Article Figures & Data
Figures
- Figure 1.
CAFs induce monocyte differentiation into proinvasive macrophages with M2-like characteristics. A, 6 × 104 THP-1 human monocytes were cocultured with 2 × 104 human OSCC CAFs in a 24 transwell system (pore size, 0.4 μm) or treated with 200 nmol/L PMA (a known inducer of differentiation) for 72 hours. Micrographs indicated that treatment with PMA or coculture with CAFs increased monocyte differentiation into adherent macrophages. Scale bar, 10 μm. The increase in differentiation was quantified by counting cells that adhered to the culture plate. n = 3; error, SD. B, qPCR analysis of CD68 (pan-macrophage marker); IL12p40 and iNOS (M1 markers); IL10, CD206, and TGFβ (M2 markers) in the adherent macrophages. ACTB (human β-actin) was used to normalize gene expression. n = 3; error, SE. C, Schematic diagram of the protocol to measure the effect of CAF-induced macrophages on cancer cell invasion. n = 3; error, SE. D, Invasion assay for 2 × 104 YD-10B cancer cells alone (A) or with the following cells types: THP-1 monocytes (B), macrophages produced by treating THP-1 monocytes with PMA (C), and macrophages produced by coculturing THP-1 monocytes with CAFs (D). E, Representative micrographs of crystal violet–stained invaded cells. Scale bar, 200 μm; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05; ##, P < 0.01; ###, P < 0.001.
- Figure 2.
Cancer cells potentiate CAF-induced monocyte differentiation into M2-like macrophages. A, Schematic diagram of the transwell systems used to investigate the effect of cancer CM-stimulated CAFs on monocyte differentiation. B, A total of 2 × 105 THP-1 monocytes treated with the following stimuli: (i) YD-10B CM (5% final concentration); (ii) coculturing with 2 × 104 CAFs; (iii) coculturing with 2 × 104 CAFs plus YD-10B CM (5% final concentration), underwent differentiation into adherent macrophages. Addition of cancer cell CM to CAF–monocyte cocultures induced significantly higher levels of monocyte differentiation compared with coculturing without cancer CM (scale bar, 10 μm). C, Quantification of macrophages differentiated by the above conditions. n = 3; error, SE. D, Effect of HCT116 colon carcinoma or MDAMB231 breast carcinoma cell CM on CAF-induced THP-1 monocyte differentiation. THP-1 was treated with cancer CM alone or cocultured with CAFs in the absence or presence of cancer CM for 72 hours. n = 9; error, SD. E, Western blotting for M2 marker expression (CD206, 166 kDa and IL10, 17 kDa) in the differentiated macrophages. THP-1 monocytes were treated with a mixture of YD-10B cancer cell CM and CAF CM, or CM from YD-10B CM stimulated CAFs, for 72 hours. F, Quantification of CD206 and IL10 expression in the differentiated macrophages. n = 4; error, SD. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05; ###, P < 0.001.
- Figure 3.
GM-CSF and IL6 are the factors secreted by cancer cell–stimulated CAFs that induce monocyte differentiation. A, Human cytokine array for CAF culture supernatant, CAF culture supernatant after YD-10B cancer CM stimulation, and YD-10B CM. Cytokines upregulated in cancer-stimulated CAFs are indicated with red boxes. B, Representative photographs of THP-1 monocyte differentiation into macrophages after treatment with 12.5 ng/mL IL7, CXCL5, GM-CSF, or IL6 for 72 hours (scale bar, 100 μm for low-magnification pictures and 10 μm for high-magnification pictures). C, Quantification of THP-1 monocyte differentiation into macrophages after cytokine treatment. n = 3; error, SE. D, GM-CSF and IL6 secretion by CAFs after treatment with CM from different cancer cell types. CAFs were treated with CM from YD-10B OSCC cells, HCT116 colon carcinoma cells, or MDAMB231 breast carcinoma cells for 24 hours. GM-CSF or IL6 secretion was measured by ELISA. n = 3; error, SD. E, Invasion assay for YD-10B cells alone (control) or YD-10B cells cocultured with macrophages derived from THP-1 monocytes by treatment with 12.5 ng/mL IL6, 12.5 ng/mL GM-CSF, or 12.5 ng/mL IL6 + 12.5 ng/mL GM-CSF (scale bar, 100 μm). n = 3; error, SD. ns, not significant; ***, P < 0.001; #, P < 0.05; ###, P < 0.001.
- Figure 4.
GM-CSF and IL6 induce BMDM differentiation into proinvasive M2-like macrophages. A, Schematic diagram of the protocol assessing the effect of GM-CSF and/or IL6 on BMDM differentiation. B, Micrographs of 2 × 104 BMDMs after treatment with 12.5 ng/mL GM-CSF and/or 12.5 ng/mL IL6 for 72 hours. Scale bar, 100 μm. The bar chart shows quantification of BMDM differentiation by counting adherent macrophages. The effect of 20 ng/mL M-CSF treatment (a recommended protocol for monocyte purification from bone marrow cells; ref. 15) was also assessed. n = 3; error, SE. C, Flow cytometry analysis of CD86 (M1 marker), CD11b (M1/M2 marker), and CD206 (M2 marker) expression in the BMDMs after cytokine treatment. The bar chart shows the quantification of the CD11b+, CD86+, and CD206− (M1) population and CD11b+, CD86−, and CD206+ (M2) populations. n = 3; error, SD. **, P < 0.01 and ***, P < 0.001 (compared to untreated control w/o M-CSF); ###, P < 0.001 (compared to untreated control with M-CSF).
- Figure 5.
CAFs enhance cancer cell invasion, facilitate metastasis, and increase serum/tumor levels of IL6 and GM-CSF in an orthotopic, syngeneic colon carcinoma model. A, Schematic diagram showing the isolation of primary CAFs. B, Schematic diagram of the protocol to investigate the effect of CAFs on CT26 murine colon cancer cell metastasis and IL6 and GM-CSF expression levels. C, IVIS images of representative mice during the 5 weeks’ time course posttransplantation. D, Photon flux detected from cancer cells in the transplanted mice. n = 8 mice/group; error, SE. E, Representative photographs of the tumors at 6 weeks’ posttransplantation. F, Bar chart of primary tumor size. n = 8 mice/group; error, SE. G, ELISA for serum levels of IL6 and GM-CSF. n = 6; error, SE. ns, not significant; **, P < 0.01; ***, P < 0.001; ##, P < 0.01; ###, P < 0.001.
- Figure 6.
IL6 and GM-CSF blockade synergistically reduce the recruitment of TAMs and inhibit metastasis. A, IVIS images of representative mice during the 3-week period of antibody blockade. B, Photon flux from cancer cells in the transplanted mice with or without antibody blockade. n = 8 mice/group; error, SE. C, Primary tumor weight in the transplanted mice. n = 8 mice/group; error, SE. D, IVIS images of representative dissected organs [primary tumor (upper), liver (middle) and lung (lower)] from tumor-bearing mice for metastasis. Mice were sacrificed after the 6-week period of antibody blockade. E, Metastasis rate for lung and liver in the transplanted mice with or without antibody blockade. n = 10 mice/group; error = SE. F and G, Immunohistochemical analysis of CD206 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 < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05; ##, P < 0.01; ###, P < 0.001; Ж Ж, P < 0.01.
Additional Files
Supplementary Data
- Figure S1 to S22 - Supplementary Figure S1. (A) Immunofluorescence staining of DAPI and α-SMA expression in primary human normal fibroblasts (NF; passage<5) and three different human cancer-associated fibroblasts (CAF1, CAF2, and CAF3). Scale bar=100 μm. Bar chart shows quantification of α-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 μm. 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-1α (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 μm). 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-β (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. ¶¶=p<0.01; ¶¶¶=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 μm. 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 α-SMA expression in primary murine normal fibroblasts (mNFs; passage 3), cancer-associated fibroblasts (mCAFs; passage 4) and CT26 colon carcinoma cells. Scale bar=100 μm. (B) Bar chart showing quantification of α-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 μm. 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 μm). 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 μg/mL IL-6 and/or 0.2 μg/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 μg or 200 μg 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-1α 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.
- Supplementary Table 1 - Oncomine data
- Supplementary Table 2 - Antibodies used in this study
- Supplementary Table 3 - Primers used in this study
Volume 24, Issue 21
