Life Sciences Reporting Summary

Transcription

1 Life Sciences Reporting Summary Corresponding Author: Johanna A. Joyce Date: Jun 13, 2017 Nature Research wishes to improve the reproducibility of the work we publish. This form is published with all life science papers and is intended to promote consistency and transparency in reporting. All life sciences submissions use this form; while some list items might not apply to an individual manuscript, all fields must be completed for clarity. For further information on the points included in this form, see Reporting Life Sciences Research. For further information on Nature Research policies, including our data availability policy, see Authors & Referees and the Editorial Policy Checklist. Experimental design 1. Sample size Describe how sample size was determined. 2. Data exclusions Describe any data exclusions. 3. Replication Describe whether the experimental findings were reliably reproduced. 4. Randomization Describe how samples/organisms/participants were allocated into experimental groups. 5. Blinding Describe whether the investigators were blinded to group allocation during data collection and/or analysis. Sample sizes were chosen based on power of 0.9 to detect a difference of >1.5 standard deviations between LF vs. HF means with 95% confidence. However, in most cases, previous data was sufficient to inform sample size for subsequent experiments. Exclusion criteria were not needed. All data included in the study are reproducible: all flow cytometry experiments were repeated 3 or more times with similar results; all animal trials were repeated with at least n=4 in at least 2 independent cohorts with similar results; all in vitro assays were repeated in at least 3 independent experiments with similar results. For most experiments, a method for randomization was not needed since experimental groups were pre-determined by diet/weight. In cases where randomization was required (e.g. Figure 7), average weight was used to ensure balanced representation between experimental groups. For all experiments, automated quantitative methods were used. We chose to analyze data in this manner to avoid investigator bias. Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used. 1

2 6. Statistical parameters n/a For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or the Methods section if additional space is needed). Confirmed Software The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.) A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same sample was measured repeatedly. A statement indicating how many times each experiment was replicated The statistical test(s) used and whether they are one- or two-sided (note: only common tests should be described solely by name; more complex techniques should be described in the Methods section) A description of any assumptions or corrections, such as an adjustment for multiple comparisons The test results (e.g. p values) given as exact values whenever possible and with confidence intervals noted A summary of the descriptive statistics, including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range) Clearly defined error bars Policy information about availability of computer code 7. Software See the web collection on statistics for biologists for further resources and guidance. Describe the software used to analyze the data in this study. GraphPad Prism Pro5 was used for all data analysis. For all studies, we encourage code deposition in a community repository (e.g. GitHub). Authors must make computer code available to editors and reviewers upon request. The Nature Methods guidance for providing algorithms and software for publication may be useful for any submission. Materials and reagents Policy information about availability of materials 8. Materials availability Indicate whether there are restrictions on availability of unique materials or if these materials are only available for distribution by a for-profit company. 9. Antibodies Describe the antibodies used and how they were validated for use in the system under study (i.e. assay and species). All materials used in this study are available from commercial sources. Detailed information on antibody vendors, catalog and clone numbers and dilutions used can be found in Supplementary Table 3. All antibodies used in this study were titrated for each lot, and an optimal dilution was selected (see also Supplementary Table 4 for each type of assay). Mouse antibodies included: CD45 A700 and APC, Rat anti-mouse; Gr1 FITC and PerCP-Cy5.5, Rat anti-mouse; CD11b PE-Cy7 and PE, Rat anti-mouse/ human; Ly6G PE, Rat anti-mouse; Ly6C BV421 and APC-Cy7, Rat antimouse; CD3 PE-Cy7, Rat anti-mouse; CD4 BV605, Rat anti-mouse; CD8A FITC, Rat anti-mouse; NK1.1 PerCP-Cy5.5, Rat anti-mouse; CD107a APC, Rat anti-mouse; SiglecF APC, Rat anti-mouse IL5ra A488, Rat anti-mouse. Human antibodies included: CD45 BV605, Rat anti-mouse/human; CD11B BUV395, Mouse anti-human; CD14 FITC, Mouse anti-human; CD16 A700, Mouse anti-human; CD66B PE-Cy7, Mouse anti-human; IL5RA PE, Mouse anti-human. 2

3 10. Eukaryotic cell lines a. State the source of each eukaryotic cell line used. Breast tumor cell lines were isolated from the MMTV-PyMT mouse model, backcrossed into the BL6 background for >10 generations. These cell lines were subsequently selected for their capacity to grow in the mammary fat pad (7x105 cells injected/mouse) of WT BL6 animals within a reasonable time period (<2 months). Three cell lines were selected for subsequent experiments, including 99LN, 86R2, and 91R2. All breast tumor cell lines in culture were maintained in DMEM supplemented with 10% FBS, and were validated to be mycoplasma-free. For assays involving immune cell culture, primary cells were isolated from peripheral sources (i.e. blood or bone marrow) by FACS and used immediately for functional assays, for example NK cell co-culture or T cell CFSE. b. Describe the method of cell line authentication used. Cell line authentication was not needed - all cell lines from this study were generated in-house for immediate downstream application. c. Report whether the cell lines were tested for mycoplasma contamination. d. If any of the cell lines used in the paper are listed in the database of commonly misidentified cell lines maintained by ICLAC, provide a scientific rationale for their use. All cell lines were confirmed to be mycoplasma negative. No cell lines used in this study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample. 3

4 Animals and human research participants Policy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines 11. Description of research animals Provide details on animals and/or animal-derived materials used in the study. Diet-induced obesity (DIO) model: To model obesity with diet, 5w-old female BL6 mice (Jackson Laboratory) were enrolled on either high fat (HF; 60% kcal, Research Diets D12492) or low fat (LF; 10% kcal, Research Diets D12450) irradiated rodent diet for 15w. After 15w, animals were either sacrificed for flow cytometry, or injected with tumor cells. For the dietswitch model (HF-LF), 5w-old female BL6 mice were fed for 15w with HF diet, and then switched to LF diet for 7w prior to sacrifice. Ob/ob model: To control for the effects of adipose tissue content in mice, 4w-old female B6.Cg-Lep-ob (ob/ob; Jackson Laboratory) mice were purchased and maintained on normal rodent diet. These mice gain weight due to a homozygous mutation in the leptin (Lep) gene that causes excessive eating and rapid weight gain. Weight was monitored over time beginning at 5w-old, and mice were euthanized when they reached >40g. This time period was significantly shorter (6w) than that of the DIO model (15w). After 6w, animals were sacrificed for flow cytometry analysis of myeloid cell populations in the lung, or injected with tumor cells for 48h metastasis assays. Balb/c obesity-resistant model: To control for the effects of nutrient content in diet, 5w-old WT female Balb/c mice (Jackson Laboratory) were enrolled on either HF or LF diet (Research Diets, see DIO model ) for 15w. Balb/c animals do not gain weight in response to HF feeding. After 15w, animals were sacrificed for flow cytometry analysis of myeloid cell populations in the lung. Immune compromised mouse models: Two immune compromised mouse models were used in this study, athymic nude (lack T cells) and NOD-scid IL2r-gamma null (NSG; lack mature T, B and NK cells). In both cases, 5w-old female mice (Jackson Laboratory) were used. Mice were treated for 5d with recombinant antibodies, and then sacrificed for flow cytometry analysis of myeloid populations in blood and lung. Preparation of mouse samples for flow cytometry or FACS: Mice were anesthetized with avertin, blood was collected by submandibular bleeding, and cardiac perfusion with PBS was performed. All tissues were mechanically dissociated and filtered through a 40 um mesh to generate a single cell suspension, and red blood cells were lysed (Pharm Lyse; BD Biosciences). Preparation of mouse serum: To collect serum from mice, blood was collected by submandibular bleeding into eppendorf tubes and allowed to clot at room temperature for ~20 minutes. Samples were centrifuged at 2000 x g, 4 C, 10 min. Supernatant was transferred to a polypropylene tube either individually or pooled. For pooled serum, three individual mouse samples were combined and stored at -80 C for downstream applications. 4

5 Policy information about studies involving human research participants 12. Description of human research participants Describe the covariate-relevant population characteristics of the human research participants. Human blood samples: Blood collection from human donors was approved by the Institutional Review Board of Rockefeller University, fully compliant with all relevant ethical regulations regarding research involving human participants, and obtained with informed consent. Fresh whole-blood samples were obtained from healthy female donors (including 5 lean donors (BMI=18-25), and 2 obese donors with BMI equal to or more than 35; donors were all postmenopausal) at Rockefeller University. Human serum samples: Human serum samples were obtained from consenting healthy female donors, and banked as individual or pooled. Serum samples were pooled from 9 lean (BMI=18-25) or 10 obese (BMI>35) postmenopausal women (median age=56 years old; age range 45-66) and stored at -80 C for downstream application. For collection of matched human weight loss serum samples, the clinical trial was conducted at Rockefeller University under ClinicalTrials.gov identifier NCT Sample collection was approved by the Institutional Review Board of Rockefeller University (New York, NY). Mean BMI before weight loss= /-3.4 sd; mean BMI after weight loss= /-3.0 sd; mean age= 60.6 years +/- 3.6 sd. 5

6 Flow Cytometry Reporting Summary Form fields will expand as needed. Please do not leave fields blank. For all flow cytometry data, confirm that: Data presentation 1. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC). Corresponding Author: Johanna A. Joyce Date: Jun 13, The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers). 3. All plots are contour plots with outliers or pseudocolor plots. 4. A numerical value for number of cells or percentage (with statistics) is provided. Methodological details 5. Describe the sample preparation. For flow cytometry of mouse samples, mice were anesthetized with avertin, blood was collected by submandibular bleeding, and cardiac perfusion with PBS was performed. All tissues were mechanically dissociated and filtered through a 40 um mesh to generate a single cell suspension and red blood cells were lysed (Pharm Lyse; BD Biosciences). Cells were counted, incubated with Fc block (1h; BD Biosciences; 1:100/10^6 cells), incubated with fixable live/dead stain (30 min; Invitrogen), and then incubated with conjugated antibodies (1h). Alternatively, DAPI was used for dead cell exclusion instead of fixable live/dead stain. Mouse neutrophils were defined as CD45+CD11b+Gr1+/hi or CD45+CD11b +Ly6CloLy6G+. CD45+CD11b+Gr1lo cells were determined to be CD45+CD11b+Ly6Chi monocytes, and were therefore excluded from all neutrophil gating. OneComp ebeads (ebioscience) or ArC Amine Reactive Compensation Beads (Invitrogen) were used for compensation. 6. Identify the instrument used for data collection. A BD LSRFortessa was used for flow cytometry, and a BD FACSAria III was used for FACS. 7. Describe the software used to collect and analyze the flow cytometry data. 8. Describe the abundance of the relevant cell populations within post-sort fractions. FlowJo was used for all flow cytometry and FACS data analysis, and for generating representative flow plots. In all cases, post-sort purity was confirmed to be greater than >90% for downstream applications, including qrt-pcr and cytospin. 9. Describe the gating strategy used. Mouse gating strategy: In all cases, dead cells and debris were excluded from analyses using FSC x SSC, a live/dead stain and/or DAPI. CD45+ was used as a marker for total leukocytes, CD11b+ was used as a marker for myeloid cells. Neutrophils were further defined as CD45+CD11b+Gr1+/hi or CD45+CD11b+Ly6CloLy6G+. CD45+CD11b+Gr1lo cells were determined to be CD45+CD11b +Ly6Chi monocytes, and were therefore excluded from all neutrophil gating. Eosinophils were defined as CD45+CD11b+ nature research flow cytometry reporting summary June

7 myeloid cells with high side scatter, and Siglec-f+. In some cases, further gating on IL5ra+ or Ki67+ populations was performed for eosinophils, monocytes, and neutrophils as defined here. For CFSE assays, bulk T cells were gated as CD45+CD3+ and then further gating according to positive CD4 or CD8 status for helper and cytotoxic T cells, respectively. For NK cell cytotoxicity assays, NK cells were defined as CD45+ with low side scatter, NK1.1+. Gating on CD107a+ populations was used to further define NK cells with cytotoxic function. Human gating strategy: In all cases, dead cells and debris were excluded from analyses using FSC x SSC and DAPI. CD45+ was used as a marker for total leukocytes, CD11b+ was used as a marker for myeloid cells. Gating strategy using cell surface markers were as follows: Peripheral blood neutrophils (CD45+CD11b+CD66b+CD16 +CD14lo), eosinophils (CD45+CD11b+CD66b+CD16-CD14lo), nonclassical monocytes (CD45+CD11b+CD66b-CD16+CD14lo), intermediate monocytes (CD45+CD11b+CD66b-CD16+CD14hi), and classical monocytes (CD45+CD11b+CD66b-CD16-CD14+). Gating on IL5R+ cells was also included in analysis for each population. Eosinophils were used as a positive gating control for IL5R positivity as this is a canonical marker/signaling pathway for this cell type. Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information. nature research flow cytometry reporting summary June