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>

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

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

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

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

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

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

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

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

transfer-manipulated-tumor-associated-neutrophils-into-tumor-bearing

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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis and to identify novel therapeutic targets. To investigate molecular events that drive the progression from primary cancer to metastasis, we have developed a bitransgenic mouse model, RIP-Tag; RIP-tva. In this mouse model, the rat insulin promoter (RIP) drives the expression of the SV40 T antigen (Tag) and the receptor for subgroup A avian leukosis virus (tva) in pancreatic &#946; cells. The mice develop pancreatic neuroendocrine tumors with 100% penetrance through well-defined stages that are similar to human tumorigenesis, with stages including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. Because RIP-Tag; RIP-tva mice do not develop metastatic disease, genetic alterations that promote metastasis can be identified easily. Somatic gene transfer into tva-expressing, proliferating pancreatic &#946; premalignant lesions is achieved through intracardiac injection of avian retroviruses harboring the desired genetic alteration. A titer of &#62;1 x 108 infectious units per ml is considered appropriate for in vivo infection. In addition, avian retroviruses can infect cell lines derived from tumors in RIP-Tag; RIP-tva mice with high efficiency. The cell lines can also be used to characterize the metastatic factors. Here we demonstrate how to utilize this mouse model and cell lines to assess the functions of candidate genes in tumor metastasis.

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

We present a protocol to demonstrate a novel somatic gene transfer system utilizing RIP-Tag; RIP-tva mouse model to study the function of genes in metastasis. The avian retroviruses are delivered intracardiacally to ensure gene transfer into pre-malignant, noninvasive lesions of pancreatic &#946; cells in adult mice.

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.