Fig. 1: Tumor-infiltrating VAT boosts the metastatic potential of CR-CSphCs.

a Forest plot of survival changes in high (>30, obesity) versus low (≥18.5 and <25, lean) BMI CRC patients. Data represent the risk ratio ± 95% CI. Statistical significance was calculated by a Random-effect meta-analysis model. b Kaplan–Meier of progression-free survival (PFS) curve in a cohort of 511 CRC patients, based on BMI status. Healthy weight indicates 18,5<BMI < 30, and obesity BMI > 30. Statistical significance was calculated using the log-rank (Mantel–Cox) test. c H&E analysis and CDX2 expression on primary and liver metastasis in CRC patients with healthy weight or affected by obesity. Black arrow heads indicate tumor-infiltrating adipose cells. Li: liver; T = tumor. d Immunohistochemical analysis of CD34 (brown color), CD31 (green color), and CD45 (red color) in tissues as in c. For c, d one representative of 9 independent experiments is shown. e Phase-contrast analysis of CMS2 cells (CSphC #9) treated with medium or V-ASC CM. For (c–e) scale bars, 100 µm. One representative of three independent experiments is shown. f ELDA software analysis of the clonogenic activity in CMS2 CR-CSphCs following treatment with medium or V-ASC CM. g Clonogenic assay of CMS2 CR-CSphC lines TOP–GFP^high and TOP–GFP^low (15% highest/lowest TOP–GFP levels) treated with medium or V-ASC CM. For (f–g) statistical significance was calculated using the two-tailed t test and data are mean ± standard error of three independent experiments performed with CR-CSphCs isolated from three different CRC patients (CSphC #8, #9). h Percentage of TOP–GFP positive cells, in CMS2 cells treated with medium or V-ASC CM (left panel). Box plots show min-to-max values, with line indicating the mean value. Flow cytometry analysis of TOP–GFP (black color indicates Wnt^- cells; green color scale indicates low, intermediate, and high Wnt⁺ cells) (right panel). Statistical significance was calculated using the paired two-tailed t test. Data are mean ± standard error of independent experiments performed with different CR-CSphCs (#1, #4, #5, #8, #9, #11, #21). i Number of mouse tumor xenografts generated by subrenal capsule injection of 10, 100, 1000, or 10,000 CR-CSphCs, alone or in combination with 50,000 V-ASCs (upper panel). Percentage of cancer-initiating cell (CIC) and its fold increase of cells (lower panels). Data are mean ± standard error (95% confidence interval) of 12 independent experiments performed with CR-CSphCs injected as described above. Statistical significance was calculated by ELDA software (http://bioinf.wehi.edu.au/software/elda/). j In vivo imaging and CK20 immunohistochemistry analysis of xenograft tumor formation obtained by subrenal capsule injection of 100 CR-CSphCs alone or together with V-ASCs at the indicted time points. Photon signal of all metastatic sites (kidney, liver, and lungs) at 12 weeks. A yellow dotted line indicates a tumor xenograt lesion. Tumor (T), kidney (K), liver (Li), and lung (Lu) are indicated. One representative of 12 independent experiments is shown. Scale bars, 100 µm.