Fig. 2

CRC tumor cells in tumor buds recruit fibroblasts via CCL5. a Heatmap of the differentially expressed proteins in the cytokine array of the CM samples from FHC (owning a weak recruitment ability) and four human CRC tumor cell lines (HCT-8, HCT116, HCT-15, SW620, owning a strong recruitment ability). The cytokine array detected 1000 cytokines. b The differentially expressed proteins in the cytokine were analyzed by GO biological process functional enrichment analysis. c Part of the cytokine array including CCL5 showing the expression of CCL5 in the CM samples from FHC and HCT116, HCT-8, HCT-15, SW620. d Relative expression of CCL5 mRNA in FHC and eight human CRC tumor cell lines. Data, mean ± SD; n = 3. e Supernatant CCL5 levels in the CM samples from FHC and eight human CRC tumor cell lines. Data, mean ± SD; n = 3. f Supernatant CCL5 levels in the CM samples from FHC, CCD-18Co and human primary normal colorectal fibroblasts (NF4, NF5, NF6, NF7). Data, mean ± SD; n = 3. g Representative CCL5 IHC staining images of human CRC tissue at the invasive front without (the left column) or with (the right four columns) tumor budding. The red triangles indicate the tumor buds. Scale bar, 50 μm. h Model diagram of recruitment assay of CCL5 to fibroblasts. i Quantification of cell numbers of CCD-18Co and human primary normal colorectal fibroblasts (NF1, NF4, NF5, NF6, NF7, NF8, NF9) recruited by 40 ng/ml CCL5. Data, mean ± SD; n = 5. j Model diagram of recruitment assay of CCL5 knockdown and overexpressing human CRC tumor cell lines to fibroblasts. k Quantification of cell numbers of CCD-18Co recruited by HCT-8/siCtrl and HCT-8/siCCL5, SW620/siCtrl and SW620/siCCL5. Data, mean ± SD; n = 5. l Quantification of cell numbers of CCD-18Co recruited by RKO/Vec and RKO/CCL5, LS174T/Vec and LS174T/CCL5. Data, mean ± SD; n = 5. m Model diagram of recruitment assay of the CM samples from CCL5 knockdown and overexpressing human CRC tumor cell lines to fibroblasts. n Quantification of cell numbers of CCD-18Co recruited by the CM samples from HCT-8/shCtrl and HCT-8/shCCL5, SW620/shCtrl and SW620/shCCL5. Data, mean ± SD; n = 5. o Quantification of cell numbers of CCD-18Co recruited by the CM samples from RKO/Vec and RKO/CCL5, LS174T/Vec and LS174T/CCL5. Data, mean ± SD; n = 5. ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Student’s t test (d, e, f, i, k, l, n, o)