Name: evaluation of the in vivo antitumor activity of polyanhydride il-1&#945; nanoparticles
Uploaded: 2021-06-28T14:30:00
Description: Watch this Scientific Journal Video about Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1ÃŽÂ± Nanoparticles at JoVE.com

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-1ÃŽÂ± Nanoparticles at JoVE.com

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1ÃƒÅ½Ã‚Â± 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 ...

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