Supplementary Figure S1. a.a. days Cell number Doubling time =14 hours CT26CT26GFP isotype CXCR4 isotype CXCR7 isotype CXCR4CXCR7 FSC b.b. Supplementary Figure S1. Characterization of GFP gene labelling on CT26 cells and expression of receptors for CXCL12. a. Isolation and characterization of CT26GFP from a single-cell clone. The doubling time of CT26GFP cells was similar to that seen in parent CT26 cells. b. FACS analyses showing the low expression of CXCR4 and intermediate expression of CXCR7 on CT26 cells. Supplementary Figure S2. a. b. c. Supplementary Figure S2. Role of hyaluronidase receptor CD44, integrins and collagens on spheroid formation of murine (a-c) and human (d) colon cancers. a. FACS analyses showing the high expression of CD44 and integrin 1 and 5, but not 1, 2, and 4, on CT26 cells. b. Sphere formation assay in vitro for CT26 cells under fresh-RPMI and PLCF-RPMI. Neither hyaluronidase nor anti-CD44 neutralizing antibody affected the spheroid formation. c. Sphere formation assay in vitro for CT26 cells under anti-integrin 1(anti- 1), anti-integrin 4(anti- 4), anti-integrin 5(anti- 5) neutralizing antibodies, and their combination. Their corresponding control antibodies were also involved. No effect was observed. d. PLCF-facilitated spheroid formation of 4 different human colon cancer cell lines is sensitive to collagenase. EDTA was used as positive control. hyaluro- nidase hyaluro- nidase ( - ) anti-CD44 Isotype IgG 1h 2h 3h 4h 5h 6h 16h 24h 48h 72h Hours after floating culture fresh -RPMI PCLF -RPMI ( - ) anti- 1 IgM (isotype) anti- 4 IgG (isotype) anti- 5 IgG + IgM (isotype) anti- 5 anti- 1 d. DLD-1 LoVo Colo-201 SW620 RPMI 5mM EDTA collage- nase hyaluro- nidase a. b. Cell count CXCR4 DMSO (solv.) HIF-inhibitor PBS DMSO (solv.) HIF-inhibitor PBS isotype Normoxia 21% O 2 Hypoxia 2% O 2 CXCR4 expression (monolayer) Cell count CXCR4 pFN cFN pFN + cFN PBS isotype Cell count CXCR4 (-) 8 nM 10 M isotype CXCR4 expression (sphere) FAK inhibitor ) Supplementary Figure S3. Relative expression 24h / 0h (normalized to -tubulin) Average S.E. n=3, each *P