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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.
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
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.....
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
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.......
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 .......
Our work was supported by grants from Deutsche Krebshilfe, Grant Number: 111647, and German Research Council (DFG), Grant Number: JA 2461/2-1.
....Name | Company | Catalog Number | Comments |
15 ml tubes | Sarstedt AG & Co., Nümbrecht, Germany | 62,554,502 | |
50 ml tubes | Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany | 227261 | |
5ml / 10ml / 25ml sterile tipps for the automatic pipette | Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany | 6006180 / 607180 / 760180 | |
6 well flat-bottom cell culture plates | Sarstedt AG & Co., Nümbrecht, Germany | 833,920 | |
96 well flat-bottom cell culture plates | Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany | 655180 | |
96 well U-bottom cell culture plates | Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany | 65018 | |
AMG EVOS fl digital inverted microscope | AMG, Bothel, U.S. | ||
anti-mouse CD11b | BD Pharmigen, Becton Dickinson, Franklin Lakes, U.S. | 553312 | clone M1/70, APC-conjugated, 0.2mg/mL |
anti-mouse Ly6G | BioLegend, California, U.S. | 127608 | clone 1A8, PE-conjugated, 0.2mg/mL |
BD FACS AriaII | BD Biosciences, Becton Dickinson, Franklin Lakes, U.S. | cell sorter | |
Caliper | Vogel Germany, Kevelaer, Germany | ||
Casy cell counter | Innovatis, Roche Innovatis AG, Bielefeld, Germany | ||
Cell Trics 50µm / 100 µm sterile filters | Sysmex Partec GmbH, Goerlitz, Germany | 04-004-2327 / 04-004-2328 | |
Centrifuge Rotina 420 R | Andreas Hettich, Tuttlingen, Germany | 4706 | |
Collagenase D | Sigma-Aldrich/Merck, Darmstadt, Germany | 11088858001 | |
DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) | BioLegend, California, U.S. | 422801 | Stock: 5mg/ml |
Dispase I | Sigma-Aldrich/Merck, Darmstadt, Germany | D4818-2MG | |
DMEM | Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S. | 41966-029 | DMEM complete: DMEM + 10% FBS + 1% penicillin-streptomycin |
DMSO (Dimethylsufoxide) | WAK-Chemie Medical GmbH, Steinbach, Germany | WAK-DMSO-10 | CryoSure-DMSO |
DNase I | Sigma-Aldrich/Merck, Darmstadt, Germany | DN25-100MG | |
DPBS | Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S. | 14190-094 | |
Endothelial cell growth medium | PromoCell, Heidelberg, Germany | c-22010 | |
FBS (Fetal Bovine Serum) | Biochrom, Berlin, Germany | S0115 | |
Fc-block (Anti-mouse CD16/32) | BD Pharmingen, Becton Dickinson,Becton Dickinson, Franklin Lakes, U.S. | 553142 | clone 2.4G2, Stock: 0.5mg/mL |
FK 866 hydrochloride | Axon Medchem, Groningen, Netherlands | Axon 1546 | Stock: 100 mM |
Goat Anti-Rabbit IgG H&L | Abcam, Cambridge, U.K. | ab97075 | Cy3-conjugated, Stock: 0.5 mg/mL |
Heracell 240i CO2 Incubator | Thermo Fisher Scientific, Waltham, U.S. | 51026334 | |
IMDM | Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S. | 12440-053 | IMDM complete: IMDM + 10% FBS + 1% penicillin-streptomycin |
Isis GT420 shaver | B. Braun Asculap, Suhl, Germany | 90200714 | |
Matrigel Matrix basement membrane | Corning Life Sciences, Amsterdam, Netherlands | 7205011 | |
Microtome Cryostat Microm HM 505 N | Microm International GmbH, Walldorf, Germany | ||
Monoclonal Anti-Actin, α-Smooth Muscle | Sigma-Aldrich/Merck, Darmstadt, Germany | F3777 | FITC-conjugated, no information about stock concentration |
Needles 0.4 mm x 16 mm | BD Microlance, Becton Dicson, Becton Dickinson, Franklin Lakes, U.S. | 302200 | |
Neomount | Merck, Darmstadt, Germany | HX67590916 | |
Normal goat serum | Jackson ImmunoResearch Laboratories, West Grove, U.S. | 005-000-121 | |
Penicillin Streptomycin | Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S. | 15140-122 | |
Pipetus automatic pipette | Hirschmann Laborgeräte, Eberstadt, Germany | 9907200 | |
ProLong Gold Antifade Mountant with DAPI | Invitrogen, Thermo Fisher Scientific, Massachusetts, U.S. | P36935 | |
rabbit anti mouse Laminin gamma 1 chain | Immundiagnostik, Bensheim, Germany | AP1001.1 | No information about stock concentration |
StemPro Accutase | Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S. | A1105-01 | |
Sterile disposal scalpel (no. 15) | MedWare, Naples, U.S. | 120920 | |
Syringes 1 ml | BD Plastipak, Becton Dickinson, Franklin Lakes, U.S. | 303172 | |
Syringes 10 ml | BD Discardit II, Becton Dickinson, Franklin Lakes, U.S. | 309110 | |
T75 sterile cell culture flasks | Sarstedt AG & Co., Nümbrecht, Germany | 833,911,302 | |
Tissue-Tek O.C.T. Compound | Sakura Finetek, Torrance, U.S. | 4583 | |
Zeiss AxioObserver.Z1 Inverted Microscope with ApoTome Optical Sectioning | Carl Zeiss, Oberkochen, Germany |
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