Figure S1 to S22 from Cancer-Stimulated CAFs Enhance Monocyte Differentiation and Protumoral TAM Activation via IL6 and GM-CSF Secretion
Authors: Cho Haaglim , Seo Youngha , Loke Kin Man , Kim Seon-Wook , Oh Seong-Min , Kim Jun-Hyeong , Soh Jihee , Kim Hyoen Sik , Lee Hyunju , Kim Jin , Min Jung-Joon , Jung Da-Woon , Williams Darren Reece 
Published: April 2023

<p>Supplementary Figure S1. (A) Immunofluorescence staining of DAPI and Î±-SMA expression in primary human normal fibroblasts (NF; passage<5) and three different human cancer-associated fibroblasts (CAF1, CAF2, and CAF3). Scale bar=100 Î¼m. Bar chart shows quantification of Î±-SMA expression. n=4; error=SD. (B) Invasion of YD-10B human OSCC cells cocultured with human normal fibroblasts (NF) or three different primary CAFs (CAFs 1, 2, and 3). Scale bar=100 Î¼m. n=3; error=SD. (C) ELISA for human IL-6 and GM-CSF in the culture media from YD-10B-stimulated fibroblasts (cell types: human normal fibroblasts (NF) vs three different primary human CAFs). n=4; error=SD. ***=p<0.001. /// Supplementary Figure S2. q-PCR analysis of STAT1 and STAT3 expression in THP-1 human monocytes treated for 72 h with 200 nM PMA or the CM from CAFs after activation by culturing with a 1:20 dilution of YD-10B OSCC CM. n=3; error=SE; ns=not significant; ***=p<0.001; ###=p<0.001. /// Supplementary Figure S3. The log fold-change in expression of the human cytokine array for CAF culture supernatant and CAF culture supernatant with YD-10B cancer CM stimulation (cancer-stimulated CAFs vs CAFs). n=3; error=SE. Diamond=greater than 5 fold-increase in cytokine expression. /// Supplementary Figure S4. Human cytokine array for untreated CAFs or CAFs treated with IL-1Î± (50 pg/mL) for 24 h. Cytokines upregulated in cancer-stimulated CAFs are indicated with red boxes. The log fold-change in expression is shown in the graph. Diamond=greater than 5 fold-increase in cytokine expression. /// Supplementary Figure S5. (A) Concentration-dependent effect of IL-7, CXCL5, GM-CSF or IL-6 on THP-1 differentiation into adherent macrophages. Cells were treated with cytokines for 72 h. n=3; error=SD. (B) Representative photographs of THP-1 monocyte differentiation into macrophages after treatment (micrograph scale bar = 10 Î¼m). ns=not significant; *=p<0.05; **=p<0.01; ***=p<0.001. /// Supplementary Figure S6. Time course study of the effects of 12.5 ng/mL GM-CSF and/or 12.5 ng/mL IL-6 on THP-1 differentiation into adherent macrophages. n=3; error=SD. *=p<0.05; **=p<0.01; ***=p<0.001 /// Supplementary Figure S7. (A) q-PCR analysis of CD68 (pan macrophage marker), IL-12p40, iNOS (M1 markers), CD206, Arg-1 and TGF-Î² (M2 markers) in macrophages differentiated from THP-1 monocytes after treatment with 12.5 ng/mL IL-6, 12.5 ng/mL GM-CSF or 12.5 ng/mL IL-6 + 12.5 ng/mL GM-CSF for 72 h. n=3; error=SE. (B) Flow cytometry analysis of CD86 (M1 marker), CD11b (M1/M2 marker) and CD206 (M2 marker) expression in CD14+ human PBMC after treatment 12.5 ng/mL IL-6, 12.5 ng/mL GM-CSF or 12.5 ng/mL IL-6 + 12.5 ng/mL GM-CSF for 72 h. The populations of CD11b+CD86+ CD206- and CD11b+ CD86- CD206+ cells are shown as blue and red bars on the bar chart, respectively. n=3; error=SD. ns=not significant; *=p<0.05; **=p<0.01; ***=p<0.001; #=p<0.05; ##=p<0.01; ###=p<0.001. /// Supplementary Figure S8. q-PCR analysis of Il-10 and Arg-1 (M2 markers), Il-12p40 and iNOS (M1 markers), and F4/80 (murine macrophage marker) in BMDM treated with GM-CSF and IL-6. The Arg-1/iNOS ratio was also calculated. Marker expression in M1 and M2 macrophages are included for comparison. n=3; error=SE. ns=not significant; *=p<0.05; **=p<0.01; ***=p<0.001; #=p<0.05; ##=p<0.01; ###=p<0.001. Â¶Â¶=p<0.01; Â¶Â¶Â¶=p<0.001; Ò-=p<0.05; Ò-Ò-=p<0.01; Ò-Ò-Ò-=p<0.001. /// Supplementary Figure S9. q-PCR analysis of Stat1 and Stat3 expression in BMDM treated with 12.5 ng/mL GM-CSF and/or 12.5 ng/mL IL-6 for 72 h. n=3; error=SE. #=p<0.05; **=p<0.01; ***=p<0.001. /// Supplementary Figure S10. The increased cancer cell invasion was similar to that produced by M2-type macrophages. Scale bar=100 Î¼m. n=3; error=SE. ns=not significant; *=p<0.05; **=p<0.01; ***=p<0.001; #=p<0.05; ##=p<0.01; ###=p<0.001; Ò-Ò-Ò-=p<0.001. /// Supplementary Figure S11. (A) Immunofluorescence staining of DAPI and Î±-SMA expression in primary murine normal fibroblasts (mNFs; passage 3), cancer-associated fibroblasts (mCAFs; passage 4) and CT26 colon carcinoma cells. Scale bar=100 Î¼m. (B) Bar chart showing quantification of Î±-SMA expression. n=3; error=SE. (C) Invasion of CT26 colon carcinoma cells cocultured with normal fibroblasts (NF) or three different primary CAFs lines (mCAFs 1, 2 and 3). Scale bar=100 Î¼m. n=8; error=SE. ***=p<0.001 (compared to untreated control); ###=p<0.001 (compared to mNF). (D) ELISA for IL-6 and GM-CSF in the culture media from CT26 colon carcinoma cells, primary NFs or primary CAFs stimulated for 48 h with CT26 cancer cell CM (1:5 dilution) n=6; error=SD. ***=p<0.001; ###=p<0.001. /// Supplementary Figure S12. Representative photographs of liver and spleen at 6 weeks' post-transplantation (scale bar=1cm). The bar chart shows organ weight. Data are presented as mean {plus minus} SE (n=6) of organ weight. ns=not significant; **=p<0.01; ***=p<0.001; ###=p<0.001. /// Supplementary Figure S13. (A) IVIS-based detection of metastases rate in the transplanted mice. Red rings denote areas of metastases. Metastatic tumors could also be visually observed in the dissected liver tissue (designated with black arrows). (B) Incidence of metastasis in the transplanted mice. n=13 mice/group; error=SE. ns=not significant; *=p<0.05; #=p<0.05. /// Supplementary Figure S14. ELISA for IL-6 and GM-CSF in the tumor tissues. n=6; error=SE. ns=not significant; **=p<0.01; ***=p<0.001; ###=p<0.001. /// Supplementary Figure S15. Immunofluorescence staining for CD68 (pan-macrophage marker) and CD206 (M2 marker) in the primary tumor tissue (scale bar=100 Î¼m). The rate of double-stained cells was determined from 10 randomly selected fields of view per section and normalized for double stained cells by DAPI counter staining. The bar chart shows quantification of the merged, double-stained cells. n=5; error=SE. ***=p<0.001; ##=p<0.01. /// Supplementary Figure S16. q-PCR analysis of the effect of 10 Î¼g/mL IL-6 and/or 0.2 Î¼g/mL GM-CSF neutralizing antibodies on CD68, Arg-1, and IL-10 expression in THP-1-derived macrophages. CAFs were stimulated by co-culture with cancer CM for 72 h with or without the antibodies. The supernatant was collected and added to the THP-1 cultures for macrophage induction. 'Untreated' refers to THP-1 culture without CM treatment. n=3; error=SE. ***=p<0.001; #=p<0.05. /// Supplementary Figure S17. Schematic diagram of the protocol to assess the effect of IL-6 and GM-CSF blockade on tumorigenesis in the orthotopic, syngeneic colon carcinoma model. /// Supplementary Figure S18. (A) Primary tumor size in the transplanted mice. n=8 mice/group; error=SE. (B) Representative photographs of the tumor-bearing colon tissue and primary tumors in the treated mice. Scale bar=1 cm.. **=p<0.01; ##=p<0.01; Ò- Ò- =p<0.01. /// Supplementary Figure S19. Schematic diagram of the rate of lung and liver metastasis. /// Supplementary Figure S20. Immunohistochemical analysis of CD86 and CD68 positive cells in the tumor tissue. The rate of double-stained cells was determined from 10 randomly selected fields of view per section and normalized for double stained cells by DAPI counter staining. The bar chart shows quantification of the merged, double-stained cells. n=6; error=SE.; ***=p<0.001. /// Supplementary Figure S21. Effect of 50 Î¼g or 200 Î¼g IL-6 and GM-CSF antibody blockade on tumorigenesis in the orthotopic, syngeneic colon carcinoma model. n=9-10 mice/group; error=SE. (A) Survival graph. (B) Photon flux from cancer cells in the transplanted mice and IVIS images of representative mice at 1, 2 and 3 weeks. (C) IVIS images of representative dissected mice after sacrifice at the end of the experiment and detection of liver metastasis. ns=not significant; *=p<0.05; **=p<0.01; ***=p<0.001. /// Supplementary Figure S22. Schematic diagram of the major findings in this study. Cancer cells activate CAFs to upregulate secretion of IL-6 and GM-CSF, which induce monocyte differentiation into M2-like pro-invasive TAMs. The TAMs facilitate cancer cell invasion, leading to increased metastasis. IL-1Î± from cancer cells further activates CAF to secrete IL-6 and GM-CSF. These positive effects of CAFs and IL-6/GM-CSF on TAMs infiltration into tumors and tumorigenesis/metastasis were confirmed in an orthotopic, syngeneic model of colon carcinoma.</p>