generating anti-angiogenic tumor-associated neutrophils

Generating Anti-Angiogenic Tumor-Associated Neutrophils

For tumor-associated neutrophil isolation, place five tumors per well into individual wells of a sterile six-well plate, as they are harvested, and use sterile scissors to mince the tumors into 2- to 3-millimeter pieces. Next, digest the tumor fragments with 1 milliliter of disk-based collagenase, DNase I solution for 45 minutes at 37 degrees Celsius and 5% carbon dioxide with humidity, mixing the samples with a 10-milliliter syringe every 15 minutes.
At the end of the incubation, filter the cells through 100-micrometer strainers into one 15-milliliter tube per well to remove the undigested fibers, and add PBS to a final volume of 15 milliliters. Collect the dissociated cells by centrifugation, and lyse the red blood cells with 1 milliliter of lysis buffer per tube.
After mixing, combine the contents from each tube into a single 15-milliliter tube, stopping the reaction after two minutes with 11 milliliters of complete 4 degrees Celsius medium. After centrifugation, resuspend the pellet in 1 milliliter of PBBS, and block any nonspecific binding with three microliters of FC-blocked antibody for 15 minutes on ice.
At the end of the incubation, label the cells with anti-CD11b and Ly6G antibodies and any other antibodies of interest plus DAPI for a 30-minute incubation on ice protected from light. At the end of the incubation, wash the cells in 14 milliliters of PBS, and resuspend the pellets at a 1 x 107 cells per milliliter of fresh complete medium concentration on ice.
Then, sort the CD11b-positive Ly6G-high DAPI-negative neutrophils on a fluorescence-activated cell sorter according to standard sorting protocols, checking the purity of the isolated neutrophils on the flow cytometer at the end of the sort. For in vitro tumor-associated, neutrophil inhibition, seed 1.5 times 10 to the fifth sorted neutrophils into each of two wells of a 96-well U-bottom plate, and add FK866 to a final concentration of 100 nanomolar in complete medium into one well and an equal volume of complete medium alone to the other well. After two hours in the cell culture incubator, wash the cells two times with PBS, and resuspend the pellets in PBS at the appropriate concentration for the subsequent injection.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
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&#39;s Modified Dulbecco&#39;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-phycoerythrin (Ly6G-PE, stock 0.2 mg/mL) and 10 &#956;L of CD11b-allophycocyanin (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).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 ml tubes</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
			<td>6,25,54,502</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>8,33,920</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>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>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>83,39,11,302</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Pylaeva, E., et al. <a href="https://app.jove.com/v/59807">Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to generate anti-angiogenic tumor-associated neutrophils (TANs). Upon taking a murine melanoma tumor containing pro-angiogenic TANs, the single cells are isolated using fluorescence-activated cell sorting (FACS) and treated with an inhibitor of nicotinamide phosphoribosyl transferase (NAMPT) to reduce the capacity to stimulate angiogenesis.

<img alt="Figure 1" src="/files/ftp_upload/22272/22272_Figure1.jpg" /> 
Figure 1. The scheme of the protocol. Step 1. Preparation of B16F10 melanoma cell line; 2. Allogenic tumor model in mice; 3. Isolation of TANs from the tumors; 4. Inhibition of NAMPT in TANs in vitro; 5. Estimation of angiogenic properties of TANs in the aortic ring assay; 6. Adoptive transfer of treated neutrophils in the allogenic tumor model; 7. Tumor growth monitoring, histological examinatio...

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE: The overall scheme of the ...

Take a murine melanoma tumor comprising tumor-associated neutrophils, or TANs.
TANs display elevated activity of the enzyme nicotinamide phosphoribosyltransferase, or NAMPT, stimulating angiogenesis, the proliferation of blood vessels in tumors, and facilitating tumor growth.
Mince the tissue and add enzymes to degrade the tissue matrix, loosening the cells.
Filter the suspension through a strainer and centrifuge to pellet the cells.
Add a lysing buffer to lyse the red blood cells, then centrifuge and discard the supernatant containing cell debris.
Introduce antibodies to block cellular Fc receptors, thereby avoiding non-specific immunostaining.
Introduce a fluorophore-labeled antibody cocktail specific for surface markers on TANs and a dye to discriminate dead cells.
Using FACS, isolate the fluorophore-labeled viable TANs and transfer them onto a microplate.
Add an inhibitor blocking NAMPT activity to selected wells and incubate.
Use the cells to assess their angiogenesis-stimulating capacity. The inhibitor-treated TANs show reduced angiogenesis compared to the untreated TANs.

51 Views. Generazione di neutrofili anti-angiogenici associati al tumore

Video: Generazione di neutrofili anti-angiogenici associati al tumore - Experiment

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Un modello di Xenotrapianto a parte del paziente di melanoma

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

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Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

Impianto ortotopico a ultrasuoni dell'adenocarcinoma dugalese Murine

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

Analisi del microambiente epatico durante la progressione precoce del carcinoma epatocellulare associato alla steatosi epatica non alcolica nel pesce zebra

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

Generating Anti-Angiogenic Tumor-Associated Neutrophils

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Generating Anti-Angiogenic Tumor-Associated Neutrophils