Mice

C57BL/6, Ly5.1, Rag1−/−, Ldlr−/−, and B6.Cg-Aw-J EdaTa-6J+/+ArTfm/J mice were purchased from Jackson Laboratory. Age and sex matched controls were used for all of our studies. The mice were housed in a 12-h light: dark cycle at 22 °C–25 °C temperature with relative humidity of 50–70 percent, and given ad-libitum access to food and water for the duration of the study. Mice were housed in specific-pathogen-free conditions and cared for in accordance with US National Institutes of Health guidelines, and all procedures were approved by the Cedars-Sinai Animal Care and Use Committee. The number of animals used in various experiments ranged between 5 and 13 in each group, as specified in the legend of each figure.

Cells and tumor models

The mouse melanoma cell line B16F10 was obtained from ATCC® (CRL-6475) and was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml). The mouse melanoma cell lines YUMM1.7 and YUMM3.1 were a generous gift from Marcus Bosenberg (Yale University, New Haven, CT). These cell lines were created from the genetically engineered mouse model of inducible melanoma harboring the following mutations: BrafV600E; Cdkn2a−/−; (YUMM1.7 and YUMM3.1) and Pten−/− (YUMM1.7 only). These mice, B6.Cg-Braftm1Mmcm Ptentm1Hwu Tg(Tyr-cre/ERT2) 13Bos/BosJ, are available for purchase at Jackson Laboratory. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml).

For lung metastatic models, B16 (2 × 105), YUMM1.7 (5 × 105), or YUMM3.1 (4 × 105) cells were injected via tail vein and were enumerated visually facilitated by the use of a magnifying glass and a lamp or by extracted melanin determined by spectrophotometry (for B16 only). To extract melanin, the entire left lobe was homogenized and digested in 300 μL of PBS. After centrifugation for 10 min at max speed, the supernatant was removed. The samples were then processed in 1 mL of lysis buffer containing 1 M tris-Hcl, 10%SDS, 0.5 M EDTA, 10 μg/mL Proteinase K, and water in a 56 °C shaker until completely dissolved. This process can take up to 48 h and was aided by the addition of additional proteinase K and processing through an 18-gauge needle. Once the melanin was dissolved, samples were centrifuged for 10 min at max speed and the supernatant discarded so that a black pellet remained at the bottom. 200 μL of 2 N NaOH was added and the sample placed in a shaker at 95 °C overnight or until completely re-dissolved. Then, 100 μL of 2 chloroform:1 methanol was added and the sample mixed well before being centrifuged for 10 min at max speed. 100 μL of the top layer was read on a SpectraMax spectrophotometer: OD405-OD570. Resulting values were analyzed directly. For YUMM1.7 and YUMM3.1 tumors, lungs were injected with India ink and then destained using Fekete’s solution so that tumors appeared white in color. For subcutaneous tumors, 2 × 105 B16 or 2.5 × 105 YUMM1.7 cells were injected into the right flanks of C57BL mice and were measured with calipers; size was expressed as one-half the product of perpendicular length and square width in cubic millimeters. For survival studies, mice were euthanized when endpoint criteria of a Body Condition Score <2 were met. For high fat diet studies, 6-week old Ldlr−/− mice were placed on a Western Diet containing 21% fat and 0.2% cholesterol with 42% of calories coming from fat (Harlan Teklad, TD88137) consumed ad libitum for 12 weeks prior to inoculation of tumor cells and continued until sacrifice. Serum cholesterol levels were quantified using an enzymatic colorimetric test of total cholesterol according to the manufacturer’s protocol (Cholesterol E, Wako, 439-17501).

Histopathological analysis

Lungs were fixed in formalin, parrafin-embedded, and sectioned. For immunohistochemistry of neutrophils, slides were stained with anti-Ly6G monoclonal antibody (mAb) (Clone 1A8, BP0075, Bio X Cell) or control rat IgG (14131, Sigma) at 1:50 overnight at 4 °C. Slides were washed with PBS 3× and stained with donkey-anti-rat IgG (AlexaFluor594, Thermofisher Scientific, A-21209) at 1:500 for 1 h at room temperature. Slides were then washed 3× with PBS and were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen, P36931). Images were obtained on a Keyence BZ-9000 microscope using ×20 magnification (Keyence). Neutrophils were identified by positive Ly6G staining and the distance from the neutrophil to the closest B16 tumor edge was calculated using the Keyence software. Neutrophils within 100 µM of a tumor nodule were included65. Corresponding H&E stains (MilliporeSigma, hematoxylin HHS32 and eosin 230251) of sequential tissue samples were used to confirm the location of B16 tumor nodules.

Cell specific depletions

In vivo neutrophil depletion was performed by intraperitoneal (i.p.) injection of 500 μg sterile anti-Ly6G monoclonal antibody (Clone 1A8, BP0075, Bio X Cell) or control rat IgG (14131, Sigma) to both female and male C57BL/6 mice beginning one day prior to tumor inoculation and was continued to maintain depletion throughout the tumor study (days-1, 1, 4, 8, and 11). Depletion of neutrophils (Gr1hi) in lungs was confirmed by flow cytometry at the time of sacrifice.

NK cell depletion was performed by i.p. injection of 200 μg sterile anti-NK1.1 mAb (Clone PK136, BE0036, Bio X Cell) or control mouse IgG (I5381, Sigma-Aldrich) to both female and male C57BL/6 mice beginning one day prior to tumor inoculation and was continued to maintain depletion throughout the tumor study (days-1, 1, 4, 8, and 11). Depletion of NK1.1 cells in lungs (NK1.1+CD49b+) was confirmed by flow cytometry at the time of sacrifice.

Ovariectomy

Four weeks prior to tumor inoculation, 8-weeks-old C57BL/6 mice underwent ovariectomy or sham-surgery. Mice were maintained on inhalation anesthesia (1.5% Isoflurane) via nose-cone. Prior to the start of the surgery, carprofen (5 mg/kg body weight) was administered subcutaneously. The area below the ribs was shaved and cleaned with betadine followed by alcohol. This area was then lifted with forceps to make a small 2 mm horizontal cut. Resorbable vicryl sutures were used to clamp the horn beneath the ovary and each ovary was removed using forceps and scissors. The uterine horns were then placed back into the body and the peritoneal cavity was closed using interrupted resorbable vicryl sutures. The skin was closed with interrupted nylon sutures. Following spontaneous movement, buprenorphine (0.5 mg/kg body weight) and 300 µL of warm saline were administered subcutaneously. In sham operated mice, both the skin and inner skin membrane were incised. The ovaries were externalized and returned to the abdominal cavity and the wound was sealed with interrupted nylon sutures.

Castration and testosterone implantation

Four weeks prior to tumor inoculation, C57BL/6 mice of 8 weeks of age were castrated or sham-operated. Mice were maintained on inhalation anesthesia (1.5% Isoflurane) via nose-cone. Prior to the start of the surgery, carprofen (5 mg/kg body weight) was administered subcutaneously. The area between the penis and the anus was shaved and cleaned with betadine followed by alcohol to disinfect the scrotum. The area between the penis and the anus were lifted to make a small 1 mm horizontal cut. To remove the testes, a small 1 mm cut into the inner skin membrane enclosing the testicles was made and the testicles were exteriorized. Testicular arteries were tied off using resorbable vicryl sutures prior to removing the testes. Once the testes were removed, the wound was sealed with two nylon sutures. Following spontaneous movement, buprenorphine (0.5 mg/kg body weight) and 300 µL of warm saline were administered subcutaneously. In sham operated mice, both the skin and inner skin membrane between the penis and anus were incised. The testes were drawn out and placed back and the wound was sealed with interrupted nylon sutures.

For hormone replacement, at the time of castration, testosterone pellets (1.5 mg or 50 mg dose, 60 day release, SA-151; Innovative Research of America) or placebo pellets (1.5 or 50 mg dose, 60 day release, SC-111; Innovative Research of America) were implanted subcutaneously. For the flutamide studies, 8 week old male and female mice were implanted with either a placebo pellet or a flutamide pellet (100 mg, 21 day release, A-152; Innovative Research of America) one week prior to B16 implantation. Hair from a small area behind the neck was removed with electric clippers and cleaned with betadine followed by alcohol. A small incision was made and a pellet (1 cm × 1 cm in size) containing testosterone or biodegradable matrix (placebo) were implanted subcutaneously on the lateral side of the neck between the ear and shoulder. The wound was closed with interrupted nylon sutures.

Bone marrow transfer

C57BL/6 or Ly5.1 mice at 8 weeks of age were lethally irradiated once with 950 rads using a 137Cesium gammacell source (Gammacell 40 Cs γ-irradiation; GammaCell Bio-Technologies). Four hours post irradiation, 1 × 107 cells were injected via tail vein under brief anesthesia while in a mouse restraint. The bone marrow cells were collected by flushing the femurs of donor mice (C57BL/6, ArTfm, or Ly5.1) under sterile conditions and were prepared as a single cell suspension using a 70 μM nylon filter (BD, Falcon, 352250). The immune system was allowed to reconstitute for 8–12 weeks before tumor cells were injected. Complete bone marrow reconstitution was confirmed by flow cytometry analysis of the expression of CD45.2/Ly5.2 (from C57BL/6 mice) and CD45.1/Ly5.1 (from Ly5.1 mice).

Complete blood count

Fifty microliter of mouse blood was collected via retro-orbital bleeding in an EDTA coated tube and was processed the same day. A complete blood count/differential was performed by the Division of Laboratory Medicine at UCLA (Los Angeles, CA) on a Drew Scientific HemaVet hematology analyzer.

Testosterone level quantification

Mouse serum was obtained at the time of sacrifice and testosterone levels were assayed using a commercially available Testosterone EIA kit (582701; Cayman Chemical) according to the manufacturer’s protocol.

Quantitative real-time PCR

Total RNA was extracted by RNeasy extraction kit (Qiagen) and reverse transcribed using PrimeScript RT Reagent Kit (Takara Bio) according to the manufacturer’s protocols. Real-time polymerase chain reaction (qPCR) was performed using SYBR Green Master Mix (Takara Bio) and was run on a CFX96 Touch Real-Time PCR Detection System (BioRad). GAPDH or beta actin served as a loading control. The following primer sequences were used for cDNA quantification: AR fwd 5’-GCCTCCGAAGTGTGGTATCC-3’, rev 5’-TGGTCCCTGGTACTGTCCAA-3’; Cxcr2 fwd 5’-GGTGGGGAGTTCGTGTAGAAG3’, rev 5’-CTACTACACAGGGATCAGGGC-3’; Cxcr4 fwd 5-GCCATGGAACCGATCAGTGTGA-3’, rev 5’-GATGAAGTAGATGGTGGGCAGG-3’; L-sel (CD62L) fwd 5’-TACTGGGGCTCGAGGAACAT-3’, rev 5’-TCCCAGTTCATGGGCTTTTCA-3’; Nfkb2 fwd 5’-CGCCTCTCTTCACCTTAGGC-3’, rev 5’-CAGGCCTGGATCGTAGCAAT-3’; Rab4a fwd 5’-GAGAAACCAAGAACTGCCCCT-3’, rev 5’-AGCGCATTGTAGGTTTCTCG-3’; Rel fwd 5’-GAGAAACCAAGAACTGCCCCT’3’, rev 5’-AACTCCTGAAGACCTGGGCA; Stat3 fwd 5’-ATGGATGCGACCAACATCCT-3’, rev 5’-CAATGGTATTGCTGCAGGTCG-3’; VLA4 fwd 5’-TTGGCTACTCGGTGGTGCTG-3’, rev 5’-ATGTCTTCCCACAAGGCTCTC-3’.

RT2 profiler PCR array

Neutrophils from lungs or prostate lobes were isolated from sham male and castrated males and were pooled with 5–6 mice per group. RNA was extracted using an miRNA extraction kit (miRNeasy, Qiagen, 217004). Synthesis of cDNA was performed using the RT2 First Strand Kit (Qiagen, 330401) and cDNA was amplified using the RT2 PreAMP cDNA synthesis kit (Qiagen, 330451) and AR pathway primer mix (Qiagen, PBM-142Z) for neutrophils. The RT2 Profiler PCR Array Mouse Androgen Receptor Signaling Targets (Qiagen, 330231, PAMM-142ZD-2) plates were run on a CFX96 Touch Real-Time PCR Detection System (BioRad). All steps were performed according to the manufacturer’s protocol.

Western blot

B16, YUMM1.7, and LnCaP cells (ATCC, clone FGC, CRL-1740) were pelleted and lysed in ice-cold Lysis buffer consisting of Tris (25 mM; Bio Rad; catalog 161-0719), Triton x-100 (1%; Sigma: catalog T8787), Glycerol (10%; Fisher Scientific; catalog BP229-1), EGTA (4 mM; Sigma; catalog E-4378), pH adjusted to 7.8 with dilute o-phosphoric acid. The lysates were passed 5 times through a 25-gauge needle for complete homogenization. Mouse testes were mechanically homogenized using an 18-gauge needle in ice-cold lysis buffer. Testes homogenates were then centrifuged at 10,000×g for 10 min at 4 °C to remove unbroken cells and debris. Protein quantification was performed on supernatants using bicinchoninic copper assay (ThermoFisher Scientific, catalog 23225). Equal masses of protein were loaded in 10% SDS page gels and transferred overnight at 30 V to a PVDF membrane (ThermoFisher Scientific, catalog 88518) at 4 °C. The membrane was cut below the 63 kDa and anti-AR (Santa Cruz Biotechnology, catalog sc-7305; top portion of membrane) and anti-β-Actin (Sigma, catalog A5316; bottom half of membrane) antibodies were diluted at a concentration of 1:500 and 1:10,000, respectively, in blocking buffer consisting of 5% nonfat dry milk (Bio Rad, catalog 1706404) dissolved in TBS-T. HRP-conjugated goat anti-mouse (Jackson ImmunoResearch, catalog 115-035-003) secondary antibodies were diluted at a concentration of 1:10,000 in blocking buffer. Chemiluminescent substrate (ThermoFisher Scientific, catalog 34577) was applied to membranes, and bands were imaged via Bio-Rad Chemidoc (Bio-Rad).

Human study population

Informed consent and blood samples were collected under Cedars‐Sinai Medical Center Institutional Review Board-approved Pro00042197. For this study, a separate IRB-approved protocol (Pro00047412) was obtained to specifically study neutrophils. Patient ages ranged from 52-80 years (median 72). The patients were white (n = 16), black (n = 5), other (n = 2), Asian (n = 5), Hispanic (n = 1), and American Indian (n = 1). Patient samples were classified into two categories: patients not receiving any current treatment and patients undergoing active androgen deprivation therapy. Supplementary Table 1 indicates clinical characteristics of each patient sample used in this study.

Neutrophil and NK cell isolation

To isolate murine neutrophils or NK cells from the bone marrow, spleen, or lungs, an EasySep Positive PE Selection Kit (19762 A) or Negative Selection Mouse Neutrophil Enrichment Kit (18557) was used according to the manufacturer’s protocols (Stemcell Technologies). The PE murine antibody used was Ly6G (1 A8; BioLegend, 127608) or NK (PK136; BioLegend, 108707). Samples were pooled from 3-5 mice for each independent experiment and purity was confirmed if >93% by flow cytometry or by Siemens Diff-Quik Stain Set (B4132-1A, Fisher Scientific).

For human neutrophil isolation, peripheral blood mononuclear cells (PBMC) were removed using SepMate tubes and the Lymphoprep density gradient according to the manufacturer’s protocol (85450;07801 StemCell Technologies). Once the PBMCs were removed, the pelleted blood was transferred to a new tube and the red blood cells were lysed using 1X RBC lysis buffer (eBioscience). Cells were stained using the Siemens Diff-Quik Stain Set to confirm >94% purity. Cells were maintained in Endothelial Basal Medium-2 (EBM2, CC-3156, Lonza) which was confirmed to not activate neutrophils.

Cytotoxicity assays

B16 cells were plated 24 h prior to the experiment at 5 × 103 per well in a 96 well flat bottom plate. Neutrophils or NK cells were isolated from the lungs of B16 tumor-bearing mice (pooled 3 mice/sample) and were co-cultured with the B16 cells for 4 h at effector:target ratios of 3:1 and 6:1 with blank wells, effector only or target only serving as controls. All supernatant was removed and remaining adherent B16 cells were fixed with 4% paraformaldehyde overnight at 4 °C. Fixing solution was then discarded and 100 µL/well of crystal violet stain was added and plate was placed on a rocker for 30 min. Plates were then washed under running DI water for 15 min. The crystal violet was dissolved by adding 100 µL/well of 10% acetic acid with rocking and plate was read at absorbance 600 on a SpectraMax M2 spectrophotometer.

Neutrophil scoring

Isolated neutrophils were transferred to slides using a Cytospin 4 centrifuge (Thermo Fisher Scientific) and were stained with the Siemens Diff-Quik Stain Se (Thermo Fisher Scientific) to visualize neutrophil maturation based on nucleus morphology25. Each subpopulation of neutrophils was assigned an arbitrary number of 1 having a banded nuclei (immature) or 2 having a segmented nuclei (mature). Images were obtained on a Keyence BZ-9000 microscope using 40x magnification (Keyence). Greater than 150 neutrophils per sample were scored by two investigators and scores were only included if both investigators were in agreement.

Flow cytometry

The same lobe of the lung was manually digested in a lysis buffer containing HBSS, collagenase Type IV (2 µg/mL; 17104019; Thermo Fisher Scientific), and DNAseI (25 units/mL; 10104159001; Sigma-Aldrich) with two 10 min incubations at 37 °C. Samples were centrifuged for 5 min at 3000 RPM and the red blood cells were lysed using 1X RBC lysis buffer (eBioscience). Single cell suspensions were obtained from the digested lung and were incubated with FcBlock (CD16/CD32, Clone 2.4G2; 70-0161-M001; Tonbo Biosciences). The following murine antibodies against the respective antigens were used for flow cytometry at 1:50 concentration: neutrophils: Ly6G (1A8; PE; BioLegend, 127608) or Ly6G (Gr-1; PE; eBioscience, 12-5931-82); macrophages: CD11b (M1/70; PerCP-Cy5.5; Tonbo Biosciences, 35-0112) and F4/80 (BM8.1; APC; Tonbo Biosciences, 20-4801); dendritic cells: CD11b and CD11c (N418; vF450; Tonbo Biosciences, 75-0114); CD4 T cells: CD4 (RM4-5; APC; Tonbo Biosciences, 35-0042); CD8 T cells: CD8a (Ly-2; PerCP-Cy5.5; eBioscience, 15-0081-82); natural killer cells: NK1.1 (PK136; APC; eBioscience, 17-5941-82) or CD49b (DX5; PE; eBioscience); CD69 activation marker: CD69 (H1.2F3; PE; eBioscience, 12-0691-83); BMT confirmation: CD45.1 (A20; PerCp-Cy5.5; eBioscience, 45-0453-80) and CD45.2 (104; FITC; Tonbo Biosciences, 35-0454) and cells were fixed with 2% paraformaldehyde. For intracellular protein staining, the cells were washed and resuspended in permeabilization buffer (eBioscience, 00-5523) and stained by antibodies in the permeabilization buffer for 1 h on ice in the dark. The cells were then pelleted and resuspended in the flow cytometry staining buffer for flow cytometry analysis.

For the analysis of human neutrophils, FcR blocking antibody (130-059-901, MACS Miltenyi Biotec) was used at a concentration of 20 μL/107 cells. The following human antibodies were used: Cd11b (ICFR44; PE, Tonbo Biosciences, 50-0118-T100), CD14 (HCD14;APC/Cy7, BioLegend, 325620), CD15 (MMA; APC, eBioscience, 17-0158-42), CD16 (CB16; Alexa Fluor 700, eBioscience, 56-0168-42), CD33 (P67.6; eFluor 450, eBioscience, 48-0337-42), CD45 (HI30; violetFluor 450, Tonbo Biosciences, 75-0459-T100), CD66b (G10F5; APC, eBioscience, 17-0666-42), HLA-DR (L243; FITC, Tonbo Biosciences 35-9952-T100), Mouse IgG1 K IsoControl (P3.6.2.8.1; eFluor 450, eBioscience, 48-4714-82), and MPO (MPO455-8E6; eFluor 450, eBioscience, 48-1299-42).

For ROS staining, cells were incubated with 0.5 μg/mL dihydrorhodamine 123 (DHR123, CAS 109244-58-8; Santa Cruz Biotechnology) for 5 min with agitation at 37 °C and either remained unstimulated or were stimulated with 0.3 μM of phorbol myristate acetate (PMA) for 30 min at 37 °C. Following incubation, cells were washed and stained with CD11b and Ly6G (1A8) antibodies for murine cells or CD11b, CD45, CD66b and CD16 for human cells and were assessed by flow cytometry. Stained cells were analyzed on an LSRII Fortessa (BD Biosciences) and data were processed using FlowJo, version 10.5 (Tree Star Inc.).

Phagocytosis assay

Neutrophils from the lungs of B16-tumor bearing mice were plated at 2.5 × 105/well in a 24 well plate and 5 µL/well of FITC-labeled zymosan A S. cerevisiae beads (ThermoFisher Scientific, Z2841) were added. Following a brief spin, cells were incubated for 30 min at room temperature. The media was removed and the wells were washed with PBS. The cells were lifted by incubating for 15 min in PBS/5 mM EDTA/1 mM sodium azide plus proteinase K to cleave any beads on the surface of the cells. The cells were then stained for neutrophil markers as above and analyzed by flow cytometry.

NETosis staining

NETosis was induced in 2.5 × 104 neutrophils suspended in 250 μL of EBM2 media in 8-well Millicell EZ glass chamber slides (Millipore, PEZGS0816). Briefly, unstimulated cells were maintained in a 37 °C incubator for 1 and 3 h and then fixed with 4% paraformaldehyde. The slides were washed with sterile PBS three times for 5 min each and then stained with 1:2000 SYTOX orange (ThermoFisher Scientific, S11368) and 1:500 MPO (Abcam, ab9535) for 1 h at 37 °C. Slides were then washed 3× with PBS for 5 min each and then stained with 1:500 Goat pAb to Rb IgG (Abcam, ab 150077) for 1 h at room temperature. Slides were washed with PBS 3×, the chamber slide was removed according to the manufacturer’s protocol and the slides were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen, P36931). Four images of each time point were obtained on a Keyence BZ-9000 microscope at ×40 magnification. The total number of cells was automatically calculated with the Keyence BioAnalyzer software and the number of neutrophils was counted by a blinded scientist. The percentage of NETosing neutrophils was calculated as an average of the number of NETs divided by total number of cells/per image.

Invasion and migration assay

Corning transwells polyester membranes (6.5 mM, 3.0 μM pore size, Sigma Aldrich, CLS3472) were coated with human fibronectin at 10 μg/mL (Sigma Aldrich, F089S) for 1 h at 37 °C. Wells were then washed 2× with sterile PBS and dried overnight. For invasion/migration assays, the bottom chamber contained 600 EBM2 media μL with or without 100 nM N-formyl-met-leu-phe (Sigma Aldrich, 47729-10mg-f) as a chemoattractant. Neutrophils (2.5 × 104) suspended in 200 µL of EBM2 media were placed in the upper chamber and were allowed to migrate for 2 h at 37 °C. Membranes were carefully aspirated, washed twice with PBS and stained with crystal violet for 10 min and rinsed with water until clear. Five images of each transwell were obtained on a Keyence BZ-9000 microscope at ×40 and the area of crystal violet was analyzed using the Keyence Bioanalyzer software. The results from 5 images were averaged together and a percent change in migration was calculated by dividing the percent area of transwell stimulated with a chemoattractant divided by percent area without chemoattractant for each individual patient.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 6.0. Data are presented as mean ± SEM. For selection of appropriate statistical tests, recorded experimental data were subjected to the D’Agostino-Pearson omnibus test to detect normal, Gaussian distribution. 2-tailed Student’s t test or 1-way ANOVA (Tukey’s post-test) were used for comparisons of parameters among two or three groups, respectively. The non-parametric Mann-Whitney U test was substituted for the student’s t test when data did not follow a normal distribution. For neutrophil maturation studies, the t test for two independent proportions was used. For patient data, a multivariable linear regression analysis with adjustments for presence of prostatectomy, age, race and current status was performed by the Biostatistics Core at Cedars Sinai. White blood cell count was not available for analysis and thus was not controlled for. The value of p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001. Statistical parameters can be found in the figure legends.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.