Abstract: 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

Euler, Maximilien

Hoffmann, Markus

Janowitz, Tobias

Jones, James O.

Martins, Carla P.

Munir, Hafsa

Shields, Jacqueline D.

Welsh, Sarah J.

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

Jones, James O

Martins, Carla P

Welsh, Sarah J

Shields, Jacqueline D

Apollo (Cambridge)

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

Cold Spring Harbor Laboratory Institutional Repository

SUPPLEMENTARY INFORMATION FILE  Stromal-driven and Amyloid β-dependent induction of neutrophil extracellular traps modulates tumour growth  Hafsa Munir1, James O. Jones1,2, Tobias Janowitz3,4,5, Markus Hoffmann6, Maximilien Euler6, Carla P. Martins1, Sarah J. Welsh2, Jacqueline D. Shields1*.   A.C.D. E.Skin FBs Skin CAFs Lung FBs Lung CAFs Pancreatic FBs Pancreatic CAFsUnstained ctrlCD45 EpCAMThy1PodoplaninPDGFRaB.F.MPO   DAPI MPO    CitH3    DAPIUntreated FB CMedCAF CMedPMA051015Neutrophil treatment%7-AAD+Annexin V+ neutrophilsPancreaticUntreated FB CMedCAF CMedPMA051015Neutrophil treatment%7-AAD+Annexin V+ neutrophilsLungNone FB CAF050010001500CMed treatmentgMFI CD11b*None FB CAF050010001500CMed treatmentgMFI CD18None FB CAF02004006008001000CMed treatmentgMFI CD18None FB CAF0500100015002000CMed treatmentgMFI CD62LNone FB CAF0200040006000800010000CMed treatmentgMFI CD62L None FB CAF0200040006000800010000CMed treatmentgMFI CD11b*LPS treatedUntreatedUntreated CAF CMedPMA0123Neutrophil treatmentrelative gMFI DCFDA***Untreated CAF CMedPMA0246Neutrophil treatmentrelative gMFI  beads***                                    Sup Fig. 1. Neutrophil activation and function are unaltered by short term stimulation with CAF-derived factors. (A) Characterization of skin, lung and pancreatic FBs and CAFs by flow cytometry showing that the cells express the classic CAF markers (Thy1, Podoplanin, PDGFR) on the surface and lack expression of tumor cell (EpCAM) and immune cell (CD45) markers. (B) Quantification of neutrophil death after treatment with FB CMed, CAF CMed from lung or pancreas or PMA based on expression of Annexin V and 7-AAD by flow cytometry. (C) Quantification of CD11b, CD18 and CD62L expression on neutrophils after treatment with lung FB or CAF CMed with and without simulation with LPS by flow cytometry. (D) Quantification of ROS production by neutrophils after treatment with lung CAF CMed or PMA based on the levels of DCFDA by flow cytometry. (E) The phagocytic capacity of neutrophils after lung CAF CMed or PMA treatment based on uptake of fluorescent 1µm beads assessed by flow cytometry. (F) Representative confocal image of neutrophils (arrows) and NETs (arrowheads) in murine lung cancer, scale bar 50µm. Data are mean ± SEM; * = p<0.05, ** = p<0.01 and *** = p<0.001 using (C) paired t-test (comparing each condition) and (D-E) one-way ANOVA with a Dunnett post hoc test. Assays were performed on (A) n=1, (B) n=2, (C) n=4, (D) n=8 (Untreated and CAF CMed) and n=5 (PMA), (E) n=5 and (F) Representative of n=3 independent experiments.                          Sup Fig. 2. CAF-induced NETosis is a ROS-dependent, autophagy independent response. (A) Schematic of NETosis assay (B) Representative phase contrast images of SYTOX green stained dead neutrophils and NETs after 3h treatment with PMA or pancreatic CAF CMed. Inset; zoomed image of a NET (green haze) outlined with a dashed white line. Quantification of the relative NET coverage of neutrophils treated with lung CAF CMed with or without treatment with (C) Chloroquine (Cl), (D) Diphenyleneiodonium (DPI), (E) Trolox and (F) Vitamin C. Data are mean ± SEM; * = p<0.05, ** = p<0.01 and *** = p<0.001 using (C-F) one-way ANOVA with a Tukey post hoc test. Assays were performed on (B) Representative of n=7, (C) n=5, (D) n=4, (E-F) n=2 (in triplicate) independent experiments. Scale bars correspond to 50µm.    A. B.C.No tumour Tumour051015% dead neutrophils***PancreaticNo tumour Tumour01020304050% dead neutrophilsLungNo tumour Tumour0102030% dead neutrophils*SkinBasal mediaFB CMedCAF CMed0204060Media I/V infused% dead neutrophils***LungBasal mediaFB CMedCAF CMed010203040Media I/V infused% dead neutrophils******PancreaticCAF CMed CAF CMed+Cl-amidine0102030% dead neutrophils***                  Sup Fig. 3. CAF-derived factors drive NETosis systemically. (A) Quantification of the percentage death of bone marrow neutrophils taken from mice with pancreatic, lung or skin tumors. (B) Quantification of percentage death of bone marrow neutrophils taken from wild type mice intravenously infused with pancreatic or lung FB or CAF CMed 24h before performing the NETosis assay.  (C) Quantification of percentage death and relative number of bone marrow neutrophils taken from mice intravenously infused with lung CAF CMed with or without pre-treatment with Cl-amidine for 24h. Data are mean ± SEM; * = p<0.05, ** = p<0.01 and *** = p<0.001 using (A and C) paired t-test and (B) one-way ANOVA with Dunnett post hoc test. Assays were performed on (A) n=4 (in duplicate), n=7 (in quadruplet) and n=7 for pancreatic, lung and skin tumor bearing mice respectively, (B) n=8, 4 and 8 (Pancreatic for basal media, FB CMed and CAF CMed treatment, respectively. All performed in triplicate) and n=9, 6 and 9 (Lung for basal media, FB CMed and CAF CMed treated, respectively. Performed in duplicate for basal media and CAF CMed and only a single time for FB CMed treated), (C) n=8 (in triplicate) independent experiments.    Vehicle Cl-amidine0102030% CD45+ (% live cells)Vehicle Cl-amidine05101520% Ly6G+ (% CD45+)Vehicle Cl-amidine020406080100% CD11b+ (% CD45+)Vehicle Cl-amidine0204060% F4/80+ (% CD45+)A. B.C.F.1 2 3 4 5 6 7 8012345Treatment dayRelative tumour volume Vehicle Cl-amidine treated******Vehicle Cl-amidine0204060% Thy1+ (% live cells)Vehicle Cl-amidine0204060% Podoplanin+ (% live cells)Vehicle Cl-amidine05101520% PDGFRa+ (% live cells)Vehicle Cl-amidine treated050100150Plasma fibrinogen (mg/ml)Vehicle Cl-amidine treated01020304050Plasma vWF (ng/ml)D.Vehicle Cl-amidine treated GSK484 treated0h24h48hVehicle GSK4840204060% Thy1+ (% live cells)Vehicle GSK4840204060% Podoplanin+ (% live cells)Vehicle GSK48405101520% PDGFRa+ (% live cells)E.SSC-AFSC-HLive/deadCD45CD11bCD11bCD11bFSC-A FSC-A FSC-A CD11cLy6GF4/80FSC-ASSC-AFSC-HLive/deadCD45CD31FSC-A FSC-A FSC-A FSC-AFSC-AFSC-AFSC-AFSC-AThy1PodoplaninPDGFRα                                Sup Fig. 4. Treatment of skin tumor bearing mice with a broad-spectrum PAD inhibitor reduces tumor growth. (A) Relative volume of skin tumors on mice treated with vehicle or Cl-amidine over 8d. (B) Flow cytometric analysis of the percentage immune cells (CD45+), myeloid cells (CD11b+), macrophages (F4/80+) and neutrophils (Ly6G+) in the tumors treated with Cl-amidine. (C) Flow cytometric analysis of CAF populations in the tumors treated with Cl-amidine based on the percentage Thy1, Podoplanin and PDGFRα positive cells. (D) The levels of clotting factors (fibrinogen and von Willerbrand factor; vWF) in the plasma of skin tumor bearing mice treated with vehicle or Cl-amidine. (E) Flow cytometric analysis of CAF populations in the tumors treated with GSK484 based on the percentage Thy1, Podoplanin and PDGFRα positive cells. (F) Representative phase contrast images of skin tumor cells after treatment with vehicle, 100µM Cl-amidine or 10µM GSK484 over 48h. Bar graphs are mean ± SEM; * = p<0.05 and ** = p<0.01 using (A) a Mann-Whitney test. Box and whiskers graph-line: median, box: upper and lower quartiles, whiskers: maxima and minima. Assays were performed on (A and B) n=7 (Vehicle) and n=8 (Cl-amidine), (C) n=8 (Vehicle) and n=6 (Cl-amidine) (D) n=6 (both conditions) for Fibrinogen and n=7 (both conditions) for vWF, (E) n=8 (Vehicle) and n=4 (GSK484) in duplicate for all markers, (F) n=3 (Vehicle) and n=2 (Cl-amidine and GSK484) (in duplicate) independent experiments.  Vehicle Cl-amidinetreated0100200300KPC treatmentPlasma fibrinogen (ug/ml)Vehicle Cl-amidine treated050100150KPC treatmentPlasma vWF (ug/ml)B.A.KrasLSL.G12D/+; p53R172H/+; PdxCretg/+Ultrasound tumourUltrasound tumourDay 03-6mmDay 8CullCl-amidine treatmentVehicle Cl-amidine0.00.51.01.52.02.5TreatmentRelative CSAVehicle Cl-amidine1.01.11.21.31.41.5TreatmentRelative diameterC.D.E.Vehicle Cl-amidine05101520% Thy1+ (% live cells)Vehicle Cl-amidine05101520% Podoplanin+ (% live cells)Vehicle Cl-amidine01020304050% PDGFRa+ (% live cells)Vehicle Cl-amidine0204060% CD45+ (% live cells)Vehicle Cl-amidine020406080100% Ly6G+ (% CD45+)Vehicle Cl-amidine020406080100% CD11b+ (% CD45+)Vehicle Cl-amidine05101520% F4/80+ (% CD45+)UltrasoundtumourUltrasoundtumourDay 03-6mmDay 8CullCl-amidine treatment                                 Sup Fig. 5. Treatment of pancreatic tumor bearing mice with a broad-spectrum PAD inhibitor has no effect on tumor growth. (A) Schematic of Cl-amidine treatment regime for pancreatic tumor bearing mice. (B) Relative cross-sectional area (CSA) and diameter of tumors treated with vehicle or Cl-amidine over 8d. (C) The levels of clotting factors (fibrinogen and von Willerbrand factor; vWF) in the plasma of pancreatic tumor bearing mice treated with vehicle or Cl-amidine. (D) Flow cytometric analysis of the percentage immune cells (CD45+), myeloid cells (CD11b+), macrophages (F4/80+) and neutrophils (Ly6G+) in the tumor. (E) Flow cytometric analysis of CAF populations in the tumor based on the percentage Thy1, Podoplanin and PDGFRα positive cells. Assays were performed on n=4 independent experiments. Bar graphs are mean ± SEM. Box and whiskers graph-line: median, box: upper and lower quartiles, whiskers: maxima and minima.                                       Sup Fig. 6. Inhibiting t-NETosis suppresses growth of subcutaneous (s.c.) pancreatic tumors in vivo. (A) Schematic of GSK484 treatment regime for s.c. pancreatic tumor-bearing mice. (B) Relative endpoint volume of s.c. pancreatic tumors in mice treated with vehicle or GSK484 after 8d (volume compared to the pre-treatment tumor volume – indicated by dotted line). Red dots represent mice reaching legal endpoint prior to the end of the 8d treatment (within 5-7d of treatment). (C) Representative confocal images of neutrophils and CAFs in vehicle or GSK484 treated s.c. pancreatic tumors (MPO, green; PDPN, blue; DAPI, white). (D) Representative confocal images of NETting neutrophils and CAFs in vehicle or GSK484 treated s.c. pancreatic tumors (MPO, green; CitH3, red; PDPN, blue; DAPI, white). Scale bars are 50µm. (E) Quantification of relative NET coverage, percentage death and relative number of bone marrow neutrophils taken from mice with s.c. pancreatic tumors. (F) Relative endpoint volume of s.c. pancreatic tumors in PAD4-/- or WT littermate mice after 8d (compared to the tumor volume at day 10 post-induction indicated by dotted line). Red dots indicate mice reaching legal endpoint prior the end of the 8d (within 5-7d). Data are mean ± SEM; * = p<0.05, ** = p<0.01 and *** = p<0.001 using (B, E-F) unpaired t-test. Assays were performed on (B) n=8 mice, (C-D) Representative of n=3, (E) n=2 or 3 vehicle and GSK484 treated mice respectively (in duplicate) and (F) n=6-7 mice. Scale bars are 50µm.                                                Sup Fig. 7. Enlarged heatmap for differentially secreted proteins in pancreatic FB and CAF CMed analyzed by mass spectrometry. The relative intensity of each protein is indicated by the coloured bar under the heatmap. The assay was performed on n=3 samples.                                             Sup Fig. 8. Amyloid β is sufficient to drive NETosis in vitro. (A) Quantification of the relative NET coverage and percentage death of neutrophils treated with pancreatic CAF CMed with or without a fibronectin inhibitor (FNi). (B) Log2(reads per kilobase transcript per million map reads; RPKM+1) expression of the app gene in pancreatic (pan) CAFs normalized to pancreatic (pan) stellate cells from publicly available data (2). (C) Quantification of the relative NET coverage and percentage death of neutrophils treated with recombinant Amyloid α (Aα), recombinant Amyloid β (Aβ) or PMA for 3h. Data are mean ± SEM; * = p<0.05, ** = p<0.01 and *** = p<0.001 using (A) paired t-test and (C) one-way ANOVA with a Dunnett post hoc test. Assays were performed on (A) n=2 (in duplicate), (C) n=3 (in duplicate or triplicate) independent experiments.   CAF CMed CAF CMed+BACEirAmyloid β020406080100Media I/V infusedRelative area NET coverage (mm2)******CAF CMed CAF CMed+BACEirAmyloid β01020304050Media I/V infused% dead neutrophils******B.A.C.CAF CMed Amyloid βVehicleD.MPO CitH3 PDPN DAPI50umUntreated  CAF CMedCAF CMed+BACE1-2i05101520% dead neutrophils**PancreaticUntreated  CAF CMedCAF CMed+BACE1-2i0102030% dead neutrophils*LungUntreated CAF CMedCAF CMed+αCD11b01020304050Neutrophil treatment% dead neutrophils **Untreated CAF CMedCAF CMed+αTLR201020304050Neutrophil treatment% dead neutrophils *                                     Sup Fig. 9. Inhibition of Amyloid β secretion by CAFs suppresses NETosis. (A) Quantification of percentage death of neutrophils treated with pancreatic and lung CAF CMed generated with or without 24h pre-treatment of the CAFs with β-secretases 1 and 2 (BACE1-2) inhibitor.(B) Quantification of the percentage death of bone marrow neutrophils taken from wild type mice 24h after intravenous infusion with pancreatic CAF CMed taken from cells pre-treated with or without BACE1-2 inhibitor for 24h or recombinant Amyloid β. (C) Quantification of cell death after 3h treatment with pancreatic CAF CMed with or without CD11b or TLR2 blocking antibodies. (D) Representative confocal images showing neutrophils (myeloperoxidase (MPO; in green), CAFs (podoplanin, in blue) and counterstained with DAPI (in white) of orthotopically implanted B16.F10 tumor cells with vehicle, CAF CMed or recombinant Amyloid β treatment. Data are mean ± SEM; * = p<0.05, ** = p<0.01 and *** = p<0.001 using (A-B) one-way ANOVA with a Tukey post hoc test and (C) one-way ANOVA with a Dunnett post hoc test. Assays were performed on (A) n=6 (in triplicate), (B) n=7 (in duplicate or triplicate), (C) n=3 (in triplicate) and (D) Representative of n=3 independent experiments. Scale bars are 50µm.    Melanoma brain metMPO CitH3 PDPN DAPIA. B.Normal Melanoma0200040006000Plasma Amyloid beta (pg/ml)               Sup Fig. 10. Amyloid β driven t-NETosis is conserved in the human disease. (A) Representative confocal images of NETting neutrophils and CAFs in a human brain metastasis (MPO, green; CitH3, red; podoplanin, blue; DAPI, white). (B) Quantification of Amyloid β in plasma from melanoma patients and healthy controls. Data are mean ± SEM. Assays were performed on (A) n=1 brain metastasis and (B) n=10 (Normal) and n=19 (Melanoma) independent experiments. Scale bars are 50µm.  

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

http://repository.cshl.edu/id/eprint/39946/1/2021.Munir.tumorgrowth.pdf

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

Abstract

Similar works

Full text

Available Versions

Apollo (Cambridge)

Cold Spring Harbor Laboratory Institutional Repository

Apollo (Cambridge)