Name: transfer of manipulated tumor-associated neutrophils into tumor-bearing mice to study their angiogenic potential in vivo
Uploaded: 2019-07-20T13:00:00.000+00:00
Duration: 8 min 19 s
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.
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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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...

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 ...

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Research

JoVE Journal

Cancer Research

5.9K Views.  University of Duisburg-Essen. 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.

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 ...

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

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Biology

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 ...

Medicine

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.

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

Detecting Migration and Infiltration of Neutrophils in Mice

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.

Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. ...

Study of Angiogenesis Using Scratch Wound Migration and Spheroid Sprouting Assays

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

Author Spotlight: Investigating Angiogenesis Through Challenges and Innovations in Assay Development

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

Monitoring Neutrophil Recruitment Dynamics and Bacterial Burden at the Infection Site in a Murine Model

Source: Anderson, L. S., et al. <a href="https://app.jove.com/v/59015">A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection.</a> J. Vis. Exp. (2019).
This video demonstrates in vivo imaging to study the neutrophil recruitment dynamics at an infection site in an immunocompromised, transgenic mouse with GFP-expressing neutrophils. It involves injecting luminescent bacteria to cause an infection. This leads to neutrophil recruitment to the infection site, whose tracking is done by imaging.