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A standard protocol is described to study the antitumor activity and associated toxicity of IL-1α in a syngeneic mouse model of HNSCC.
Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1α) 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-1α would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1α systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1α-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-1α-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.
One of the emerging areas of cancer immunotherapy is the use of inflammatory cytokines to activate patients' immune system against their tumor cells. Several proinflammatory cytokines (i.e., interferon-alpha (IFNα), 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-1α) 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 ....
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.......
In this study, the antitumor activity of polyanhydride IL-1α in a syngeneic mouse model of HNSCC was investigated. Recombinant IL-1α (rIL-1α) 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-1α-NPs did not induce a significant antitumor effect compared to saline or blank-NPs (Figure 1A) and was.......
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 .......
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.
....Name | Company | Catalog Number | Comments |
Bio-Plex 200 Systems | Bio-Rad | The system was provided from the Flow Cytometry Facility University of IOWA Health Care | |
Bio-Plex Pro Mouse Cytokine 23-plex Assay | Bio-Rad | M60009RDPD | |
C57BL/6J Mice | Jakson Labs | 664 | 4 to 6 weeks old |
DMEM (Dulbecco's Modified Eagle Medium) | Thermo Fisher Scientific | 11965092 | |
DMEM/Hams F12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) | Thermo Fisher Scientific | 11320033 | |
EGF | Millipore Sigma | SRP3196-500UG | |
Fetal Bovine Serum | Millipore Sigma | 12103C-500ML | |
Gentamycin sulfate solution | IBI Scientific | IB02030 | |
gentleMACS Dissociator | Miltenyi biotec | ||
Hand-Held Magnetic Plate Washer | Thermo Fisher Scientific | EPX-55555-000 | |
Hydrocortisone | Millipore Sigma | H6909-10ML | |
Insulin | Millipore Sigma | I0516-5ML | |
Ketamine/xylazine | Injectable anesthesia | ||
MEERL cell line | Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells | ||
Portable Balances | Ohaus | ||
Scienceware Digi-Max slide caliper | Millipore Sigma | Z503576-1EA | |
Sterile alcohol prep pad (70% isopropyl alcohol) | Cardinal | COV5110.PMP | |
Transferrin Human | Millipore Sigma | T8158-100MG | |
Tri-iodothyronin | Millipore Sigma | T5516-1MG |
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