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.
1. Preparation of B16F10 melanoma cell line
<ol>
	<li>Prepare mycoplasma-negative cells grown to a 90% confluent monolayer (approximately 10 x 106 cells/T75 flask) in complete Iscove&#8217;s Modified Dulbecco&#8217;s Medium (IMDMc: IMDM + 10% Fetal Bovine Serum (FBS) + 1% penicillin-streptomycin).</li>
	<li>Remove the medium, and rinse the cells with phosphate buffered saline (PBS). Apply 6 mL of a cell detachment solution containing proteolytic and collagenolytic enzymes (see the Table of Materials), and incubate at 37 &#176;C for 2 min.</li>
	<li>Knock the flask gently to mobilize remaining adherent cells from the bottom. Collect the cell suspension in 15 mL tubes and centrifuge at 300 x g for 7 min and 20 &#176;C.</li>
	<li>Remove the supernatant, and resuspend the pellet well in 1 mL of PBS. Add 14 mL of PBS and centrifuge (300 x g and 20 &#176;C for 7 min).</li>
	<li>Remove the supernatant and resuspend the pellet in 1 mL of PBS.</li>
	<li>Count the cells, and resuspend them to the concentration of 3 x 106/mL PBS (for the step 2) or 6 x 106/mL PBS (for the step 6) for injection. Keep cells on ice for a maximum of 30 min.</li>
</ol>
2. Allogenic tumor model in mice
<ol>
	<li>Use 10 female Ifnar1-/- mice 8-12 weeks old that are kept under specific-pathogen-free (SPF) conditions. 
	NOTE: Female mice are preferable in a subcutaneous model of tumor growth, since males are more aggressive and thus prone to infractions of the tumor site, which influences tumor growth.</li>
	<li>Shave the skin of the mouse on the flank with an electrical shaver, and disinfect the skin with tissue wet with 70% ethanol.</li>
	<li>Collect the prepared B16F10 melanoma cells at a concentration of 3 x 106/mL PBS (see the step 1) in a 1 mL syringe and 0.4 x 19 mm needle. Inject 100 &#956;L of the suspension subcutaneously.
	<ol>
		<li>Mix the cells well before every injection. Use needles not less than 0.4 mm in diameter as to not disturb tumor cells.</li>
	</ol>
	</li>
	<li>Place up to 5 mice in one cage, and control tumor size (length, width and depth) with a caliper for 14 days. 
	NOTE: According to the animal regulations, the tumor size should not exceed 15 mm in diameter, mice with bigger or necrotic/open tumors should be sacrificed beforehand.</li>
	<li>At day 14, sacrifice the mice in the CO2 chamber.</li>
	<li>Disinfect the skin with 70% ethanol and remove tumors with scissors and forceps in a sterile Petri dish. Keep tumors in a 50 mL tube in complete Dulbecco&#39;s Modified Eagle Medium (DMEMc: DMEM + 10% FBS + 1% penicillin-streptomycin) on ice.</li>
</ol>
3. TAN isolation
<ol>
	<li>Place tumors into sterile 6-well plates, 5 tumors per well. Cut tumors into 2-3 mm pieces with sterile scissors.
	<ol>
		<li>Digest with 1 mL of dispase/collagenase D/DNase I solution (0.2 mg/0.2 mg/100 mg in 1 mL of DMEMc) per tumor. Incubate at 37 &#176;C, 5% CO2 in a humid incubator, and mix with a 10 mL syringe without a needle every 15 min 3 times.</li>
	</ol>
	</li>
	<li>To remove undigested fibers, mesh cells through 100 &#181;m filters into 15 mL tubes (one well per filter per tube). Add PBS to 15 mL, centrifuge tubes at 460 x g, 4 &#176;C for 5 min, and remove the supernatant.</li>
	<li>Lyse erythrocytes with a lysis buffer (NH4Cl 150 mM, KHCO3 10 mM, EDTA 0.1 mM, pH 7.3, 20 &#176;C) by adding 1 mL into each tube. Mix well, and combine the solution from all tubes into one. Stop the reaction after 2 minutes with 11 mL of ice-cold (4 &#176;C) DMEMc.</li>
	<li>Centrifuge at 460 x g, 4 &#176;C for 5 min, and remove the supernatant. Resuspend the pellet with 15 mL of cold PBS. Centrifuge at 460 x g, 4 &#176;C for 5 min, and remove the supernatant.</li>
	<li>Resuspend the pellet in 1 mL of PBS. Add 3 &#956;L of Fc-block antibodies (CD16/CD32, stock 0.5 mg/mL), and incubate on ice for 15 min.</li>
	<li>Add antibodies: 10 &#956;L of Ly6G-PE (stock 0.2 mg/mL) and 10 &#956;L of CD11b-APC (stock 0.2 mg/mL). Add 20 &#956;L of 6-Diamidin-2-phenylindol viability dye (DAPI, stock 5 mg/mL) and incubate on ice in darkness for 30 min. 
	NOTE: Another combination of viability dyes and fluorescent conjugates of antibodies can be used.</li>
	<li>Add PBS up to 15 mL, centrifuge at 460 x g, 4 &#176;C for 5 min, and remove the supernatant.</li>
	<li>Resuspend the pellet in DMEMc to the concentration approximately 10 x 106/mL, and keep on ice.</li>
	<li>Sort CD11b+ Ly6Ghi alive (DAPI-negative) neutrophils with a fluorescence-activated cell sorter (gating strategy see Figure 2). 
	NOTE: Keep the tube with cell suspension and the tube with DMEMc for sorted cells at 4 &#176;C. Use the following optimal sorting settings: a 70 &#956;m nozzle, a threshold rate of maximal 22,000 events/second and a flow rate of 1-3.</li>
	<li>Check the purity of the sorted neutrophils using a cytometer for a recommended purity of &#62;95%.</li>
	<li>Centrifuge sorted neutrophils at 460 x g, 4 &#176;C for 5 min, and remove the supernatant. Resuspend the sorted cells in DMEMc to the concentration of 1 x 106/mL. 
	NOTE: The expected number of neutrophils in one 14-day B16F10 tumor (10 mm diameter) is approximately 3 x 104 cells.</li>
</ol>
4. NAMPT inhibition in TANs in vitro
<ol>
	<li>Prepare FK866 (NAMPT inhibitor) stock in dimethylsulfoxide (DMSO) at a final concentration of 100 mM.</li>
	<li>Seed sorted neutrophils (step 3.11) into 2 wells of a 96-well U-bottom plate (1.5 x 105 neutrophils/well). Add FK866 into the intervention well (final concentration of 100 nM), and an equal amount of DMEMc with DMSO into the control well. Incubate for 2 h at 37 &#176;C, 5% CO2 in a humid incubator.</li>
	<li>Centrifuge at 460 x g, 4 &#176;C for 5 min, and remove the supernatant. Resuspend in 200 &#956;L of PBS in each well. Repeat 2 times.</li>
	<li>Centrifuge at 460 x g, 4 &#176;C for 5 min, and remove the supernatant. Resuspend in commercial endothelial cell growth medium (supplemented with 4 &#181;L/mL endothelial cell growth supplement, 0.1 ng/mL recombinant human epidermal growth factor, 1 ng/mL recombinant human basic fibroblast growth factor, 90 &#181;g/mL heparin and 1 &#181;g/mL hydrocortisone) to a final concentration of 0.2 x 106 cells/mL (in 0.75 mL) (for step 5) or in PBS to the final concentration of 0.6 x 106 cells/mL (in 0.25 mL) (for step 6).</li>
</ol>
5. Estimation of angiogenic properties of TANs using the aortic ring assay
<ol>
	<li>Dissect the thoracic aorta from a male C57BL/6J (WT) mouse. Clean and cut into 0.5 mm width rings. Place all rings in a well of a 24-well plate with 1 mL of supplemented endothelial cell growth medium. Incubate overnight at 37 &#176;C, 5% CO2 in a humid incubator. 
	NOTE: The use of young (younger than 8 weeks) male mice for aorta dissection is preferable, since they give a more robust angiogenic response15.</li>
	<li>Fill the wells of the 96-well flat-bottom plate with 50 &#181;L of solubilized basement membrane matrix, let the gel set for 30 min in a 37 &#176;C, 5% CO2 humid incubator to allow the matrix to polymerize. Prepare at least 3 wells per condition.</li>
	<li>Embed the rings in solubilized basement membrane matrix by placing an aortic ring on the top of the solid matrix layer, 1 ring in the center of each well. Add another 50 &#181;L of solubilized basement membrane matrix to cover each ring.
	<ol>
		<li>Place the plate in a 37 &#176;C, 5% CO2 humid incubator for another 30 min to allow the polymerization of the second matrix layer.</li>
	</ol>
	</li>
	<li>Add 150 &#956;L/well of supplemented endothelial cell growth medium and 2x104 Ifnar1-/- TANs (control and FK866-treated) (step 4.4).</li>
	<li>Incubate the plate for 14 days at 37 &#176;C, 5% CO2 in a humid incubator.</li>
	<li>Image using a standard phase-contrast microscope and estimate the endothelial branching. Quantitative assessment of vessel morphometric and spatial parameters including branching index can be performed automatically using the image processing program designed for scientific images. 
	NOTE: Representative results are depicted in the Figure 3.</li>
</ol>
6. Adoptive transfer of treated neutrophils in the allogenic tumor model
<ol>
	<li>Prepare B16F10 melanoma cells (step 1.6) in PBS at a concentration of 6 x 106 cells/mL.</li>
	<li>Prepare 2 types of neutrophils: FK866-treated neutrophils and control untreated neutrophils (step 4.4) in PBS at a concentration 6 x 105 cells/mL. Mix neutrophils with B16F10 melanoma cells (the final neutrophil to tumor cells ratio 1:10) to have 2 types of cell mixtures.</li>
	<li>Take 10 female WT mice 8-12 weeks old, 5 in each group. Shave the skin on the flank with an electrical shaver, and disinfect with 70% ethanol.</li>
	<li>Inject 100 &#181;L of the cell suspension (step 6.2) subcutaneously with an insulin syringe and a 0.4 mm diameter needle, to both groups of mice. Place 1-5 mice from the same group in one cage.</li>
	<li>At day 2, prepare 2 types of neutrophils: FK866-treated neutrophils and control untreated neutrophils (step 4.4) in PBS at a concentration 6 x 105 cells/mL. Inject 100 &#181;L of cell suspension (step 6.4) i.v. into the tail vein with an insulin syringe and a 0.4 mm diameter needle, to both groups of mice. Place mice back to the cage.</li>
</ol>
7. Tumor growth measurement, histological examination
<ol>
	<li>Monitor tumor growth every other day. Evaluate the tumor size with calipers and calculate the tumor volume with the formula V=4/3*&#960;*(h*w2)/8 (h=height, w=width, depth= width).</li>
	<li>Sacrifice mice at the day 14 in the CO2 chamber. Remove tumors and measure the tumor weights.</li>
	<li>Freeze tumors in optimum cutting temperature compound in liquid nitrogen, and store at -80 &#176;C.</li>
	<li>Thaw the samples to -20 &#176;C and prepare 5 &#956;m sections using a cryotome. Let the cryocuts dry for 30 min at 20 &#176;C. Fix the sections in -20 &#176;C cold acetone for 2 min and let them dry for 30 min at 20 &#176;C.</li>
	<li>Block with Fc-block antibodies (CD16/CD32, stock 0.5 mg/mL 1:500) in PBS for 1 h at 20 &#176;C.</li>
	<li>Stain with rabbit anti mouse Laminin gamma antibody (1:1500 in PBS, 200 &#181;L) for 1 h at 20 &#176;C. Wash with PBS three times.</li>
	<li>Stain with secondary goat anti-rabbit antibody (stock 0.5 mg/mL, 1:400 in PBS,), anti-mouse &#945;SMA (1:500 in PBS) and 2 &#181;L of DAPI (stock 5 mg/mL, 1:100 in PBS) in a final volume of 200 &#181;L of antibody solution. Incubate for 1 h at 20 &#176;C in darkness. Wash with PBS three times.</li>
	<li>Dry slides for 20 minutes at 20 &#176;C in darkness. Mount with anhydrous mounting medium for microscopy and cover with a coverslip. Let it dry 1 h in 37 &#176;C.</li>
	<li>Perform microscopical examination. Quantify the vascularization by counting the total number (optionally area) of Laminin+ vessels and the number (area) of SMA+ developed vessels. 
	NOTE: To perform image analysis, take all images under the same conditions (light, contrast, magnification). In this case, processing parameters are fixed, and image processing becomes completely automatic. Representative results are depicted in the Figure 4 and Figure 5.</li>
</ol>

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	<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. -. 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The authors have nothing to disclose.

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 &amp;amp; Co., N&amp;uuml;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 &amp;amp; Co., N&amp;uuml;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&amp;micro;m / 100 &amp;micro;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&#039;,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&amp;amp;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, &amp;alpha;-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&amp;auml;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 &amp;amp; Co., N&amp;uuml;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...

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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 ...

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Research

JoVE Journal

Cancer Research

5.9K Views.  University of Duisburg-Essen. Ons protocol biedt een nieuw hulpmiddel voor het evalueren van het angiogene potentieel van tumor-geassocieerde primaire neutrofielen in vivo zonder de noodzaak om kunstmatige neutrofiele cellijnmodellen te gebruiken. Directe remming van nicotinamide fosforibosyltransferase, kort NAMPT, bij tumor-geassocieerde neutrofielen met hun daaropvolgende adoptieoverdracht in tumordragende dieren maakt het mogelijk neutrofielen te manipuleren zonder systemische toxische bijwerkingen. We zullen het ther...

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

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Immunology and Infection

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Medicine

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

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

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