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1 Supporting Information Demaria et al /pnas SI Materials and Methods Study Approval. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Lausanne. Human studies were approved by the institutional review board of the University of Lausanne and were performed in accordance with the guidelines of the Declaration of Helsinki. Melanoma metastasis explants were obtained with informed consent from patients by punch biopsies and were defined according to standard clinical and histopathological criteria. Mice. C57l6 mice were purchased from Harlan. OT-I (TCR transgenic mice specific for the K b -SIINFEKL peptide of ovalbumin), IFNAR /, TLR9 /, and CD11c-DTR mice were obtained from the SwimmR mice repository in Zurich. STINGdeficient (Golden ticket or STING gt/gt ) and hdca2-dtr mice were purchased from The Jackson Laboratories. Tumor Cell Lines. 16F1 cells were obtained from the ATCC. 16F1 and 16-OVA cells were cultured in RPMI supplemented with 1% (vol/vol) FCS and 1 mm penicillin/streptomycin. MC38 cells (provided by Jeffrey Schlom, NIH) were cultured in DMEM supplemented with 1% (vol/vol) FCS and 1 mm penicillin/streptomycin. Tyr::N-ras (Q61K) INK4A / melanoma cell line was cultured in DMEM 1% FCS, 1 mm penicillin/streptomycin, and M β-mercaptoethanol. -raf (V6E/+) PTEN / CDKN2A / melanoma cell line was cultured in DMEM 1% (vol/vol) FCS, 1 mm penicillin/streptomycin, M β-mercaptoethanol, glutamine, 1 mm sodium pyruvate, and 1 mm Hepes. Tumor cells were grown in monolayer at 37 C in a humidified CO 2 incubator. efore reaching confluence, tumor cells were harvested with.5% trypsin, washed, and suspended in PS for injection. Tumor Implantation, Treatment, and Growth Measurement. A total number of tumor cells were injected s.c. into the flank of 7- to 12-wk-old mice. At day 5 and day 1, progressing tumors were injected with 1 μl of PS solution containing 1 μg of3 3 -cgamp (c[g(3-5 )pa(3-5 )p]) (Invivogen) complexed with 3 μl of Lipofectamine 2 (Invitrogen). Tumor size was measured in two dimensions using a caliper. Tumor volume was calculated using the modified ellipsoid formula V = (Lxl 2 )/2, where L is the widest and l the smallest diamater. In Vivo Antibody Treatments. depleting antibody (clone ) was purchased from ioxcell and injected intraperitoneally at the dose of 2 μg per mouse twice a week. IFNAR1 blocking antibody (clone MAR1-5A3) was purchased from ioxcell and injected at the dose of 2 μg per mouse at day 1,, +1 of intratumoral cgamp injection. PD1 (clone J43) and CTLA4 (9H1) blocking antibodies were purchased from io- Xcell. Anti-CTLA4 and anti-pd1 were injected intraperitoneally twice a week at the dose of 2 μg per mouse after intratumoral cgamp injection. Metastasis Model. A total number of tumor cells were given i.v. to generate lung metastases in mice bearing a 5-d-old s. c. tumor. At the same time, s.c. tumors were treated with intratumoral cgamp. After 1 d, mice were killed and the number of lung metastases was counted macroscopically. Ex Vivo Treatment of Human Melanoma Explants. Fresh melanoma metastases were harvested by punch biopsies from a patient with in-transit skin metastases. Fresh tumor explants were injected with 1 μl PS solution containing 1 μg of cgamp (c[g(2-5 )pa(3-5 )p]) (Invivogen) complexed with 3 μl of Lipofectamine 2 and incubated in a 96-well plate at 37 C in a humidified CO 2 incubator. After 5 h, tumors were harvested and snap frozen for histology and RNA extraction. Generation and Analysis of Tumor Cell Suspensions. Tumors were harvested, rinsed with PS, and passed through a 7-μm cell strainer to obtain a single cell suspension. Cells were stained with antibodies against (145.2C11), a (53-6-7), CD19 (1D3), NKp46 (29A1.4), CD11C (HL3), Ly6G (1A8), CD11 (M1/7), CD45 (3F11), 1 (MEC13.3), CD144 (ebiov13), and 9 (Flk-1) purchased from D Pharmingen or eioscience. SIINFEKL tetramer was purchased from ecton Dickinson. Data were acquired on a Gallios cytometer (eckman-coulter), LSRII (ecton Dickinson), or FACS Calibur (ecton Dickinson), and analyzed with Flow-Jo V1 software. Gene Expression. Tumors were homogenized in TRIzol (Invitrogen). RNA isolation was performed with chloroform and further isopropanol precipitation. cdna was synthesized from 1 to 2 μg of total RNA using SuperScript II reverse transcriptase (Invitrogen) according to manufacturer protocol. Gene expressions were analyzed using specific Taqman probes (Life Technologies; mouse Gapdh, Mm _g1; human GAPDH, Hs _g1; mouse Ifnβ1, Mm439552_s1; human IFNβ1, Hs _s1; mouse Mx2, Mm m1; mouse Irf7, Mm516793_g1; mouse Isg15, Mm _s1), and Taqman gene expression master mix (Life Technologies). Amplifications were performed using the Step One Real-Time PCR system (Applied iosystem). Relative gene quantifications were expressed as 2 -ΔCt using Gapdh as endogenous control. Type I IFN Activity Measurement. Type I IFN activity was measured with 16 IFNα/β reporter cells (Invivogen). For assessment of type I IFN activity in tumors: Tumors were homogenized in 3 μl culture medium supplemented with protease inhibitor mixture (Roche). Proteins were quantified with a CA assay (Pierce). 16 IFNα/β reporter cells were seeded in 96-well plates and stimulated with 3 μg/ml of tumor protein extracts for 16 h. Detection of type I IFNs was monitored with Quanti-blue (Invivogen) according to manufacturer instructions and absorbance at 62-nm read with a spectrophotometer. For assessment of type I IFN activity in culture supernatant: STING-knockdown 16 IFNα/β reporter cells were incubated with 4 μl of samples for 24 h prior to revelation with Quanti-blue (Invivogen). IFN-β Detection by Immunofluorescence and Flow Cytometry. The 5-d-old tumors were injected with 1 μl of cgamp solution plus refeldin A (eioscience). After 5 h, tumors were harvested and snap frozen for histology or processed to generate a single cell suspension for flow cytometry analysis. For immunofluorescence staining, sections (2 μm) were cut with a cryostat, thawed and mounted on Superfrost plus slides, air dried, and stored at 2 C for further use. Sections were rehydrated in PS, fixed with 4% (vol/vol) PFA, permeabilized with PS.3% Triton-X, and incubated with blocking buffer [PS, 5% (vol/vol) donkey serum,.5% SA,.3% Triton-X] for 3 min at room temperature. Sections were incubated with rat anti-mouse IFN-β FITC (RMM-1, PL, dilution 1/25) or rabbit anti-human IFN-β (PA5-239, Thermo Scientific, dilution 1/5) diluted in blocking buffer at 4 C overnight. Anti-mouse 1-biotin (MEC13.3, Pharmingen), anti-mouse (Flk-1, R&D), and anti-human 1of1

2 1 (JC7A, DAKO) were incubated for 2 h at room temperature in blocking buffer. Anti-mouse IFN-β FITC signal was amplified with an anti-rat Alexa 488 secondary antibody. Secondary antibodies (Molecular Probes, Invitrogen) were incubated for 1 h at room temperature in blocking buffer. Fluorescent images were acquired using ZEISS LSM 51 laser scanning confocal microscope and analyzed with ZEN 21 software. For flow cytometry, harvested tumors were processed as described above to generate a single cell suspension. Single cell suspension was first stained with antibodies targeting cell surface markers. Intracellular detection of IFN-β was performedwithintracellularfixationandpermeabilizationbufferset (eioscience). After surface staining, tumor cell suspensions were fixed, washed twice with permeabilization buffer, and incubated overnight with anti-mouse IFN-β FITC (RMM, PL, dilution 1/25) at 4 C. Tumor cells were analyzed on a FACS Calibur (ecton Dickinson)andanalyzedwithFlow-JoV1software. Enrichment of CD11c DCs and pdcs. For CD11c DC enrichment, spleens were treated with DNase1 (1 μg/ml) and Collagenase type 2 (2 mg/ml) for 3 min at 37 C and passed through a 7-μm cell strainer to generate a single cell suspension. T and cells were removed by magnetic separation using -biotin antibody (145-2C11), CD19-biotin antibody (1D3), and antibiotin lyophilized microbeads (Miltenyi). The negative fraction was stained with anti-cd11c-biotin (N418) and antibiotin lyophilized microbeads and positively selected on magnetic column. For pdc enrichment, spleens were treated with DNase1 (1 μg/ml) and Collagenase type 2 (2 mg/ml) for 3 min at 37 C and passed through a 7-μm cell strainer to generate a single cell suspension. pdcs were enriched from spleen by negative selection using magnetic beads (Plasmacytoid Dendritic Cell Isolation kit, Miltenyi) according to manufacturer instructions. MS-1 Endothelial Cell Line and Generation of STING-Deficient MS-1 Endothelial Cells. MS-1 endothelial cell line was obtained from the ATCC. MS-1 cells were cultured in DMEM supplemented with 1% FCS and 1 mm penicillin/streptomycin. STING knockdown MS-1 cells were generated using plko.1-puro plasmids expressing mouse STING specific shrna or nonrelevant shrna from Sigma (SHCLNG-NM_28261 and SHC2, respectively). MS-1 cells were cultured with lentivirus-containing supernatants for 24 h before replacing the medium. After 72 h, puromycin (1 μg/ml) was added to the culture to select infected cells. STING knockdown efficiency was verified by Western blot analysis of total cell extract using the anti-sting antibody 3337S (Cell Signaling Technology). IFNγ ELISPOT. IFNγ producing cells were quantified using mouse IFN gamma ELISPOT Ready-SET-Go assay from eioscience according to manufacturer instructions. Statistical Analysis. All statistical analyses were performed with GraphPad Prism 6. software. Test, group sizes, and P values are given in the corresponding figure legends. P values <.5 were considered statistically significant. A Tyr::N-Ras (Q61K) INK4a -/- melanoma model % of T cells Injected tumor volume (mm 3 ) Raf V6E/+ PTEN -/- CDKN2A -/- melanoma model % of T cells * Injected tumor volume (mm 3 ) * Fig. S1. Intratumoral cgamp injection promotes T-cell responses and efficiently delays growth of several melanoma models. (A and ) Tyr::N-Ras (Q61K) INK4a / melanoma cells (A) or -Raf V6E/+ PTEN / CDKN2A / melanoma cells () were implanted s.c. in mice. cgamp () or Lipofectamine alone () was injected into tumors at day 5 and day 1 after engraftment. Depicted are: representative flow cytometry dot plots of T cells infiltrating tumors, quantification of tumor infiltrating T cells (*P <.5, P <.1 by unpaired t test), and tumor growth analysis (represented as mean tumor volume ± SEM with n = 5. *P <.5, P <.1 by two-way ANOVA). Data are combined from two independent experiments. 2of1

3 Gated on TILs 6 CD62L CD44 CD62L CD44 T cells from spleen T cells infiltrating tumors PMA/IONO + PMA/IONO + PMA/IONO IFN No antibody Perforin % of T IFN + gated on TILs % of T Perforin+ gated on TILs * 4 2 C 16-OVA 16-OVA % + V D V 2 SIINFEKL tetramer 16-OVA 16-OVA % of CD 3+ SIINFEKL t etramer : OVA OVA Fig. S2. Intratumoral cgamp injection promotes the generation of Ag-specific cytotoxic T cells that infiltrate the tumors. 16- or 16-OVA (if indicated) tumor cells were implanted s.c. into mice. (A and ) cgamp () or Lipofectamine alone () was injected into tumors at day 5 and day 1. (A) Flow cytometry analysis of CD44 and CD62L expression gated on T cells infiltrating tumors at day 15. T cells from spleen of naive mice were used as control. () Tumor cell suspensions were cultured with refeldin A for 5 h in addition of PMA/Ionomycin if indicated. Data represent flow cytometry detection and quantification of IFNγ or Perforin expressing- T cells in tumor-derived cell suspension. *P <.5 by unpaired t test. (C and D) At day 4, OT-I splenocytes were transferred intravenously. cgamp () or Lipofectamine alone () was injected into the tumor at day 5 and day 1. Flow cytometry analysis and quantification of OVA-specific T cells in tumors at day 15 detected with Vα2 (C) or SIINFEKL tetramer staining (D). P <.1 by unpaired t test. 3of1

4 Injected tumor Contralateral tumor CD4 16F1 melanoma model % of CD4 T cells in injected tumors % of CD4 T cells in contralateral tumors * Tyr::N-Ras (Q61K) INK4a -/- melanoma model CD % of CD4 T cells * -Raf V6E/+ PTEN -/- CDKN2A -/- melanoma model CD % of CD4 T cells * Fig. S3. Intratumoral cgamp injection induces high numbers of tumor-infiltrating CD4 T cells. (A) 16F1 were implanted s.c. into two opposite flanks of mice. At day 5 and day 1, cgamp () or Lipofectamine alone () was injected into only one tumor. Data represent flow cytometry analysis and quantification of CD4 T cells infiltrating injected tumor and noninjected tumor (contralateral) at day 15. *P <.5, P <.1 by unpaired t test. () Tyr::N- Ras (Q61K) INK4a / melanoma cells or -Raf V6E/+ PTEN / CDKN2A / melanoma cells were implanted s.c. into mice. At day 5 and day 1, cgamp (cgampinj) or Lipofectamine alone () were injected into the tumor. Data represent flow cytometry analysis and quantification of CD4 T cells infiltrating injected tumors. *P <.5, P <.1 by unpaired t test. 4of1

5 % of tumor volume 1 5 / 2/2 1/1 5/5 αctla4 (μg) / αpd1 (μg) Fig. S4. Increasing doses of actla4/apd1 treatment improve intratumoral cgamp efficacy. 16F1 cells were implanted s.c. into mice. cgamp () or Lipofectamine alone () was injected into the tumors at day 5. Anti-CTLA4/anti-PD1 treatment was injected intraperitoneally twice a week at the indicated dose. Data represent the percentage of tumor volume compared with ected tumor at day 18. Importantly, as in Fig. 1, anti-ctla4/anti-pd1 alone showed significantly less activity than anti-ctla4/anti-pd1 plus cgamp (not shown). n = 5 mice per group. P <.1 by two-way ANOVA. MC38 colon carcinoma model Injected tumor volume (mm 3 ) Controlateral tumor volume (mm 3 ) * Fig. S5. Intratumoral cgamp injection induces potent direct and systemic antitumor activity in the MC38 colon cancer model. MC38 colon cancer cells were implanted s.c. into two opposite flanks of mice. cgamp () or Lipofectamine alone () was injected into one tumor at day 5. Data represent tumor growth of injected tumors and noninjected contralateral tumors, shown as the mean tumor volume ± SEM with n = 4 5. *P <.5, P <.1 by twoway ANOVA. 5of1

6 C IFNγ spots / 1 6 cells MX2 relative expression * STING gt/gt * IFNAR -/- Irf7 relative expression /- IFNAR * 16: OVA OVA OVA mice: STING gt/gt IFNAR -/- * 8 STING gt/gt D Tumor volume (mm 3 ) IFNAR -/ isg15 relative expression % of + T cells (day15) STING gt/gt IFNAR -/- IFNAR -/- * IFNAR -/ Fig. S6. Spontaneous antitumor immunity in melanoma is IFNAR dependent. 16F1 or 16-OVA (if indicated) tumor cells were implanted s.c. in, STING gt/gt,or IFNAR / mice. (A) Gene expression analysis of Mx2, Irf7, and Isg15 in tumors harvested at day 15 after engraftment. Data are combined from three independent experiments. () Flow cytometry analysis and quantification of tumor-infiltrating + T cells at day 15 after engraftment. Data are representative of two independent experiments. (C) At 11 d after engraftment, tumor draining lymph node cells were restimulated with 1 μg/ml SIINFEKL peptide for 24 h. The frequency of OVA-specific IFNγ-producing cells was evaluated by ELISPOT. Data are representative of three independent experiments. (D) 16F1 Tumor growth analysis in IFNAR / compared with mice. (A C) *P <.5, P <.1, *P <.5, by unpaired t test. (D) P <.1 by two-way ANOVA. 6of1

7 % of cell expansion tumor lysate anti-ifnar1 IFNβ Ctrl cgamp Ctrl cgamp Fig. S7. Tumor extracts from ected tumor inhibit the growth of 16F1 cells in a type I IFN-dependent manner. Protein extracts were prepared from tumors lysed after they have been injected in vivo for 4 h with cgamp or Lipofectamine (Ctrl). 16F1 cells were incubated with 3 μg/ml of tumor lysate proteins. Type I IFN signaling was blocked by adding 1 μg/ml of anti-ifnar1 antibody (+) in the culture medium. As positive control, 1 units/ml of murine recombinant IFN-β was added to the culture (+). Data represent the percentage of cellular expansion compare with Ctrl conditions after 3 d of culture. Data are representative of at least three independent experiments. P <.1 by unpaired t test. 7of1

8 IFN IFN STING gt/gt STING gt/gt STING gt/gt IFN IFN IgG2a IgG2a 1 STING gt/gt % of vessels expressing IFN Non injected ected Fig. S8. Endothelial cells represent the main source of IFN-β in tumors upon therapeutic or spontaneous STING activation. (A) 16F1 cells were implanted into or STING gt/gt mice. At day 5, tumors were treated with i.t. injection of cgamp plus refeldin A for 5 h. Data represent confocal imaging of injected tumor cryosections stained with rat anti-mouse IFN-β, rat IgG2a isotype control, and goat anti-mouse antibodies. () 16F1 cells were implanted into or STING gt/gt mice. At day 5, tumors were treated for 5 h with refeldin A alone (noninjected) or refeldin A plus cgamp (ected) before being embedded in OCT. Data represent the quantification by confocal microscopy of 1+ vessels in tumors that express IFN-β. Each symbol represents an independent tumor. P <.1, P <.1 by unpaired t test. 8of1

9 Ifnβ1 relative expression ns Ctrl cgamp cgamp DCdepleted Tumor volume (mm 3 ) pdcko pdcko 1 ns Fig. S9. IFN-β induction in response to intratumoral cgamp is not altered in tumor of DC-depleted mice and antitumor activity induced by intratumoral cgamp is retained in pdc-depleted mice. (A) 16F1 cells were engrafted into or CD11C-DTR transgenic mice. At day 4, 12 ng of diphteria toxin were i.p. injected in all mice. At day 5, 4 h after i.t. injection of cgamp (cgamp) or Lipofectamine alone (Ctrl), tumors were harvested for RNA extraction. Ifnβ1 gene expression was performed by quantitative PCR. ns, not significant by unpaired t test. () 16F1 cells were implanted s.c. into and hdca2-dtr mice. pdc depletion was achieved by systemic injection of 12 ng of diphteria toxin at day 4 and day 9. cgamp or Lipofectamine (control) was injected into tumors atday 5 and 1. Tumor growth was monitored over time. Data show the mean tumor volume ± SEM with n = 4 5. ns, not significant by two-way ANOVA. 9of1

10 IFNAR -/-.25 CD11C CD45 % of CD45+ CD11C IFNAR -/- 6 * 2 6 Geo Mean 4 2 Geo Mean IFNAR -/- IFNAR -/- Fig. S1. Type I IFNs increase the number of infiltrating DCs in ected tumors and stimulate DC maturation in lymph node draining ected tumors. 16F1 cells were implanted into or IFNAR / mice. At day 5, tumors were injected with cgamp () or Lipofectamine alone (). (A)At day 7, CD11c+ DC number was analyzed in tumors by flow cytometry. () At day 7, CD11C+ DCs from tumor draining lymph nodes were analyzed by flow cytometry for 6 and expression. P <.1, *P <.1, P <.1 by unpaired t test. 1 of 1