This work was supported in part by Merit Review Award #I01BX004829 from the United States (U.S.) Department of Veterans Affairs, Biomedical Laboratory Research and Development Service and supported by the Mezhir Award Program through the Holden Comprehensive Cancer Center at the University of Iowa.

All the in vivo procedures used in this study were approved by the Institutional Animal Care and UseCommittee (IACUC) of the University of Iowa.
1. Preparation and maintenance of HNSCC cell line
NOTE: In this study, the murine oropharyngeal epithelial cell line stably transformed with HPV E6 and E7 together with hRas and luciferase (mEERL) will be used. This cell line was developed from C57BL/6J mouse strain and was a gift from Dr. Paola D. Vermeer (Departmen...

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This protocol will allow any investigator to study the antitumor activity and some of the underlying mechanisms of immunomodulatory drugs in an in vivo tumor mouse model system. Here, a syngeneic subcutaneous tumor model was used, which has several advantages over orthotopic models, including its technically straightforward protocol, easy monitoring of tumor growth, less animal morbidity, and higher producibility. Subcutaneous tumor models can also be modified to a bilateral tumor model by injecting tumor cells ...

One of the emerging areas of cancer immunotherapy is the use of inflammatory cytokines to activate patients&#39; immune system against their tumor cells. Several proinflammatory cytokines (i.e., interferon-alpha (IFN&#945;), interleukin-2 (IL-2), and interleukin-1 (IL-1)) can mount significant antitumor immunity, which has generated interest in exploring the antitumor properties as well as the safety of cytokine-based drugs. Interleukin-1 alpha (IL-1&#945;) in particular, is a proinflammatory cytokine known as the master cytokine of inflammation1. Since the discovery of this cytokine in the late 1970s, it has been investigated as an anticancer agent as well as a hematopoietic drug to treat the negative effects of chemotherapy2. During the late 1980s, several preclinical and clinical studies were conducted to determine the anticancer effects of IL-1&#945;3,4,5,6. These studies found promising antitumor activity of recombinant IL-1&#945; (rIL-1&#945;) against melanoma, renal cell carcinoma, and ovarian carcinoma. However, toxicities, including fever, nausea, vomiting, flu-like symptoms, and most severely dose-limiting hypotension were commonly observed. Unfortunately, these dose-related toxicities dampened the enthusiasm for further clinical use of rIL-1&#945;.
To attempt to address the critical issue of IL-1&#945;-mediated toxicities, polyanhydride nanoparticle (NP) formulations that allow for the controlled release of IL-1&#945; by surface erosion kinetics will be investigated. These NP formulations are intended to reap the benefits of the antitumor properties of IL-1&#945; while reducing dose-limiting side effects7. Polyanhydrides are FDA-approved polymers that degrade through surface erosion resulting in nearly zero-order release of encapsulated agents8,9,10,11,12. Amphiphilic polyanhydride copolymers containing 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis-(p-carboxyphenoxy) hexane (CPH), have been reported to be excellent delivery systems for various payloads in oncology and immunology-based research8,12. In the following protocol 20:80 CPTEG:CPH NPs loaded with 1.5 wt.% rIL-1&#945; (IL-1&#945;-NPs) will be used to study the antitumor activity and toxicity of this cytokine in a mouse model of HNSCC.
The overall goal of the following procedures is to assess the antitumor activity of IL-1&#945;-NPs on HNSCCs. The procedures described, including assessing tumor growth and survival, can be applied to any immune-modulatory agent of interest. These procedures should be performed in a syngeneic mouse model with an intact immune system13 to maximize clinical relevancy. IL-1&#945;-NP toxicity will also be assessed by measuring changes in circulating levels of proinflammatory cytokines and animal weight. There are many methods to determine in vivo drug toxicity; however, the most widely used methods involve the measurement of serum enzymes for organ toxicity and histological changes in those organs. However, to perform histological analyses, the animal needs to be sacrificed, which will affect the survival curves of the experiment. Therefore, this protocol will include a protocol for the collection of blood from live mice for the measurement of cytokines in serum samples. The collected serum can be used for the measurement of any desired serum analytes for organ toxicity. Multicolor flow cytometry will be used to understand the changes in the immune cell population in the tumor microenvironment and immune cell migration to the lymph node. Other methods can be utilized to identify immune cells, including immunohistochemistry and/or immunofluorescence of preserved sections14. However, these techniques can be time-consuming and tedious to perform on a large number of animals. Overall, the following methods will allow investigators to study the antitumor immune response and potential mechanisms of immunostimulatory agents for cancer treatment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bio-Plex 200 Systems</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>The system was provided from the Flow Cytometry Facility University of IOWA Health Care</td>
		</tr>
		<tr>
			<td>Bio-Plex Pro Mouse Cytokine 23-plex Assay</td>
			<td>Bio-Rad</td>
			<td>M60009RDPD</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J Mice</td>
			<td>Jakson Labs</td>
			<td>664</td>
			<td>4 to 6 weeks old</td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/Hams F12 (Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Millipore Sigma</td>
			<td>SRP3196-500UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Millipore Sigma</td>
			<td>12103C-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin sulfate solution</td>
			<td>IBI Scientific</td>
			<td>IB02030</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Dissociator</td>
			<td>Miltenyi biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hand-Held Magnetic Plate Washer</td>
			<td>Thermo Fisher Scientific</td>
			<td>EPX-55555-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Millipore Sigma</td>
			<td>H6909-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Millipore Sigma</td>
			<td>I0516-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine/xylazine</td>
			<td></td>
			<td></td>
			<td>Injectable anesthesia</td>
		</tr>
		<tr>
			<td>MEERL cell line</td>
			<td></td>
			<td></td>
			<td>Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells</td>
		</tr>
		<tr>
			<td>Portable Balances</td>
			<td>Ohaus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scienceware&#160;Digi-Max&#160;slide caliper</td>
			<td>Millipore Sigma</td>
			<td>Z503576-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile alcohol prep pad (70% isopropyl alcohol)</td>
			<td>Cardinal</td>
			<td>COV5110.PMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin Human</td>
			<td>Millipore Sigma</td>
			<td>T8158-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-iodothyronin</td>
			<td>Millipore Sigma</td>
			<td>T5516-1MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of the in vivo antitumor activity of polyanhydride il-1&#945; nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

In this study, the antitumor activity of polyanhydride IL-1&#945; in a syngeneic mouse model of HNSCC was investigated. Recombinant IL-1&#945; (rIL-1&#945;) significantly slowed mEERL tumor growth (Figure 1A), although weight loss was observed in the treated mice, which was restored after treatment withdrawal (Figure 1B). IL-1&#945;-NPs did not induce a significant antitumor effect compared to saline or blank-NPs (Figure 1A) and was...

Watch this Scientific Journal Video about Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1Î± Nanoparticles at JoVE.com

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&amp;#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

These protocols provide an effective way to study the antitumor activity of immune modulator attacks.Moreover, the analysis of the associated chances in the circulating cytokines and immune cell populations can be done using these proposed methods.The main advantage of this protocol is it's easy and a straight forward way of implanting a tumor and monitor its growth.The direct implication of this technique is to assess the efficacy of certain therapies or challenges in anti-cancer research.Additionally, some aspects of these techniques are suitable for toxicity studies.To begin, thaw a frozen vial of murine EERL cells in a preheated water bath at 37 degrees Celsius and transfer them to a 15 milliliter conical tube containing warm culture media.Centrifuge the conical tube at 277 times G for five minutes at 25 degrees Celsius to remove the media.Then re-suspend the cell palate in three to five milliliters fresh media and transfer it to a T25 cell culture flask.Let the cells grow in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide, Expand to larger flasks and passage every three days.When there are enough cells for the desired implantation into all mice, remove the flasks, discard the media and gently rinse the cells PBS.Then add four milliliters of 0.25%trypsin EDTA and incubate at 37 degrees Celsius for two minutes.Add fresh media to stop the trypsin reaction and collect the cell suspension in 50 milliliter conical tubes.Re-suspend the cells in fresh media and count the cells.Centrifuge once more, then add cold PBS to the cells to make a final concentration of 10 million cells per milliliter.Keep the cell suspension on ice before injection.Disinfect the flank area with an ethanol pad.To inject cell suspension into mice, aspirate 100 microliters of cell suspension into the syringe and remove all the bubbles and dead space from the top.Then inject the cell suspension subcutaneously in a slow and steady manner into anesthetized mice.Place the animals in their respective cages and monitor them until they recover from anesthesia.To collect blood, grasp the loose skin over the shoulder using the non-dominant hand and puncture the submandibular vein with an 18 gauge needle or lancet.Collect 200 to 300 microliters of blood into a 1.5 milliliter polypropylene micro centrifuge tube or serum separator tube.After blood collection, apply gentle pressure to the puncture site until the bleeding has stopped.Return the mice to their respective cages and observe until they recover from anesthesia.Let the collected blood to clot at room temperature for 20 to 30 minutes.Then place the tubes on ice until they are ready to be centrifuged.Centrifuge the clotted blood at 1, 540 times G for 15 minutes at four degrees Celsius, and carefully collect the serum layer from the top.Thaw the serum or plasma samples while keeping them on ice.Then, centrifuge the samples at 1, 540 times G for five minutes at four degrees Celsius, to sediment any cell debris and carefully collect the serum layer from the top.Place the multiplex kit at room temperature and perform the assay according to the manufacturer's protocol.Lay each mouse on its back and spray 70%ethanol on the abdominal area skin.Use forceps and scissors to cut the lymph node from the right side of the mice and the tumor out from the left side.If the tumor is big, cut it into small pieces and take about 500 to 600 milligrams off the tissue.Place the tissues onto the respective dissociator tubes containing three to five milliliters of RPMI media.Homogenize the tissue using an automated dissociator.After homogenization, filter the cell suspension through a 70 micrometer filter into a 50 milliliter conical tube.Centrifuge at 277 times G for five minutes at 25 degrees Celsius to remove the media.Wash and re-suspend the cells in one to two milliliter of cold PBS.Use 0.4%trypsin blue staining, to count the viable cells in a hemocytometer.Calculate the volume required to get two to three million cells and transfer them to the respective fax tube.Centrifuge counted cells at 277 times G for five minutes at 25 degrees Celsius.Decant the supernatant and re-suspend the cells into 300 microliters of PBS.Add 0.5 to one microliter of zombie dye per tube and incubate at room temperature for 20 minutes in the dark.Centrifuge the cells Wash them with one to two milliliters of fax buffer and re-suspend them into 200 microliters of fax buffer.Add FC receptor blocker according to the manufacturer's protocol and incubate the cells for 10 minutes.Centrifuge again at previously described settings and wash the cells with one to two milliliters of fax buffer.Add the antibody cocktail and incubate for 30 minutes at four degrees Celsius in the dark.After the incubation, centrifuge and wash the cells with one to two milliliters fax buffer.Add 300 to 400 microliters of 2%paraformaldehyde solution, and re-suspend for fixation.Store the cells for a couple of days at four degrees Celsius or analyze immediately by multicolor flow cytometer.In this study, the antitumor activity of polyannhydride IL-1

evaluation-vivo-antitumor-activity-polyanhydride-il-1

Research

JoVE Journal

Cancer Research

1.9K visualizações.  University of Iowa. Esses protocolos fornecem uma maneira eficaz de estudar a atividade antitumoral dos ataques imunomoduladores. Além disso, a análise das chances associadas nas citocinas circulantes e populações de células imunes pode ser feita usando esses métodos propostos. A principal vantagem deste protocolo é que é fácil e uma maneira direta de implantar um tumor e monitorar seu crescimento. A implicação direta desta técnica é avaliar a eficácia de certas terapias ou desafios na pesquisa ant...