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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

Abstract

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Introduction

Type I Interferons (IFNs) play an important role in the stimulation of host responses to neoplasias, as the lack of type I IFN signaling results in significantly elevated tumor growth1. One of the mechanisms involved in this process is the regulation of tumorigenic activity of tumor-associated neutrophils, which is controlled by colony-stimulating factor 3 receptor (CSF3R) downstream signaling2. Colony-stimulating factor 3 (CSF3), or granulocyte colony-stimulating factor, was shown to activate signaling involving nicotinamide phosphoribosyltransferase (NAMPT)3,4.....

Protocol

All the procedures including animal subjects have been approved by the regulatory authorities: LANUV (Landesamt für Natur, Umwelt und Verbraucherschutz NRW) and Regierungspräsidium Tübingen, Germany. All manipulations should be performed in sterile conditions (under laminar flow hood) using sterile reagents and instruments (syringes, scissors, forceps, disposable scalpels, Petri dishes).

NOTE: The overall scheme of the protocol is shown in the Figure 1

Representative Results

Using the procedure described here, Ifnar1-/- neutrophils were isolated from tumors and treated with NAMPT inhibitor FK866 for 2 h. Untreated Ifnar1-/- neutrophils were used as a control. The effectivity of the treatment was evaluated using the aortic ring assay, which reflects the key steps involved in angiogenesis (matrix degradation, migration, proliferation, reorganization). We could demonstrate that FK866-treated neutrophils have a significantl.......

Discussion

Despite progress in surgical and pharmacological cancer treatment, successful therapy remains a challenge. Since immune cells are known to play an important role in the regulation of tumor growth, novel methods inhibiting tumorigenicity of such cells should be established. Here we demonstrate a novel approach to suppress tumor growth via adoptive transfer of anti-angiogenic tumor-associated neutrophils. Selective targeting of pro-angiogenic NAMPT signaling in TANs, using FK866 inhibitor, prevents side effects, which are .......

Acknowledgements

Our work was supported by grants from Deutsche Krebshilfe, Grant Number: 111647, and German Research Council (DFG), Grant Number: JA 2461/2-1.

....

Materials

NameCompanyCatalog NumberComments
15 ml tubesSarstedt AG & Co., Nümbrecht, Germany62,554,502
50 ml tubesCellstar, Greiner Bio One International GmbH, Frickenhausen, Germany227261
5ml / 10ml / 25ml sterile tipps for the automatic pipetteCellstar, Greiner Bio One International GmbH, Frickenhausen, Germany6006180 / 607180 / 760180
6 well flat-bottom cell culture platesSarstedt AG & Co., Nümbrecht, Germany833,920
96 well flat-bottom cell culture platesCellstar, Greiner Bio One International GmbH, Frickenhausen, Germany655180
96 well U-bottom cell culture platesCellstar, Greiner Bio One International GmbH, Frickenhausen, Germany65018
AMG EVOS fl digital inverted microscopeAMG, Bothel, U.S.
anti-mouse CD11bBD Pharmigen, Becton Dickinson, Franklin Lakes, U.S.553312clone M1/70, APC-conjugated, 0.2mg/mL
anti-mouse Ly6GBioLegend, California, U.S.127608clone 1A8, PE-conjugated, 0.2mg/mL
BD FACS AriaIIBD Biosciences, Becton Dickinson, Franklin Lakes, U.S.cell sorter
CaliperVogel Germany, Kevelaer, Germany
Casy cell counterInnovatis, Roche Innovatis AG, Bielefeld, Germany
Cell Trics 50µm / 100 µm sterile filtersSysmex Partec GmbH, Goerlitz, Germany04-004-2327 / 04-004-2328
Centrifuge Rotina 420 RAndreas Hettich, Tuttlingen, Germany4706
Collagenase DSigma-Aldrich/Merck, Darmstadt, Germany11088858001
DAPI (4',6-Diamidino-2-Phenylindole, Dilactate)BioLegend, California, U.S.422801Stock: 5mg/ml
Dispase ISigma-Aldrich/Merck, Darmstadt, GermanyD4818-2MG
DMEMGibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.41966-029DMEM complete: DMEM + 10% FBS + 1% penicillin-streptomycin
DMSO (Dimethylsufoxide)WAK-Chemie Medical GmbH, Steinbach, GermanyWAK-DMSO-10CryoSure-DMSO
DNase ISigma-Aldrich/Merck, Darmstadt, GermanyDN25-100MG
DPBSGibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.14190-094
Endothelial cell growth mediumPromoCell, Heidelberg, Germanyc-22010
FBS (Fetal Bovine Serum)Biochrom, Berlin, GermanyS0115
Fc-block (Anti-mouse CD16/32)BD Pharmingen, Becton Dickinson,Becton Dickinson, Franklin Lakes, U.S.553142clone 2.4G2, Stock: 0.5mg/mL
FK 866 hydrochlorideAxon Medchem, Groningen, NetherlandsAxon 1546Stock: 100 mM
Goat Anti-Rabbit IgG H&LAbcam, Cambridge, U.K.ab97075Cy3-conjugated, Stock: 0.5 mg/mL
Heracell 240i CO2 IncubatorThermo Fisher Scientific, Waltham, U.S.51026334
IMDMGibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.12440-053IMDM complete: IMDM + 10% FBS + 1% penicillin-streptomycin
Isis GT420 shaverB. Braun Asculap, Suhl, Germany90200714
Matrigel Matrix basement membraneCorning Life Sciences, Amsterdam, Netherlands7205011
Microtome Cryostat Microm HM 505 NMicrom International GmbH, Walldorf, Germany
Monoclonal Anti-Actin, α-Smooth MuscleSigma-Aldrich/Merck, Darmstadt, GermanyF3777FITC-conjugated, no information about stock concentration
Needles 0.4 mm x 16 mmBD Microlance, Becton Dicson, Becton Dickinson, Franklin Lakes, U.S.302200
NeomountMerck, Darmstadt, GermanyHX67590916
Normal goat serumJackson ImmunoResearch Laboratories, West Grove, U.S.005-000-121
Penicillin StreptomycinGibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.15140-122
Pipetus automatic pipetteHirschmann Laborgeräte, Eberstadt, Germany9907200
ProLong Gold Antifade Mountant with DAPIInvitrogen, Thermo Fisher Scientific, Massachusetts, U.S.P36935
rabbit anti mouse Laminin gamma 1 chainImmundiagnostik, Bensheim, GermanyAP1001.1No information about stock concentration
StemPro AccutaseGibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.A1105-01
Sterile disposal scalpel (no. 15)MedWare, Naples, U.S.120920
Syringes 1 mlBD Plastipak, Becton Dickinson, Franklin Lakes, U.S.303172
Syringes 10 mlBD Discardit II, Becton Dickinson, Franklin Lakes, U.S.309110
T75 sterile cell culture flasksSarstedt AG & Co., Nümbrecht, Germany833,911,302
Tissue-Tek O.C.T. CompoundSakura Finetek, Torrance, U.S.4583
Zeiss AxioObserver.Z1 Inverted Microscope with ApoTome Optical SectioningCarl Zeiss, Oberkochen, Germany

References

  1. Jablonska, J., Leschner, S., Westphal, K., Lienenklaus, S., Weiss, S. Neutrophils responsive to endogenous IFN-beta regulate tumor angiogenesis and growth in a mouse tumor model. Journal of Clinical Investigation. 120 (4), 1151-1164 (2010).
  2. Andzinski, L., Wu, C. F., Lienenklaus, S., Kröger, A., Weiss, S., Jablonska, J.

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