An interleukin-17-mediated paracrine network promotes tumor resistance to anti-angiogenic therapy

Transcription

1 CORRECTION NOTICE Nat. Med. 9, (13) An interleukin-17-mediated paracrine network promotes tumor resistance to anti-angiogenic therapy Alicia S Chung, Xiumin Wu, Guanglei Zhuang, Hai Ngu, Ian Kasman, Jianhuan Zhang, Jean-Michel Vernes, Zhaoshi Jiang, Y Gloria Meng, Franklin V Peale, Wenjun Ouyang & Napoleone Ferrara In the version of this supplementary file originally posted online, the title and legend of Supplementary Figure 7 were incorrect. The errors have been corrected in this file as of September 13.

2 SUPPLEMENTARY DATA for: Interleukin-17 mediated paracrine network promotes tumor resistance to anti-angiogenic therapy Alicia S. Chung, Xiumin Wu, Guanglei Zhuang, Hai Ngu, Ian Kasman, Jianhuan Zhang, Jean-Michel Vernes, Zhaoshi Jiang, Y. Gloria Meng, Franklin V. Peale, Wenjun Ouyang, Napoleone Ferrara* Genentech, Inc., 1 DNA Way, South San Francisco, CA 98 *Corresponding author. Present address: University of California San Diego 3855 Health Sciences Drive, Mail Code 819 La Jolla, CA 993 Phone: nferrara@ucsd.edu Keywords: angiogenesis; VEGF; IL-17; T H 17; G-CSF; myeloid cells Nature Medicine doi:1.138/nm.391

3 SUPPLEMENTARY FIGURES Supplementary Fig. 1 (a-c). a. Tumor Area only b. Tumor-Associated Stromal Area. p= p= TH17.1 TH Bv Bv8 c. anti-il-17a anti-tnfa anti-il-1b -1 % G-CSF inhibition Ab conc (ng/ml) Supplementary Fig. 1 (a-c). Functional and molecular characterizations of anti-vegf treatment sensitive and resistance tumors and clinical correlates. (a,b) Level of tumor infiltrating TH17 lymphocytes significantly correlated with the level of Bv8 expressing neutrophils particularly within tumor margins in human colorectal cancer specimens. Regions of interest (ROI) were selected around the tumor area on each slide and extended out by ~ 5 microns to denote tumor margin area (tumor associated stroma). The IL-17A and Bv8 immno-positive areas (DAB) normalized to the hematoxylin area are outputed for the (a) tumor (ROI) and (b) extended (tumor associated stroma) regions. The correlation of IL-17A positive lymphocytes and Bv8 expressing granulocytes is estimated by Spearman s correlation and linear regression. (best-fit line is indicated, together with 95% confidence bands). (c) Neutralizing antibodies against other known G-CSF inducers including TNF-α and IL-1β did not significantly reduce the levels of secreted G-CSF in target fibroblasts. EL cells (1x1 6 cells/ml) and normal skin fibroblasts (NSF) (3.3x1 5 cells) were cultured together at 1:3 ratio (NSF/EL) in 6-well plates for 7 hrs in triplicate with either the indicated neutralizing or isotype control antibodies; supernatants were collected and analyzed for G-CSF by ELISA. Cells were cultured in reduced serum medium (1% FBS). Neutralization of IL-17A can account for ~7% of G-CSF expression in fibroblasts induced by EL in co-culture. Anti-IL-17A: IC5 ~.5nM (375ng/ml) Nature Medicine doi:1.138/nm.391

4 Supplementary Fig. 1 (d-e). d. 8 IL-17FF IL-17AF.5 / ng/ml IL-17FF 6 ng/ml IL-17AF / / / Naive. e. -IL-17A -IL-17F -IL-17A+ -IL-17F Tumor volume (mm^3) Days after injection Supplementary Fig 1 (d-e). Functional and molecular characterizations of anti-vegf treatment sensitive and resistance tumors and clinical correlates. (d) EL tumors express high levels of IL-17F/F and IL-17A/F in addition to IL-17A/A. (Left) Serum levels of IL-17FF in EL tumor bearing vs. Il17rc -/- mice () in response to control (anti- Ragweed, α-rw) and anti-vegf (α-vegf) treatment measured with an in-house ELISA system. (Right) Serum levels of IL-17AF in EL tumor bearing vs. Il17rc -/- mice () in response to control (anti-ragweed, α-rw) and anti-vegf (α-vegf) treatment measured with the ebioscience Platinum ELISA assay (ebioscience, San Diego, CA). (e) Growth curves of EL tumors in C57BL/6 mice treated with the control antibody (α-rw), IL-17A neutralizing antibody (α-il- 17A), IL-17F neutralizing antibody (α-il-17f), or in combination. Treatment for all groups was initiated 8 h after tumor inoculation. Data shown are mean tumor volumes ±SD. Nature Medicine doi:1.138/nm.391

5 Supplementary Fig. 1 (f-g). f. g. Relative expression to GAPDH (x1-5) B16F1 LLC EL* Tib68 Tib16 Crl17 Tib1 Tib6 Tib5 Tib5 Tumor volume (mm^3) Days Tib6-neo clone 1 Tib6-neo clone Tib6-IL17 clone 1 Tib6-IL17 clone Tib6-IL17 clone 3 Supplementary Fig. 1 (f-g). Functional and molecular characterizations of anti-vegf treatment sensitive and resistance tumors and clinical correlates. (f) Lymphoma cell lines including EL and Tib6 cells lack IL-17 receptor (IL-17RC) expression in contrast to carcinoma cell lines including B16F1 and LLC. Tumor cell lines were lysed and RNAs assessed by quantitative reverse transcription-polymerase chain reaction (qrt-pcr) for Il-17rc expression. Representative qrt-pcr assays from two independent experiments run in triplicates are represented as mean ±SEM, and asterick denotes p<.5 by Student s t-test. B16F1= melanoma; LLC= lung carcinoma; Tib68= peripheral blood leukemia; Tib16, EL= T-lymphoma; Crl17=thymic lymphoma; Tib5=pre-B lymphoblast lymphoma; Tib5= B cell lymphoma; Tib6, Tib1, Tib3 = plasmacytoma/b cell myeloma. (g) Implanted Tib6-IL-17 tumors did not exhibit significant increase in tumor growth rate compared to Tib6-neo tumors. Ten million control Tib6 cells (Tib6-neo) or Tib6 cells with enforced IL-17A (Tib6-IL-17) expression were subcutaneously injected in 1ul of growth factor-reduced matrigel (BD BioScience) in the dorsal flank of Nu/Nu mice. Tumor volumes were monitored after tumors established at ~3mm 3 every other day using the ellipsoid volume formulas (.5 L W, where L is length and W is width). Nature Medicine doi:1.138/nm.391

6 Supplementary Fig.. IL-17 promotes an alternate pro-angiogenic pathway in anti-vegf refractory tumor models Tumor volume (mm^3) a. 3 1 d Days post treatment (5mg/kg) (5mg/kg) (15mg/kg) (15mg/kg) (3mg/kg) (3mg/kg) b. Fold antibody():antigen (VEGF) c. VEGF mrna expression (relative units) 3 1 * ns pg/ml Mo IL-1a Mo IL-1b e. 1 ng/ml VEGF naive/ RW Mo IL- Mo IL-3 Mo IL- Mo IL-5 Mo IL-6 Mo IL-9 Mo IL-1 Mo IL-1(p) Mo IL-1(p7) Mo IL-13 naive/ VEGF Basal naive/ IL17A Mo IL-17 Mo Eotaxin Mo G-CSF Mo GM-CSF Mo IFN-g Mo KC Mo MCP-1 Mo MIP-1a Mo MIP-1b Mo RANTES VEGF Mo TNF-a Mo IL-15 Mo IL-18 Mo FGF-basic Mo LIF Mo M-CSF Mo MIG Mo MIP- Mo PDGF-bb Mo VEGF 1ug/mL IL-17 1ng/mL IL-17AF 15 1 ng/ml IL-17 1 ng/ml Bv8 1ug/mL IL-17AF ng/ml VEGFA Tube length (mm/mm) Time (h) IL-17AF Bv8 Nature Medicine doi:1.138/nm.391

7 Supplementary Fig.. IL-17 promotes an alternative pro-angiogenic pathway in anti-vegf refractory tumor models. (a) Dose escalation growth curves for EL tumors in C57BL/6 mice treated with the control antibody, anti-rw, or anti-vegf, at 5, 15, and 5 mg/kg. (b) Ratios of anti-vegfa mabs to VEGFA as measured in EL tumor lysates of treated and Il-17rc animals. (c) Total RNA was extracted from EL tumors from both and recipients following treatment and analyzed for expression of VEGFA188, VEGFA 165 and VEGFA 1 by qrt-pcr. (d) Administration of anti-vegf and anti-il17a does not promote a systemic pro-inflammatory response. Pro-inflammatory cytokines and pro-angiogenic factors in the plasma of naïve animals treated twice weekly with control antibody (anti-rw), anti-vegf or anti-il-17a were interrogated with Luminex assays. (e) HUVEC vessel assembly was analyzed by CellPlayer 96-well kinetic angiogenesis assay. Left panel shows quantification of tube length during the time course of the study (1 days) in response to treatment with purified cytokines. Each data point was mean of eight biological replicates + standard deviation. Arrows indicated addition of cytokines. Right panel showed representative images of HUVEC tube networks. *P<.5 Nature Medicine doi:1.138/nm.391

8 Supplementary Fig.3. a. b. pg/mg / RW / VEGF CCL (MCP-1) / RW / VEGF pg/mg 8 6 CCL3 (MIP-1a) CCL (pg/ml) 8 6 Tib6-neo/a-RW Tib6-neo/aVEGF CCL (MCP-1) Tib6-IL17A/a-RW Tib6-IL17A/a-VEGF naive CCL3 (pg/ml) CCL3 (MIP-1a) pg/mg 6 CCL (MIP-1b) pg/mg CCL5 (Rantes) pg/mg VEGF CCL (pg/ml) 3 1 CCL (MIP-1b) CCL5 (pg/ml) CCL5 (Rantes) VEGF (pg/ml) VEGF. c. # cells/, live cells 3 1 CD11b+F/8+ p=.6 # cells/, live cells CD11b+Gr1+ p=.81e-5 d. VEGF (pg/ml) TAFs p= CD11bGr CD11bGr1- Supplementary Fig. 3. IL-17 does not alter monocyte chemoattractants or macrophage profile within the tumor microenvironment. (a) Circulating levels of monocyte chemoattractants are not affected by IL- 17 paracrine signaling. Tumor monocyte chemoattractants levels in LLC tumor bearing, Il17rc -/- () and naive mice in response to control (anti-ragweed, α-rw) and anti-vegf (α-vegf) treatment. (b) Serum monocyte chemokine levels including VEGF in Tib6-neo and Tib6-IL-17 tumors treated with anti-rw and anti-vegf. Data represented as mean ±SEM, and asterisk denotes p<.5 by Student s t-test. (c) Minimal changes in the number of F/8+ macrophages found in EL tumors in and IL-17 receptor deficient hosts. GFP+EL tumor cells (. 1 6 ) in 1ul of growth factor-reduced matrigel (BD BioScience) were subcutaneously inoculated in the dorsal flank of C57BL/6 or recipient mice. Established tumors of equal size were removed and tumor cells harvested for flow cytometry analysis as described in the Experimental procedures section. (d) IL-17 does not directly induce the expression of VEGF within tumor stromal cells in eliciting an increase of local VEGF concentration. Level of secreted VEGF by FACS isolated stromal fibroblasts (Tumor-associated fibroblasts, TAFs), and stromal myeloid CD11b+Gr1+ and CD11b+Gr1- cells. Nature Medicine doi:1.138/nm.391

9 Supplementary Fig. a. Cd11bGr1hi Cd11bGr1 mid/lo Cd11bGr1lo/ Cd11bGr1hi A 1 5 B AmCyan CD5+ live Gr Cd11bGr1 mid/lo Cd11bGr1lo/- B A CD CD11b F/ CD11b b. A=CD11b+Gr1hiF/8- B=CD11b+Gr1lo/-F/8+ A=CD11b+Gr1hiF/8- B=CD11b+Gr1lo/-F/8+ Nos Adam17 Adam17 Mrc1 Arg1 Il1 Tgfb1 Lgals9 Nos Adam17 Adam17 Mrc1 Arg1 Il1 Tgfb1 Lgals9 Nos Adam17 Adam17 Mrc1 Arg1 Il1 Tgfb1 Lgals c. EL Tumor % Tumor associated Cd11b+Gr1hi 3 1 * * % Tumor associated Cd11b+Gr1 mid/lo 8 6 * % Tumor associated Cd11b+Gr1llo/-F/ p=.63 p=.88 d. EL Spleen Bone Marrow % Splenic Cd11b+Gr1 hi *** *** * % Splenic Cd11b+Gr1 mid/lo 3 1 % CD11b+Gr1+ in BM 8 6 **** ** Supplementary Fig.. IL-17 is a regulator of CD11b+Gr1+ MDSC development, function and maintenance. Nature Medicine doi:1.138/nm.391

10 Supplementary Fig.. IL-17 is a regulator of CD11b+Gr1+ MDSC development, function and maintenance. (a) Flow Cytometric analysis plots of tumor-infiltrating myeloid cells gated by Gr1 expression levels (Gr1 hi, Gr1 mid/lo, Gr1 lo/- ) in EL tumors implanted in syngenic hosts. These subpopulations are further analyzed for expression of F/8. Gr1 hi (subpopulation A) cells are negative F/8 while Gr1 lo/- (subpopulation B) are predominately F/8 positive. (b) CD11b+Gr1 hi (A) and CD11b+Gr1 lo/- (B) cells were FACS sorted and isolated cells were lysed and RNAs assessed by microarray gene-expression analysis. Non-supervised hierarchical clustering of MDSC effector genes expressed in both subpopulations is shown in the far left panel. Heat-map of expression level of MDSC effector genes in CD11b+Gr1 hi F/8- cells from vs. tumor bearing mice is shown in the middle panel and CD11b+Gr1 lo/- F/8+ cells from vs. tumor bearing animals is shown in the right panel. Black box highlights genes whose expression is indicated to be preferentially expressed in either myeloid sub-populations. (c) Quantification of flow cytometric analysis of tumor infiltrating CD11b+Gr1 hi (left) CD11b+Gr1 mid/lo (middle) MDSCs and tumor associated CD11b +Gr1 lo/- F/8+ tumor associated macrophages (TAMs) in EL tumor bearing mice treated with control antibody (anti-rw) or anti-vegf. (d) Quantification of flow cytometric analysis of CD11b+Gr1 hi and CD11b +Gr1 mid/lo MDSCs in the spleen (left) and bone marrow of EL tumor bearing mice.*p<.5, **P<.5, ***P<.5, ****P<.5 Nature Medicine doi:1.138/nm.391

11 Supplementary Fig. 5 (a-b). a. b. EL tumor/ recipient Ex Vivo EL TAF PDGFR CD31 SMA Vimentim SMA DAPI Expression relative to GAPDH PDGFRa FGFR1 CXCR TAF Mrc-1 Supplementary Fig. 5 (a-b). Characterization of tumor associated fibroblasts and a lung orthotopic tumor model. (a, left) EL tumor associated fibroblasts express PDGFR-α and additional TAF markers, including vimentim, smooth muscle actin and Fibroblast growth factor receptor- 1 (FGFR-1). Immunofluorescence of an EL tumor section implanted in C57BL6 mice. Representative image of PDGFRα (red), CD31 (green) and Smooth muscle actin (SMA) (blue). In vivo, tumor associated fibroblasts (TAFs) express PDGFRα but not SMA, seen here with a perivascular staining pattern. CD31 stains blood vessels. Scale bar = 1µm. (a, right) Freshly isolated TAFs ex-vivo adopt a characteristic fibroblast morphology and are immuno-positive for the fibroblasts markers vimentim and SMA. (b) qrt-pcr analysis of freshly isolated TAFs reveal that these cells express the fibroblast markers, PDGFRα and FGFR-1 but not the myeloid lineage markers Mrc-1 and CXCR-. Nature Medicine doi:1.138/nm.391

12 Supplementary Fig. 5 (c-d). c. d IL-17A + -IL-17A Days after implantation 6 1 anti-rw anti-vegf anti-il17af anti-vegf+ anti-il17af Tumor burden (cpm/cm) e Lung tumor weight (g) Days *** *** *. -IL17AF + -IL17aF Supplementary Fig. 5 (c-d). Characterization of tumor associated fibroblasts and a lung orthotopic tumor model. (c) Longitudinal study of a lung orthotopic tumor model demonstrates that IL-17 function is required for refractoriness to anti-vegf treatment. LLC luc+ cells were orthotopically implanted into syngeneic C57BL/6 mice that received antibody treatment beginning at day following implantation. Representative images were taken for each group taken at 6 and 1 days following tumor implantation. (d) Quantification of bioluminescence showed significant decrease in tumor burden when mice were treated with anti-il17af in combination with anti-vegf vs. anti-vegf alone. (e) Significant decrease in resected lung/tumor weights at day 13 following tumor implantation were correspondingly observed between mono- and combined treatment groups. *P<.5, ***P<.5 Nature Medicine doi:1.138/nm.391

13 3 IgG 1 Chung A.S., Wu X., Zhuang Z., et al. Supplementary Fig. 6 (a-d). a. Gated on CD3+CD+ 1 5 CD Gated on CD3+ 8.7%.5% CD % Gated on CD3+CD8+ 8.9% No. IL-17A+IL-+ per, cells 3 1 CD+ CD8+ IL IL+ b. c. CD/ a-rw a-vegf IFN- subset CD/ CD+IFN- + 3 * Ratio a-vegf/a-rw 1 Count CD+ a-rw a-vegf FoxP3 subset CD+ Ratio a-vegf/a-rw foxp3/cd ratio Count 1 CD8/ IFN CD8/ Ratio a-vegf/a-rw 3 1 CD8+IFN- + * FoxP3 d CD/ a-rw a-vegf CD/. Ratio a-vegf/a-rw 3 1 CD+CD+ ** Count CD8/ CD8/ Ratio a-vegf/a-rw 8 6 CD8+CD+ ** CD Supplementary Fig. 6 (a-d). T-cell and monocyte profiling within the tumor microenvironment. Nature Medicine doi:1.138/nm.391

14 Supplementary Fig. 6 (a-d). T-cell and monocyte profiling within the tumor microenvironment. (a) T H 17 cells infiltrating LLC tumors are predominately CD+ T cells. (Left) Flow Cytometric analysis plots of tumor-infiltrating CD3 + CD + and CD3 + CD8 + T cell populations that are also positive for IL-17 and IL- expression. Numbers inside plots refer to the boxed areas. (Right) Quantification of CD + IL-17 + IL- + and CD8 + IL-17 + IL- + cells per, live cells collected by flow cytometry indicates that the TH17 cells belong primary to the CD + T cell fraction. (b) T H 17 suppress anti-tumor T-cell activity and increase T- regulatory (Treg) cells within the LLC tumor microenvironment in a drug treatment-dependent manner. Treatment with anti-vegf increases the level of TH1 and IFN-γ + CD8 + T-cells within the tumor microenvironment in as compared to mice, as determined by flow cytometric analysis. Blue horizontal bar indicates positive gate for IFN-γ expression in CD cells (top) and CD8 + cells (bottom). (c) Analysis of tumor infiltrating CD + FoxP3 + Treg cells in and recipients either treated with anti-rw or anti-vegf. Blue horizontal bar indicates positive gate for FoxP3 expression in CD + (left) and (right) T-cells. (d) Anti-VEGF treatment increases CD expression on CD5 + CD + and CD5 + CD8 + T cells within the tumor microenvironment in compared to mice. Blue horizontal bar indicates positive gate for CD expression in CD + (top) and CD8 + (bottom) T-cells. *P<.5, **P<.5 Nature Medicine doi:1.138/nm.391

15 Supplementary Fig. 6e. e. IgG control IgG control CD5+ cells 1 CD CD11b AmCyan-Live CD5+CD11b+ cells AmCyan-Live CD11c IgG control MRC1lo/CD11c+ M-1, cd11c- subset Fold change (vs. a-rw) arw MRC1hi/CD11c MRC1hi/CD11c- (Pro-angiogenic) ** B ail17af Combo Fold change (vs. a-rw) MRClo/CD11c+ (angiostatic) ** arw B ail17af Combo MRC1 Supplementary Fig. 6e. T-cell and monocyte profiling within the tumor microenvironment. (e) IL-17AF blockade does not affect levels of pro-angiogenic and angiostatic tumor-associated macrophages in LLC lung tumors. Analysis of infiltrating macrophages in LLC lung allograft tumors. Tumorderived cell suspensions were stained with Am-Cyan Live/Dead cell dye to exclude non-viable cells from further analysis. Viable AmCyan - CD5 + hematopoietic-lineage cells were then stained with anti-cd11b to identify myeloid-lineage cells; CD5+CD11b+ were further analyzed for expression of CD11c and MRC1. The fold change of MRC1 hi CD11c (pro-angiogenic) and MRC1 lo CD11c + (angiostatic) macrophages in various treatment groups compared to control treated tumor bearing C57BL/6 mice. **P<.5 Nature Medicine doi:1.138/nm.391

16 Supplementary Fig IL-17AF -Bv8 -Bv8+ IL-17AF -Bv8+ IL-17AF + VEGF LLC Tumor volume (mm^3) 8 6 * * Days Supplementary Fig. 7. Pharmacological inhibition of IL-17 and Bv8 in combination had no significant additive effect on LLC tumor growth. Pharmacological inhibition of IL-17 and Bv8 in combination had no significant additivity on LLC tumor growth. Growth curves of LLC tumors in C57BL/6 mice treated with the control antibody (anti-rw), neutralizing antibodies against IL-17AF (anti-il17af), Bv8 (anti-bv8), and VEGF (anti-vegf). All treatment groups had n=8 and treatment for all groups was initiated h after tumor inoculation. Data shown are mean tumor volumes ±SEM. *P<.5 Nature Medicine doi:1.138/nm.391

17 Supplementary Fig. 8 Interleukin-17/T H 17 cells-mediated anti-vegf resistance Antigen presentation T-cell activation T-cell response Lymph node NK T NK T T NK T T T NK T Dendritic cells Effector T-cell responses IL-17 Antigen uptake T H 17 MMP Bv8 VEGF VEGF Tumor antigen IL-17 IL-17 G-CSF Tumor cell Tumor associated fibroblasts Myeloid cells IL-17 + lymphocytes Supplementary Fig. 8. Schematic representation of of the pleiotropic role of IL-17 in mediating anti-vegf resistance. Tumor cell destruction induced by anti-vegf treatment may cause release of tumor antigens, which in turn may lead to antigen-presentation cell mediated activation of TH17 effector function and release of its canonical cytokine, IL-17. IL-17 plays a role within the tumor microenvironment to stimulate tumorassociated fibroblasts to produce pro-inflammatory factors such as G-CSF, which promotes the mobilization and recruitment of immunosuppressive myeloid cells. IL-17 signaling also plays a role in regulation of immunosuppressive-myeloid cell effector function including the secretion of pro-angiogenic factors such as MMPs and Bv8, which in turn may promote VEGF-independent tumor angiogenesis. IL-17 may also have a role direct role in promoting tumor angiogenesis in a VEGF independent manner. Nature Medicine doi:1.138/nm.391