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 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-1α3,4,5,6. These studies found promising antitumor activity of recombinant IL-1α (rIL-1α) 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-1α.
To attempt to address the critical issue of IL-1α-mediated toxicities, polyanhydride nanoparticle (NP) formulations that allow for the controlled release of IL-1α by surface erosion kinetics will be investigated. These NP formulations are intended to reap the benefits of the antitumor properties of IL-1α 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-1α (IL-1α-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-1α-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-1α-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.
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 (Department of Surgery, University of South Dakota Sanford School of Medicine, South Dakota, USA).
2. Tumor implantation, drug treatment, and measurement
NOTE: The experimental animals were kept in the Animal Care Facility at the University of Iowa and followed appropriate aseptic procedures to handle them.
3. Blood collection and serum separation
NOTE: Blood collection from a submandibular vein is an easy and effective technique that allows blood collection from conscious animals or animals under anesthesia. For this study, blood was collected from the animals when they were under anesthesia.
4. Multiplexing of collected serum
5. Collection of tumor and inguinal lymph node and preparation of single-cell suspension
6. FACS staining of single-cell suspension
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 accompanied by some weight loss, although not as prominent as rIL-1α (Figure 1B). Mice treated with rIL-1α survived significantly longer than the other treatment groups (Figure 1C). Additionally, circulating levels of IL-1α, IL-1β, and IFN-γ were higher in rIL-1α-treated mice compared to the other treatment groups (Figure 2A–C). These results suggest that improvements in the IL-1α-NP with regard to antitumor efficacy are warranted.
Figure 1: Effect of rIL-1α on tumor growth, survival, and body weight. Male C57BL/6J mice (n = 10-11 mice/treatment group) bearing mEERL HNSCC tumors were treated i.p. on Day 1 and Day 5 with rIL-1α (3.75 µg of rIL-1α), IL-1α-NP (0.25 mg of NPs containing 3.75 µg of rIL-1α), Blank-NP (0.25 mg of NPs), and 100 µL of saline solution (CON). Shown are changes in average tumor volume (A), normalized body weights (B), and survival curves (C). Error bars represent the standard error of the mean. *p < 0.05 vs. other treatment groups. Please click here to view a larger version of this figure.
Figure 2: Effect of rIL-1α on circulating cytokines. Blood samples were collected from a subset of mice (n = 4 mice/treatment group) after the second drug administration and analyzed for circulating cytokine levels by multiplex assay. Shown are circulating levels of IL-1α (A), IL-1β (B), and IFN-γ (C). Error bars represent the standard error of the mean. Please click here to view a larger version of this figure.
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 on both the left and the right flank. In this bilateral tumor model, radiotherapy or drugs can be administered to one tumor intratumorally, and abscopal responses can be monitored. Orthotopic HNSCC mouse models, while more clinically relevant, are technically challenging to generate, difficult to monitor tumor growth, and the tumor burden in the oral cavity often results in premature euthanasia due to the inability of the mice to eat and drink.
The preparation of cells is an important step for the formation of symmetrical and similar-sized tumors in all mice. Poor preparation of cells results in reduced cell viability and greatly affects tumor generation in mice. The tumor cells are recommended to be at an early passage number and within 80%-90% confluency. Higher passage number and confluency affect cell viability and thus tumor generation. Cells should also be injected as soon after preparation as possible since viability is reduced if kept in PBS beyond 20-30 min. If a large number of mice need to be implanted with tumors, it is recommended to make a stock solution of cells kept in media and prepare injectable cell suspensions in PBS for a smaller group of animals.
There are several critical steps in the protocol that needs to be carefully maintained after preparing tumor cells for injection. Injecting tumor cells to the subdermal space could produce different tumor growth patterns and sizes compared to the subcutaneous space. Therefore, careful attention should be placed on needle placement for consistent tumor formation. Needle selection is also important. If the needle is smaller than the cancer cell, smaller needles could stress the cells resulting in less viability. If the needle is very big, it could hurt the animal and result in cell leakage from the injected site. Even for experienced researchers with the correct needle size, there may be cell leakage at the injected site resulting in a small or no tumor. It is important that researchers use the correct technique for tumor injection and optimal needle size in order to reduce tumor cell loss and increase accuracy and precision during tumor cell implantation. Tumor measurements should be carefully done using Vernier calipers (manual or electronic). The best practice is to be consistent with the direction of tumor length and width measurement to reduce variability. Tumor measurement by the same researcher throughout the study can reduce variability.
As expected, mice receiving rIL-1α lost weight during treatment, which supports previous findings15,16. Although weight loss is a simple and straightforward way of assessing toxicity, there are other toxicological endpoints that can be utilized. Assessment of blood cell counts (white blood cells, red blood cells, and platelet counts) and liver enzyme levels (aspartate transaminase, alanine aminotransferase, and alkaline phosphatase) provide valuable information about drug toxicity. Additionally, a subset of mice can be sacrificed, and histopathological analysis of organs (liver, kidneys, pancreas, lung, etc.) can be performed. Systemic inflammation is often used as an indicator of toxicity. Here, a number of circulating proinflammatory cytokines were analyzed in the mice after drug treatment by submandibular venipuncture using an 18 G needle. Submandibular venipuncture on mice requires a skill that comes from many repetitions of the procedure. If the puncture is too deep, it may cause bleeding from the ear and internal tissue damage. Whereas, if the needle is not penetrated far enough, an insufficient amount of blood may be collected. Alternatives to needles are the use of disposable bleeding lancets. There are different kinds of bleeding lancets that are commercially available that differ in their length. Researchers should use a suitable lancet size to ensure optimal blood collection and humane treatment of animals. For this procedure, results for three cytokines are shown (Figure 2A–C). It is likely that an increase in circulating proinflammatory cytokines including IL-1α, IL-1β, and IFN-γ observed in rIL-1α-treated mice may be associated with acute weight loss (Figure 1B) observed in this treatment group.
Lastly, a protocol for isolation and preparation of single-cell suspensions of mice tumor and lymph nodes is described. This method is useful for those seeking to detect changes in immune cell activation and recruitment due to drug treatment. During dissection of tumors, adipose tissue, skin, hair, and other debris should be eliminated as much as possible. Usually, the tumor volume should be greater than 30 mm3 to have enough cells for flow cytometry. However, if the tumor is very large, it may be difficult to prepare single-cell suspensions. Large tumors should be cut into small pieces before placing into the dissociator tube. The process should be done quickly to get optimal viable cells. Additionally, large tumors make finding the inguinal lymph node on the tumor side difficult. In this case, the inguinal lymph node can be collected from the opposite site. Once single-cell suspensions are obtained, they can be stained with different antibodies and analyzed by multicolor flow cytometry.
Overall, these protocols provide an effective way to study the antitumor activity of immune-modulatory drugs, and the associated changes in the circulating cytokines and immune cell populations.
The authors have nothing to disclose.
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.
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 |