences of human miRNAs for real-time PCR.

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