Transcript b 26 o
CT26 CT26GFP Doubling time =14 hours b. 1.00E+11 Cell number a. FSC Supplementary Figure S1. 1.00E+09 isotype 1.00E+07 CT26GFP CT26 1.00E+05 0 5 10 CXCR4 isotype CXCR4 CXCR7 isotype CXCR7 15 days 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. Hours after floating culture fresh -RPMI 1h 3h 4h 5h 6h 16h (-) 24h 48h 72h anti-b1 (-) hyaluronidase (-) PCLF -RPMI 2h IgM (isotype) anti-a4 hyaluronidase IgG (isotype) anti-CD44 Isotype IgG anti-a5 anti-a4 + -a5 anti-a4 + -a5 + -b1 IgG + IgM (isotype) d. RPMI DLD-1 LoVo Colo-201 SW620 5mM EDTA collage- hyaluronase nidase 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 b1 and a5, but not a1, a2, and a4, 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 b1(anti-b1), anti-integrin a4(anti-a4), anti-integrin a5(anti-a5) 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. Supplementary Figure S3. b. CXCR4 expression (monolayer) CXCR4 expression (sphere) DMSO (solv.) HIF-inhibitor Hypoxia 2% O2 PBS DMSO (solv.) HIF-inhibitor Normoxia 21% O2 PBS isotype Cell count ) FAK inhibitor pFN cFN pFN + cFN PBS isotype Cell count Cell count (-) 8 nM 10 mM isotype CXCR4 CXCR4 (-) CoCl2 3.55% 47.7% CoCl2 + NAC 5.19% Isotype 0.12% monolayer sphere monolayer d. sphere sphere + NAC isotype Serum 10% Serum free Sphere CXCR4 c. Cell count CXCR4 IGF-1Rb a. CXCR4 Forward scatter e. Supplementary Figure S3. PAX3 f. YY1 NF-kB GC-boxes NRF-1 TATA0 box Relative expression 24h / 0h (normalized to α-tubulin) -2,237 HRE 10 8 0h 6 24h 4 2 0 Average ± S.E. n=3, each *P<0.01 * a-d. Each experiment was done more than three times, and showed similar results. a. Focal adhesion kinase (FAK), a downstream regulator for integrins, is not a candidate for spheroidal expression of CXCR4. b. CXCR4 was not inducible on CT26 cells in response to either FNs (left panel), cultivation under a hypoxic condition, or the use of an inhibitor for hypoxia-inducible factor (HIF). c. The stressor CoCl2, a chemical that induces hypoxic responses, induces CXCR4 on the CT26 monolayer that can be inhibited by a radical scavenger NAC (left panels), but NAC can not inhibit the spheroid-induced CXCR4 expression (right panel) d. FACS analyses showing the upregulation of insulin-like growth factor-1 receptor (IGF-1R) and CXCR4 on a CT26 monolayer under a serum-starvation. e. and f. Schematic structure of the 5’-flanking region of CXCR4 (Tarnowski et al., 2010) (a) and mRNA expression of each transcription factor and CXCR4 at 0 h (monolayer) and 24 h after floating cultivation as assessed by quantitative real-time RT-PCR (b). Supplementary Figure S4. a. en face observation Cell count Cell count monolayer sphere isotype e. CXCR4 CXCR4 LoVo -GFP Hours after floating culture d. 0h 1h 3h 6h 9h 12h 24h 48h HCT116 PCLF PCLF + Coll-L LoVo - PCLF PCLF + Coll-L f. LoVo -GFP HCT116 -CFSE cross section HCT116 LoVo c. b. HCT116 -CFSE 7 days after tumor inoculation untreated AMG3100 Mithramycin A Supplementary Figure S4. (continued) Supplementary Figure S4. Identification of SCF+/CXCL12+ niche-like cell sheets, accelerated sphere formation, and directional tumor metastasis in human mesentery. Each experiment in a-d was performed three or more times with similar results. a and b. Representative en face (A) and cross section (B) findings of human mesentery samples obtained from patients who had undergone colectomy. a: Thin mesenteric surface sections, approximately 2-mm thick, were prepared using a surgical knife (left panel), and placed on the slide glass with adhesive followed by observation with a dissecting microscope (middle panel). These sections were immunohistochemically stained with SCF (red) and counterstained with DAPI (blue). Note that the red dots (white arrows) were mainly located on or around perivascular adipose tissue (yellow bipolar arrow). b: Cross sections identifying the localization of CAR cells. Doubly positive CAR cells (green: CXCL12; red: SCF) were frequently seen at the border area of perivascular adipose tissue (white arrows). c. Upregulation of CXCR4 expression of HCT116 by sphere formation. LoVo and HCT116 cells were maintained by monolayer (blue line) or floating (red line) culture for 7 days, and these cells were propagated and CXCR4 expression was examined by FACS analyses. Note that spontaneous expression of CXCR4 was seen in the monolayer of LoVo cells, but not in that of HCT116 cells, and CXCR4 expression was seen in both cell lines by sphere formation (arrow in sphere of HCT116). d. Mouse PCLF facilitated sphere formation of human colon cell lines in vitro. Floating cultivation of each tumor was done in fresh medium (without PLCF) as well as in medium that was used after intraperitoneal irrigation of nude mice (with PLCF). e. Mesenteric dissemination of LoVo and HCT116 was AMG3100- and mithramycin A-sensitive. Seven days after inoculation of LoVo-GFP or HCT116-CFSE into the peritoneal cavity of nude mice, the mesentery was subjected to analysis with a fluorescent microscope. Note that dissemination was mainly seen at the margin of the perivascular adipose tissue (panels, inset A and B), which was similar to the results using CT26 cells. These disseminations were significantly abrogated by AMG3100 or mithramycin A. f. SCF+-cell sheet-targeted dissemination of LoVo-GFP and HCT116-CFSE cells was assessed in an organ culture of the human mesentery samples. (panels) En face findings of the fluorescent dissecting microscopy. LoVo-GFP (the four upper panels) and HCT116-CSFE (the four lower panels) tumors (green) were observed on the SCF-positive cell sheet (red). (right graph) Percentage of tumor nodules on the SCF-positive cell sheet. Supplementary Figure S5. b. a. Hours after floating culture 0h PCLF Cell count Monolayer Sphere Isotype 1h 2h 3h 4h 5h 6h 9h 12h 24h 48h (-) (+) CXCR4 c. untreated Supplementary Figure S5. Sphere-induced spontaneous overexpression of CXCR4, acceleration of sphere formation by PCLF, and CXCR4-dependent peritoneal Dissemination is common mechanisms for HRA human ovarian cancer cells. a and b were done in triplicate, and showed similar results. Inset CFSE Inset nodules/ vascular loops AMD3100 10 Mithramycin *P<0.01 5 * * 0 untreated AMD3100 Mithramycin a. Detection of the upregulation of CXCR4 expression of HRA via spheroid formation in vitro. Twenty-four hours after floating culture, the spheroids were subjected to FACS analysis after propagation with EDTA (5 mM). b. Sphere formation assay in vitro for HRA ovarian cancer cells under fresh-RPMI and PLCF-RPMI. c. Effect of AMD3100 and mithramycin A on HRA ovarian cancer cell dissemination. Seven days after drug pretreatment andCFSE-labelled tumor cell intraperitoneal inoculation to nude mice, the mesenteric nodules were counted under the fluorescent microscope. Mesenteric metastasis of HRA cells was significantly inhibited by the pretreatment with AMD3100 or mithramycin A. Supplementary Figure S6. (key events) (key molecules) Supplementary Figure S6. Schematic representation of the whole molecular and cellular mechanisms of directional tumor dissemination as revealed in this study. After penetrating into the peritoneal cavity, which is rich with Coll-IV and pFN, through the serosa, tumor cells start to form some cell cluster by incorporating these ECMs, and then grow into spheroids within 6 h. Then, the Sp1 transcription factor begins to be activated and to upregulate CXCR4 in spheroids, which are mainly directed to the margin of the mesenteric perivascular adipose tissue to the SCF+/CXCL12+ niche-like cells to form disseminated tumor nodules. Supplementary Figure S7. adhesion molecules（E-cad, ZO-1, etc） → forming ‘shell’ on the sphere surface drug resistance hypoxia (HIF-1, etc) angiogenesis (VEGF, etc.) starvation (IGF-1, etc) anti-apoptosis (Mcl-1, etc) anaerobic metabolism (lactate, etc) invasive growth (uPA/uPAR, etc.) inflammatory reaction (NF-kB, etc) others - gain stem cell properties - Sp1 activation drug resistance CAR cell-niche directed tumor dissemination Supplementary Figure S7. Summary of biological events after the formation of cancer spheroids. Soon after forming spheroids, cancer cells start to express several adhesion molecules, including E-cadherin as well as ZO-1, to form ‘shell’ on the sphere surface, possibly resulted in drug resistance. Subsequently, based on several studies in the literature, inner cells demonstrate dynamic changes, including hypoxia (activation of HIF-1, etc.), starvation (expression of IGF-1R, etc.), anaerobic metabolism (accumulation of lactate, etc.), and inflammatory reactions (activation of NF-kB), causing possible angiogenic, antiapoptotic, and invasive properties via inducing VEGF, Mcl-1, and uPA/uPAR. Importantly, spheroid formation also facilitate cancer cells to gain stem cell-like properties, including upregulation of multi-drug resistant genes, as well as Sp1 activation causing niche-directed tumour dissemination, as revealed in this study.