Stromal-driven and Amyloid β-dependent induction of neutrophil extracellular traps modulates tumor growth

Tumors consist of cancer cells and a network of non-cancerous stroma. Cancer-associated fibroblasts (CAF) are known to support tumorigenesis, and are emerging as immune modulators. Neutrophils release histone-bound nuclear DNA and cytotoxic granules as extracellular traps (NET). Here we show that CAFs induce NET formation within the tumor and systemically in the blood and bone marrow. These tumor-induced NETs (t-NETs) are driven by a ROS-mediated pathway dependent on CAF-derived Amyloid β, a peptide implicated in both neurodegenerative and inflammatory disorders. Inhibition of NETosis in murine tumors skews neutrophils to an anti-tumor phenotype, preventing tumor growth; reciprocally, t-NETs enhance CAF activation. Mirroring observations in mice, CAFs are detected juxtaposed to NETs in human melanoma and pancreatic adenocarcinoma, and show elevated amyloid and β-Secretase expression which correlates with poor prognosis. In summary, we report that CAFs drive NETosis to support cancer progression, identifying Amyloid β as the protagonist and potential therapeutic target.

-Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. For in vitro experiments no statistical methods were used to determine sample size, however, we performed at least 3 biological replicates with multiple technical replicates for every treatment condition (as stated in figure legends). In some experiments, sample number was increased to improve statistical power.
Generally, in vivo tumour models included 7-8 mice per treatment group. For all other in vivo studies, we performed assays on at least 4 mice in each treatment group. The number of mice used in each experiment is stated in the respective figure legends. Sample sizes were calculated based on previous experience and a priori power analysis (G* Power).
No data were excluded All the experiments were replicated. The number of replicates is stated in figure legends for each figure. This includes the number of biological replicates (n number) and technical replicates performed.
For all ex vivo experiments, neutrophils were isolated from mice of different sexes and ages. For spontaneous tumor models (skin and pancreatic), mice of various ages and sexes were used and randomly allocated to treatment groups. Randomization was not possible for in vitro assays involving fibroblast and tumor cell lines.
Where possible, technicians performing the experiment were blinded to experimental groups and treatments.
For all assays, the investigators were not blinded to group allocation during experiments as data collection, analysis and outcome assessment required that the investigators were not blinded. All antibodies obtained from commercial sources, with extensive validation for each application by the vendor, and referenced in literature. Antibodies have also been tested used extensively in other studies within the lab as well as several pilot studies.
Skin CAFs and tumour cells isolated from Tyr::CreER; BrafCA; Ptenlox/lox primary skin tumours. Lung CAFs isolated from inducible LSLKrasG12D/+;p53LSL-R270H/ER primary lung tumours. Pancreatic CAFs and tumour cells isolated from LSL-KrasG12D/+;LSLTp53R172H/+;Pdx-1-Cre primary pancreatic tumours. Matched healthy fibroblasts were taken from healthy skin, lung and pancreas of mice on the same genetic background as the tumour models. Neutrophils were isolated from bone marrow of C57BL/6 mice. C57BL/6 derived B16 F10 melanoma cell line was purchased from American Type Culture Collection (ATCC)

Not performed
No mycoplasma detected in established cell lines.
No commonly misidentified cell lines were used. Tyr::CreER; BrafCA; Ptenlox/lox (Purchased from The Jackson Laboratory; stock number 013590). 8-32 week old male and female mice were used for these experiments.

April 2020
Wild animals

Field-collected samples
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Instrument Software Cell population abundance Gating strategy PAD4KO and littermate controls (provided by Markus Hoffmann). 16-20 week old female mice were used in these experiments.
No wild animals were used in this study.
This study did not involve sample collection from fields. This is included in the Methods section As described in the Methods: Tumors were minced using a razor and digested with 1mg/ml collagenase A and collagenase D and 0.4mg/ml DNase I in PBS at 37°C for 2h with rotation at 600rpm. 10mM EDTA was then added to stop the enzymatic reaction. The cell suspension was passed through a 70"m filter and stained with live/dead fixable violet stain (Thermofisher Scientific). Cells were subsequently stained with the following fluorescently conjugated antibodies; CD45 (30-F11), Ly6G (1A8), F4/80 (BM8), CD11b (M/170), CD11c (N418), Thy1 (30-H12), Podoplanin (8.1.1.), PDGFR! (APA5; all from Biolegend) and CD31 (390; eBioscience) at 1:300 dilution. Unstained and single-stained compensation beads (Invitrogen) were run alongside to serve as controls.
For bone marrow neutrophils, cells were aspirated from the femurs and tibias of C57BL/6 WT mice or tumour bearing mice and separated from the rest of the bone marrow using a Histopaque 1077/1119 gradient. The cells were harvested from the interface of the two components of the gradient and stained as above with antibodies.
Flow cytometry was performed on LSR Fortessa (BD Biosciences) analyzers.
Offline analysis was carried out on FlowJo (Treestar).

N/A -sorting was not performed
All cell populations were characterised by first excluding debris (using SSC-A and FSC-A) and then gating on single cells (using FSC-A and FSC-H to distinguish singlets and doublets). Dead cells were excluded by gating on LIVE/DEAD® Fixable Violet Cell Stain. Cell populations were distinguished using the following antibody combinations: Immune cells: CD45+