Supporting Information
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Supporting Information Voigt et al. 10.1073/pnas.1705165114 SI Materials and Methods the HTCR includes obtaining written informed consent from all Mice. BALB/c mice were purchased from Janvier. C57BL/6 mice patients with lung cancer and has been approved by the Ethics were purchased from Charles River. Mice were 5 wk old at the Committee of the Medical Faculty, LMU Munich (no. 025-12) and − − onset of experiments. Spleens from IL-1R / mice were provided by the Bavarian State Medical Association. All operations of from T. Stöger, Helmholtz Center Munich, Munich, and spleens Biobank are certified according to ISO 9001:2008 (57). Written − − from IL-23 / mice were a gift from K. Savvatis, Charité, Berlin. informed consent was obtained from all patients with breast cancer before collection of specimens, in line with the respective in- Cell Lines. The murine lung cancer cell line 1 (Line-1) was kindly stitutional policies and in accordance with to the Declaration of provided by Nejat K. Egilmez, University of Louisville, Louisville, Helsinki. Tumor specimens were obtained from patients un- KY (55), and cells were cultured in complete RPMI 1640 medium dergoing clinically indicated surgery. Histological analysis of (Lonza). If not stated otherwise, growth media used were supple- NSCLC or breast cancer was made by a pathologist as part of mented with 10% FBS, 100 μg/mL streptomycin, 1 IU/mL peni- standard of care. Ethical approval was obtained from the Ethics cillin, and 2 mM L-glutamine (Life Technologies). The 4T1murine Committee of the Medical Faculty, LMU Munich (reference breast cancer cell line was kindly provided by Maria Wartenberg, nos. 220-15 and 249-15). Jena University Hospital, Jena, Germany, and was cultured in complete DMEM (Lonza). The E0771murine breast cancer cell Preparation of Patient Tissue Samples. Tumor tissue was homoge- line was purchased from CH3 Biosystems and was cultured in nized with a scalpel and incubated with 1 mg/mL collagenase and complete RPMI 1640 medium supplemented with 10 mM Hepes. 0.05 mg/mL DNase I (Sigma-Aldrich) in RPMI medium (Lonza) The human lung carcinoma cell lines A549, HCC827, and at 37 °C for 30 min. Cell suspensions were first passed through a H1339 have been previously described (10). The human breast 100-μm and then a 30-μm cell strainer. Red blood cell lysis was cancer cell lines MCF7, MD-AMB-231, and CAMA-1 were a kind performed using BD Pharm Lyse (BD Biosciences). Cytokine gift of Udo Jeschke, Klinikum der Universität München, Munich. staining and flow cytometry are described below. Snap-frozen A549 and HCC827 cells were cultured in complete RPMI 1640 pieces of tissue were lysed by Bio-Plex cell lysis buffer (Bio- medium. H1339 cells were kept in complete RPMI 1640 medium Rad), and protein concentrations were measured by the Brad- supplemented with 20% FBS. A549, MCF7, and MD-AMB- ford protein assay (Bio-Rad). 231 cells were cultured in complete DMEM. The HEK293 cell line has been previously described (56). All cell lines were maintained Preparation of Murine Spleen, Lung, and Tumor Cells. Lung, spleen, and tumor tissue were prepared as previously described. Purified at 37 °C in a humidified atmosphere of 5% CO2. All human cancer + + + cell lines were authenticated through short tandem repeat profile T cells (CD3 ,CD4 ,andCD4 naive T cells) and immune cells analysis by the Institut für Rechtsmedizin, Division of Forensic depleted of T cells were enriched using negative magnetic bead Molecular Biology, LMU Munich. sorting (Miltenyi Biotech). The purities of the enriched T cells were 90–98%, and the fraction of T cells in the T cell-depleted Mouse Tumor Models and Treatment of Mice. Five-week-old BALB/c immune cell population was ≤2%. mice were injected s.c. in the right flank with 1.25 × 105 4T1 or 5 5 × 105 Line-1 tumor cells, and C57BL/6 mice were injected with Cytokine Secretion Assays. Splenocytes (4 × 10 per well) were 2.5 × 105 E0771 tumor cells. BALB/c mice were treated i.p. with the cultured in round-bottomed 96-well plates in RPMI 1640 me- AhR antagonist CH-223191 (100 μg per mouse; Sigma-Aldrich) or dium (Lonza). Splenocytes were stimulated via MACS with DMSO as a vehicle control at days 30 and 31 after 4T1 tumor in- 20 ng/mL IL-1α, IL-23, IL-6, IFN-γ, TNF-α, or G-CSF (Pepro- duction. For Line-1 tumor-bearing mice, CH-223191 or DMSO was Tech) in the case of separated lymphocytes, with 100 ng/mL injected daily starting 7 d before analysis. BALB/c mice were IL-1α or IL-23 (BioLegend) or 100 μL murine tumor-cell su- treated i.p. with anti–IL-1R antibody (300 μgpermouseevery2d; pernatant added to 100 μL of the splenocyte suspension. Cytokine clone CD121a; BioXCell) or polyclonal Armenian hamster IgG levels of splenocytes stimulated with 4T1 or Line-1 cell super- antibody (BioXCell) as a control. C57BL/6 mice were treated i.p. natants were analyzed with a proteome profiler array for murine daily with anakinra (1 mg per mouse; Swedish Orphan Biovitrum cytokines (array panel A; R&D). PBMCs were isolated from hu- AB) or with PBS as a control starting at day 0. Infiltrating lym- man peripheral blood by Biocoll (Biochrom) separation. PBMCs 5 phocytes were analyzed in spleen, lung, and tumor. (4 × 10 per well) were cultured in round-bottomed 96-well plates in very low endotoxin (VLE) RPMI 1640 (Lonza) with the stan- Patient Samples. The tissue samples of NSCLC and corresponding dard supplements described above in addition to 10% human clinical data used in this study were provided by the Biobank under serum (Sigma-Aldrich), 1 mM sodium pyruvate, and 1% non- administration of the HTCR Foundation at University Hospital, essential amino acids (100×; Thermo Fisher). For blocking assays, LMU Munich, Munich. Tissue samples and data from 15 patients anakinra (500 ng/mL; Swedish Orphan Biovitrum AB), CRID (50, were provided anonymized (double coded) from a database ap- 5, and 0.5 μM; Tocris Bioscience), Z-Vad (20, 2, and 0.2 μM; proved by the HTCR data-protection officer. Twelve patients had InvivoGen), P2X7R antagonist (100 μM; Sigma-Aldrich), or an adenocarcinoma, two had atypical lung carcinoma, and one had a neutralizing antibodies directed against mouse IL-1α (clone ALF- squamous cell carcinoma (for further information see Table S1). At 161; eBioscience), mouse IL-23 (clone HNU2319; eBioscience), the time of tissue collection, four of the patients had received human IL-1α (clone 7D4; InvivoGen), or human IL-1β (clone neoadjuvant therapy or other anticancer therapy. Patients with 4H5; InvivoGen) were incubated for 60 min with the tumor su- breast cancer were recruited from the department of gynecology pernatant before splenocyte or PBMC stimulation. For blockade of and obstetrics at the Klinikum Dritter Orden, Munich. Tissue transcription factors AhR or RORγt, CH-223191 (Sigma-Aldrich) samples were collected from five patients. All patients had a or SR-2211 (Biomol), respectively, were used at the indicated confirmed diagnosis of breast cancer, and two of these were clas- concentrations (58, 59). Cytokine levels were analyzed 6 d after sified as triple-negative breast cancer (Table S2). The framework of incubation of murine splenocytes or human PBMCs in the tumor- Voigt et al. www.pnas.org/cgi/content/short/1705165114 1of12 cell supernatants. Murine cytokine concentrations were measured incubated with Human TruStain FcX (BioLegend) for 15 min at 4 °C by ELISA for IL-22 (Antigenix America), IL-1α (eBioscience), IL- and were stained with fluorochrome-linked antibodies and Zombie 23, IL-17, or IFN-γ (R&D Systems). Human cytokine levels were NIR Fixable Viability Dye (BioLegend) for 30 min at 4 °C. Cells were analyzed by IL-22, IL-1α,IL-1β, IL-17, IL-23, or IFN-γ ELISA then permeabilized with IC Fixation/Permeabilization (eBioscience) (R&D Systems). and were stained for intracellular antigens. Stained cells were + analyzed with a FACSCanto II flow cytometer and FACSDiva Flow Cytometry. CD4 splenocytes (separated via MACS) (1 × software (BD Bioscience). When Counting Bright Beads (Thermo 106 per well) were cultured in 96-well plates in RPMI 1640 me- Fisher) was used, 2.5 μL of beads per tube were added. The ab- dium (Lonza) and were stimulated with 20 ng/mL IL-1α solute number of beads per tube was calculated as n = bead count/μL (PeproTech) for 4 d. PBMCs were isolated from human pe- *2.5. The absolute cell numbers were calculated as absolute cell ripheral blood and stimulated as described above. number = relative count detected in FACS/(relative count beads/ The following fluorophore-conjugated antibodies reactive to absolute number beads calculated). mouse antigens were purchased from BioLegend and used for flow cytometry analysis: anti-CD3 (clone 145-2C11); anti-CD4 (clone Human Microarray Data. Raw microarray data of total tissue RNA GK1.5); anti-CD8 (clone 53-6.1); anti-CD19 (clone 6D5); anti- from normal lung (n = 30), lung adenocarcinoma (LADC; n = 80), CD45 (clone 30-F11); anti-Nkp46 (clone 29A1.4); anti-CD11b normal breast (n = 5), and breast cancer (BRCA; n = 45) tissues (clone M1/70); anti-CD11c (clone N418); anti-Ly6C (clone hybridized to Human Gene 1.0 ST microarrays (Affymetrix) were HK1.4); anti–Gr-1 (clone RB6-8C5); anti-F4/80 (clone BM8); and retrieved from Gene Expression Omnibus series GSE43458 and anti–IL-17 (clone TC11-18H10.1). Mouse anti–IL-22 (clone GSE36295 (23, 24). Raw data from *.cel files were normalized 140301) and rat-IgG2a isotype control were purchased from R&D against array internal controls, and *.chp files were generated Systems.