Name: transfer of manipulated tumor-associated neutrophils into tumor-bearing mice to study their angiogenic potential in vivo
Uploaded: 2019-07-20T13:00:00
Description: Watch this Scientific Journal Video about Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo at JoVE.com

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

All the procedures including animal subjects have been approved by the regulatory authorities: LANUV (Landesamt f&#252;r Natur, Umwelt und Verbraucherschutz NRW) and Regierungspr&#228;sidium T&#252;bingen, 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</stro...

<ol>
	<li>Jablonska, J., Leschner, S., Westphal, K., Lienenklaus, S., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+responsive+to+endogenous+IFN-beta+regulate+tumor+angiogenesis+and+growth+in+a+mouse+tumor+model.">Neutrophils responsive to endogenous IFN-beta regulate tumor angiogenesis and growth in a mouse tumor model.</a> Journal of Clinical Investigation. 120 (4), 1151-1164 (2010).</li><li>Andzinski, L., Wu, C. F., Lienenklaus, S., Kr&#246;ger, A., Weiss, S., Jablonska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+apoptosis+of+tumor+associated+neutrophils+in+the+absence+of+endogenous+IFN-&#946;.">Delayed apoptosis of tumor associated neutrophils in the absence of endogenous IFN-&#946;.</a> International Journal of Cancer. 136, 572-583 (2015).</li><li>Rongvaux, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-B-cell+colony-enhancing+factor,+whose+expression+is+upregulated+in+activated+lymphocytes,+is+a+nicotinamide+phosphoribosyltransferase,+a+cytosolic+enzyme+involved+in+NAD+biosynthesis.">Pre-B-cell colony-enhancing factor, whose expression is upregulated in activated lymphocytes, is a nicotinamide phosphoribosyltransferase, a cytosolic enzyme involved in NAD biosynthesis.</a> European Journal of Immunology. 32, 3225-3234 (2002).</li><li>Skokowa, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NAMPT+is+essential+for+the+G-CSF-induced+myeloid+differentiation+via+a+NAD(+)-sirtuin-1-dependent+pathway.">NAMPT is essential for the G-CSF-induced myeloid differentiation via a NAD(+)-sirtuin-1-dependent pathway.</a> Nature Medicine. 15, 151-158 (2009).</li><li>Yaku, K., Okabe, K., Hikosaka, K., Nakagawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NAD+Metabolism+in+Cancer+Therapeutics.">NAD Metabolism in Cancer Therapeutics.</a> Frontiers in Oncology. 8, 622 (2018).</li><li>Audrito, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+nicotinamide+phosphoribosyltransferase+(NAMPT)+promotes+M2+macrophage+polarization+in+chronic+lymphocytic+leukemia.">Extracellular nicotinamide phosphoribosyltransferase (NAMPT) promotes M2 macrophage polarization in chronic lymphocytic leukemia.</a> Blood. 125, 111-123 (2015).</li><li>Brentano, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-B+cell+colony-enhancing+factor/visfatin,+a+new+marker+of+inflammation+in+rheumatoid+arthritis+with+proinflammatory+and+matrix+degrading+activities.">Pre-B cell colony-enhancing factor/visfatin, a new marker of inflammation in rheumatoid arthritis with proinflammatory and matrix degrading activities.</a> Arthritis &amp; Rheumatology. 56, 2829-2839 (2007).</li><li>Jablonska, J., Wu, C. -. F., Andzinski, L., Leschner, S., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR2-mediated+tumor+associated+neutrophilrecruitment+is+regulated+by+IFN-beta.">CXCR2-mediated tumor associated neutrophilrecruitment is regulated by IFN-beta.</a> International Journal of Cancer. 134, 1346-1358 (2014).</li><li>Andzinski, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+IFNs+induce+anti-tumor+polarization+of+tumor+associated+neutrophils+in+mice+and+human.">Type I IFNs induce anti-tumor polarization of tumor associated neutrophils in mice and human.</a> International Journal of Cancer. 138, 1982-1993 (2016).</li><li>Wu, C. -. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lack+of+type+I+interferon+induces+neutrophil-mediated+pre-metastatic+niche+formation+in+the+mouse+lung.">The lack of type I interferon induces neutrophil-mediated pre-metastatic niche formation in the mouse lung.</a> International Journal of Cancer. 137, 837-847 (2015).</li><li>Hansel, T. T., Kropshofer, H., Singer, T., Mitchell, J. A., George, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+safety+and+side+effects+of+monoclonal+antibodies.">The safety and side effects of monoclonal antibodies.</a> Nature Reviews Drug Discovery. 9 (4), 325-338 (2010).</li><li>Hasmann, M., Schemainda, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FK866,+a+highly+specific+noncompetitive+inhibitor+of+nicotinamide+phosphoribosyltransferase,+represents+a+novel+mechanism+for+induction+of+tumor+cell+apoptosis.">FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, represents a novel mechanism for induction of tumor cell apoptosis.</a> Cancer Research. 63, 7436-7442 (2003).</li><li>Holen, K., Saltz, L. B., Hollywood, E., Burk, K., Hanauske, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharmacokinetics,+toxicities,+and+biologic+effects+of+FK866,+a+nicotinamide+adenine+dinucleotide+biosynthesis+inhibitor.">The pharmacokinetics, toxicities, and biologic effects of FK866, a nicotinamide adenine dinucleotide biosynthesis inhibitor.</a> Investigational New Drugs. 26, 45-51 (2008).</li><li>von Heideman, A., Berglund, A., Larsson, R., Nygren, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+NAD+depleting+cancer+drugs:+results+of+a+phase+I+clinical+trial+of+CHS+828+and+overview+of+published+data.">Safety and efficacy of NAD depleting cancer drugs: results of a phase I clinical trial of CHS 828 and overview of published data.</a> Cancer Cancer Chemotherapy and Pharmacology. 65, 1165-1172 (2010).</li><li>De Rossi, G., Scotland, R., Whiteford, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+Factors+in+Measuring+Angiogenesis+Using+the+Aortic+Ring+Model.">Critical Factors in Measuring Angiogenesis Using the Aortic Ring Model.</a> Journal of Genetic Syndromes and Gene Therapy. 4 (5), (2013).</li><li>Pylaeva, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NAMPT+signaling+is+critical+for+the+proangiogenic+activity+of+tumor-associated+neutrophils.">NAMPT signaling is critical for the proangiogenic activity of tumor-associated neutrophils.</a> International Journal of Cancer. 144 (1), 136-149 (2019).</li></ol>

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 ...

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. NAMPT is a rate-limiting enzyme for nicotinamide adenine dinucleotide synthesis, which enhances glycolysis and regulates DNA repair, gene expression, and stress response promoting cancer cells survival and proliferation5. NAMPT is overexpressed in multiple cancer types, including colorectal, ovarian, breast, gastric, prostate cancer and gliomas6. NAMPT is essential not only for tumor cells, but also for a wide variety of other cell types that are present in tumors, such as myeloid cells - it drives their differentiation4, inhibits apoptosis and stimulate expression of multiple cytokines or matrix-degrading enzymes in macrophages7.
Tumor-associated neutrophils represent important modulators of tumor growth. TAN functions are strongly dependent on the type I IFN availability, as these cytokines prime anti-tumor activity of neutrophils. To the contrary, the absence of IFNs supports tumorigenic activation of these cells, especially their pro-angiogenic properties. In agreement with this, mice deficient in IFNs develop significantly larger and better vascularized tumors, which are strongly infiltrated with pro-tumoral/pro-angiogenic neutrophils1,2,8,9,10. Importantly, such pro-angiogenic TANs show elevated activity of NAMPT, suggesting its essential role in pro-tumor polarization of neutrophils.
Depletion of neutrophils using Ly6G antibody or inhibition of their migration (CXCR2 antibody) results in decreased tumor angiogenesis, growth, and metastasis1,8. Nevertheless, generated monoclonal antibodies are immunogenic, and their administration is associated with a range of life-threatening side effects11. Treatment with small molecules, such as NAMPT inhibitor FK866, that modulate neutrophil tumoriogenicity, could help to avoid such complications. Unfortunately, pharmacological systemic inhibition of NAMPT, next to its therapeutic effect on tumor growth, leads to severe side effects including gastrointestinal toxicity and thrombocytopenia. Therefore, the systemic application of NAMPT inhibitors is not feasible12,13,14.
For this reason, we suggest here a protocol where NAMPT activity is blocked directly in isolated TANs. Such anti-tumor neutrophils are then adoptively transferred into a tumor-bearing host. This protocol will help avoid systemic toxic side-effects of the compounds, while its effect on the target cells will be sustained.

<table>
	<tbody>
		<tr>
			<td>15 ml tubes</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
			<td>62,554,502</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml / 10ml / 25ml sterile tipps for the automatic pipette</td>
			<td>Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany</td>
			<td>6006180 / 607180 / 760180</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well flat-bottom cell culture plates</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
			<td>833,920</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well flat-bottom cell culture plates</td>
			<td>Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well U-bottom cell culture plates</td>
			<td>Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany</td>
			<td>65018</td>
			<td></td>
		</tr>
		<tr>
			<td>AMG EVOS fl digital inverted microscope</td>
			<td>AMG, Bothel, U.S.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD11b</td>
			<td>BD Pharmigen, Becton Dickinson, Franklin Lakes, U.S.</td>
			<td>553312</td>
			<td>clone M1/70, APC-conjugated, 0.2mg/mL</td>
		</tr>
		<tr>
			<td>anti-mouse Ly6G</td>
			<td>BioLegend, California, U.S.</td>
			<td>127608</td>
			<td>clone 1A8, PE-conjugated, 0.2mg/mL</td>
		</tr>
		<tr>
			<td>BD FACS AriaII</td>
			<td>BD Biosciences, Becton Dickinson, Franklin Lakes, U.S.</td>
			<td></td>
			<td>cell sorter</td>
		</tr>
		<tr>
			<td>Caliper</td>
			<td>Vogel Germany, Kevelaer, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Casy cell counter</td>
			<td>Innovatis, Roche Innovatis AG, Bielefeld, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Trics 50&#181;m / 100 &#181;m sterile filters</td>
			<td>Sysmex Partec GmbH, Goerlitz, Germany</td>
			<td>04-004-2327 / 04-004-2328</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Rotina 420 R</td>
			<td>Andreas Hettich, Tuttlingen, Germany</td>
			<td>4706</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Sigma-Aldrich/Merck, Darmstadt, Germany</td>
			<td>11088858001</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dilactate)</td>
			<td>BioLegend, California, U.S.</td>
			<td>422801</td>
			<td>Stock: 5mg/ml</td>
		</tr>
		<tr>
			<td>Dispase I</td>
			<td>Sigma-Aldrich/Merck, Darmstadt, Germany</td>
			<td>D4818-2MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>41966-029</td>
			<td>DMEM complete: DMEM + 10% FBS + 1% penicillin-streptomycin</td>
		</tr>
		<tr>
			<td>DMSO (Dimethylsufoxide)</td>
			<td>WAK-Chemie Medical GmbH, Steinbach, Germany</td>
			<td>WAK-DMSO-10</td>
			<td>CryoSure-DMSO</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich/Merck, Darmstadt, Germany</td>
			<td>DN25-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cell growth medium</td>
			<td>PromoCell, Heidelberg, Germany</td>
			<td>c-22010</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>S0115</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-block (Anti-mouse CD16/32)</td>
			<td>BD Pharmingen, Becton Dickinson,Becton Dickinson, Franklin Lakes, U.S.</td>
			<td>553142</td>
			<td>clone 2.4G2, Stock: 0.5mg/mL</td>
		</tr>
		<tr>
			<td>FK 866 hydrochloride</td>
			<td>Axon Medchem, Groningen, Netherlands</td>
			<td>Axon 1546</td>
			<td>Stock: 100 mM</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG H&#38;L</td>
			<td>Abcam, Cambridge, U.K.</td>
			<td>ab97075</td>
			<td>Cy3-conjugated, Stock: 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>Heracell 240i CO2 Incubator</td>
			<td>Thermo Fisher Scientific, Waltham, U.S.</td>
			<td>51026334</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>12440-053</td>
			<td>IMDM complete: IMDM + 10% FBS + 1% penicillin-streptomycin</td>
		</tr>
		<tr>
			<td>Isis GT420 shaver</td>
			<td>B. Braun Asculap, Suhl, Germany</td>
			<td>90200714</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix basement membrane</td>
			<td>Corning Life Sciences, Amsterdam, Netherlands</td>
			<td>7205011</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome Cryostat Microm HM 505 N</td>
			<td>Microm International GmbH, Walldorf, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Actin, &#945;-Smooth Muscle</td>
			<td>Sigma-Aldrich/Merck, Darmstadt, Germany</td>
			<td>F3777</td>
			<td>FITC-conjugated, no information about stock concentration</td>
		</tr>
		<tr>
			<td>Needles 0.4 mm x 16 mm</td>
			<td>BD Microlance, Becton Dicson, Becton Dickinson, Franklin Lakes, U.S.</td>
			<td>302200</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomount</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>HX67590916</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson ImmunoResearch Laboratories, West Grove, U.S.</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetus automatic pipette</td>
			<td>Hirschmann Laborger&#228;te, Eberstadt, Germany</td>
			<td>9907200</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Invitrogen, Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit anti mouse Laminin gamma 1 chain</td>
			<td>Immundiagnostik, Bensheim, Germany</td>
			<td>AP1001.1</td>
			<td>No information about stock concentration</td>
		</tr>
		<tr>
			<td>StemPro Accutase</td>
			<td>Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>A1105-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile disposal scalpel (no. 15)</td>
			<td>MedWare, Naples, U.S.</td>
			<td>120920</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes 1 ml</td>
			<td>BD Plastipak, Becton Dickinson, Franklin Lakes, U.S.</td>
			<td>303172</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes 10 ml</td>
			<td>BD Discardit II, Becton Dickinson, Franklin Lakes, U.S.</td>
			<td>309110</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 sterile cell culture flasks</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
			<td>833,911,302</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>Sakura Finetek, Torrance, U.S.</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss AxioObserver.Z1 Inverted Microscope with ApoTome Optical Sectioning</td>
			<td>Carl Zeiss, Oberkochen, Germany</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

transfer of manipulated tumor-associated neutrophils into tumor-bearing mice to study their angiogenic potential in vivo

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

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...

Watch this Scientific Journal Video about Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo at JoVE.com

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

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&#160;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 ...

Our protocol provides a novel tool for evaluating the angiogenic potential of tumor-associated primary neutrophils in vivo without the need to use artificial neutrophil cell line models. Direct inhibition of nicotinamide phosphoribosyltransferase, shortly NAMPT, in tumor-associated neutrophils with their subsequent adoptive transfer into tumor-bearing animals allows the manipulation of neutrophils without systemic toxic side effects. We will demonstrate the therapeutic potential of manipulated anti-angiogenic tumor-associated neutrophils after transfer into tumor-bearing hosts for the study of potential neutrophil-based immunotherapies in different cancer models.To set up an allogenic tumor mouse model, shave the skin of 10 eight to 12-week old female interferon alpha and beta-receptor subunit 1 knockout mice with an electric shaver and disinfect the skin with 70%ethanol, then load three times 10 to the six B16F10 melanoma cells per milliliter of PBS in a one-milliliter syringe equipped with a 0.4 by 19-millimeter needle and inject 100 microliters of the suspension subcutaneously on a rear flank into each shaved animal. For neutrophil-adoptive transfer, dilute the B16F10 melanoma cells to a six times 10 to the sixth cells per milliliter concentration in PBS and dilute neutrophils treated with NAMPT inhibitor FK866 to a six times 10 to the fifth cells per milliliter of PBS concentration. Then, mix untreated or inhibitor-treated neutrophils with the melanoma cells at a neutrophil-to-tumor ratio of 1:10 and subcutaneously inject 100 microliters of cells into up to five mice per group.After the injections, place up to five injected female mice in a single cage and use calipers to measure the tumor length, width, and depth every day for 14 days. At Day 14 after injection, disinfect the skin of each injected animal with 70%ethanol and use scissors and forceps to harvest the tumors into a 50-milliliter conical tube containing complete medium on ice. To section the tumors for histological analysis, submerge the tumors in optimum cutting temperature compound and freeze the samples in liquid nitrogen for storage at minus 80 degrees Celsius.On the day of the sectioning, thaw the samples to minus 20 degrees Celsius before using a cryotome to obtain five-micrometer sections. After fixation in non-specific binding, stain the tumor tissue sections with the primary antibodies of interest for one hour at 20 degrees Celsius, followed by three washes in PBS. After the last wash, stain the cells with an appropriate fluorescence-conjugated secondary antibody in a nuclear-staining dye like DAPI for one hour at 20 degrees Celsius protected from light.At the end of the incubation, dry the slides for 20 minutes at 20 degrees Celsius protected from light before mounting the samples with an anhydrous mounting medium, then cover each slide with a cover slip and allow the mounting medium to dry for one hour at 37 degrees Celsius before imaging the tissues by fluorescence microscopy. For tumor-associated neutrophil isolation, place five tumors per well into individual wells of a sterile six-well plate as they are harvested and use sterile scissors to mince the tumors into two to three-millimeter pieces. Next, digest the tumor fragments with one milliliter of dispase collagenase d dnase 1 solution for 45 minutes at 37 degrees Celsius in 5%carbon dioxide with humidity, mixing the samples with a 10-milliliter syringe every 15 minutes.At the end of the incubation, filter the cells through 100-micrometer strainers into one 15-milliliter tube per well to remove the undigested fibers and add PBS to a final volume of 15 milliliters. Collect the dissociated cells by centrifugation and lyse the red blood cells with one milliliter of lysis buffer per tube. After mixing, combine the contents from each tube into a single 15-milliliter tube, stopping the reaction after two minutes with 11 milliliters of complete four-degrees Celsius medium.After centrifugation, resuspend the pellet in one milliliter of PBS and block any non-specific binding with three microliters of Fc-block antibody for 15 minutes on ice. At the end of the incubation, label the cells with anti-CD11b and Ly6G antibodies and any other antibodies of interest, plus DAPI, for a 30-minute incubation on ice protected from light. At the end of the incubation, wash the cells in 14 milliliters of PBS and resuspend the pellets at a one times 10 to the seventh cells per milliliter of fresh complete medium concentration on ice, then sort the CD11b-positive, Ly6G, high DAPI-negative neutrophils on a fluorescence-activated cell sorter according to standard sorting protocols, checking the purity of the isolated neutrophils on the flow cytometer at the end of the sort.For in vitro tumor-associated neutrophil inhibition, seed 1.5 times 10 to the fifth sorted neutrophils into each of two wells of a 96-well U-bottom plate and add FK866 to a final concentration of 100 nanomolar in complete medium into one well and an equal volume of complete medium alone to the other well. After two hours in the cell culture incubator, wash the cells two times with PBS and resuspend the pellets in PBS at the appropriate concentration for the subsequent injection. NAMPT-inhibitor treated neutrophils have a significantly decreased capacity to stimulate aortic branch formation compared to untreated cells.In addition, the subcutaneous injection of inhibitor-treated anti-angiogenic neutrophils induces a significant impairment in tumor growth compared to mice injected with untreated interferon alpha and beta receptor subunit 1 knockout neutrophils. Histological examination of the extracted tumors further confirms the significant suppression of angiogenesis in tumors isolated from mice treated with inhibitor-treated tumor-associated neutrophils compared to those injected with untreated knockout neutrophils. To assess the properties of FK866-treated tumor-associated neutrophils, quantitative PCR and Western Blot can be performed to study the activation of intracellular pathways involved in neutrophil polarization.Since neutrophils are short-lived, it is necessary to perform all of the steps as quickly as possible and to keep neutrophils cold during the entire procedure. This technique paves the way for the study of the intracellular mechanisms and factors involved in the regulation of angiogenic and tumorigenic properties of tumor-associated neutrophils in vivo.

Research

JoVE Journal

Cancer Research

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