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 (Department of Surgery, University of South Dakota Sanford School of Medicine, South Dakota, USA).
<ol>
	<li>Thaw a frozen vial of mEERL cells in a pre-heated (37 &#176;C) water bath and then transfer to a 15 mL conical tube containing warm culture media (Dulbecco&#39;s Modified Eagle Medium [DMEM] supplemented with 40.5% 1:1 DMEM/Hams F12, 10% Fetal Bovine Serum [FBS], 0.1% gentamicin, 0.005% hydrocortisone, 0.05% transferrin, 0.05% insulin, 0.0014% tri-iodothyronine, and 0.005% epidermal growth factor).</li>
	<li>Centrifuge the conical tube at 277 x g for 5 min at 25 &#176;C to remove the media. Then, resuspend the cell pellet in 3-5 mL fresh media and transfer it to a T-25 cell culture flask. For optimal recovery from frozen storage, plate cells at high density. 
	NOTE: T-25 flasks were used because of their smaller size, results in faster recovery times from frozen storage when cells are in close proximity compared to T-75 flasks.</li>
	<li>Let the cells grow in a humidified incubator at 37 &#176;C and 5% CO2, expand to larger flasks (i.e., T-75 or T-150), and passage every 3 days. When there are enough cells for the desired implantation into all mice, remove the flasks, discard the media, and gently rinse the cells with phosphate buffered saline (PBS). Then, add 4 mL (if using T150 flasks) of 0.25% trypsin-EDTA, and incubate at 37 &#176;C for 2 min. Scale the amount of trypsin up or down depending on the dish/flask size being used. 
	NOTE: The type of cell line and degree of confluency may affect trypsinization times. Long trypsinization periods can damage cells resulting in low viability. Use the minimum amount of time needed for trypsinization.</li>
	<li>Under the microscope, the detached cells on trypsinization move freely. If some cells are still attached, very gently tap the flask to mobilize the remaining adherent cells. Add fresh media (scale amount of media as desired) to stop the trypsin reaction and collect the cell suspension in 50 mL conical tubes. Centrifuge at 277 x g for 5 min at 25 &#176;C to remove the media.</li>
	<li>Resuspend the cells in fresh media and count the cells. Centrifuge once more (as described above), and then add cold PBS to the cells to make a final concentration of 10 &#215; 106 cells/mL. Keep the cell suspension(s) on ice before injection to mice.</li>
</ol>
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.
<ol>
	<li>Anesthetize C57Bl/6J mice with ketamine (80 mg/kg) and xylazine (10 mg/kg) mix. Carefully shave the flank area or desired injection site with an electric razor. 
	NOTE: Remember to document the use of controlled substances as required by institutional (or other) rules and regulations.</li>
	<li>Disinfect the flank area with an ethanol pad and slowly inject 100 &#181;L (containing 1 x 106 cells) of the cell suspension subcutaneously using a 25-28 G syringe. Anesthetize the mice before injection to prevent sudden movements and cell loss. Before each injection, gently mix the cell suspension to prevent cells from settling to the bottom of the vial or conical tube.</li>
	<li>After taking up the cell suspension into the syringe, remove all the bubbles and dead space from the top. Inject the cell suspension in a slow and steady manner. Do not use the same needle on multiple mice. Always make extra cell suspensions to account for accidental loss.</li>
	<li>Place the animal in their respective cages and monitor them until they recover from anesthesia. During anesthesia, animals are at risk of hypothermia; therefore, provide supplemental heat or place the mice close to each other to keep warm (if housed in the same cage).</li>
	<li>When tumors reach 3 mm in any direction, randomize the mice (by tumor size and/or weight) in the treatment groups and then start the drug treatment. Inject the mice intraperitoneally (i.p.) with NPs15 containing 3.75 &#181;g rIL-1&#945;/mouse on Days 4 and 9. Measure and record the tumor volume ((length x width2) / 2) and the mouse weight daily or every other day until the tumor size or the mice reach euthanasia criteria. 
	&#8203;NOTE: Even with an experienced researcher, it is difficult to implant the same-sized tumor in all mice. Randomize animals into experimental groups based on the tumor size and mouse weight.</li>
</ol>
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.
<ol>
	<li>Anesthetize the mice with an injection of ketamine/xylazine mix as mentioned above.</li>
	<li>Grasp the loose skin over the shoulder using the non-dominant hand and puncture the submandibular vein with an 18 G needle or lancet, slightly behind the mandible (a white spot at that area).</li>
	<li>Puncture the vein to ensure blood flow immediately. Collect 200-300 &#181;L of blood (depending on mouse weight) into a 1.5 mL polypropylene microcentrifuge 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. 
	NOTE: Do not collect more than 1% of the mouse body weight in one collection or over a 24 h period.</li>
	<li>Let the collected blood clot at room temperature for 20-30 min. Place the tubes on ice until they are ready to be centrifuged.</li>
	<li>Centrifuge the clotted blood at 1540 x g for 15 min at 4 &#176;C.</li>
	<li>Collect the upper layer (serum) without disturbing the red blood cells. Store at -80 &#176;C until use.</li>
</ol>
4. Multiplexing of collected serum
<ol>
	<li>Thaw the serum or plasma samples while keeping them on ice.</li>
	<li>Centrifuge the samples at 1540 x g for 5 min at 4 &#176;C to sediment any cell debris and carefully collect the serum layer from the top.</li>
	<li>Bring out the multiplex kit at room temperature. 
	NOTE: There are several commercially available multiplex kits that are designed for specific cytokines, chemokines, or growth factors. Also, it is possible to customize the kit based on the protein of interest.</li>
	<li>Perform the assay as per the manufacturer&#39;s protocol to detect cytokines. 
	&#8203;NOTE: Most of the multiplex kits use magnetic beads to bind the captured antibody. So, it is essential to use an automated or handheld magnetic washer during washing. Otherwise, the magnetic beads will wash off from the plate, and one will not have enough events to take the reading.</li>
</ol>
5. Collection of tumor and inguinal lymph node and preparation of single-cell suspension
<ol>
	<li>Anesthetize the mice with ketamine/xylazine mix. To ensure complete sedation, use pedal reflex (firm toe pinch). If the mice are not responsive, euthanize the mice by cervical dislocation.</li>
	<li>Lay each mouse on its back and spray 70% ethanol on the abdominal area skin. Use forceps and scissors to cut the tumor out from the left side of the mice and the lymph node from the right side. If the tumor is big, cut into small pieces and take 500-600 mg of the tissue. For the lymph node, collect the whole organ. 
	NOTE: There are two lymph nodes on both sides of the inguinal region. Depending on the experimental goals, the lymph node and the tumor can be isolated from the same side. However, if the tumor becomes very large, it will not be easy to collect the lymph node from the same side.</li>
	<li>Place the tissues onto the respective dissociator tubes containing 3-5 mL of RPMI media. Homogenize the tissue using an automated dissociator. 
	NOTE: Other automated or handheld homogenizers can be used.</li>
	<li>After homogenization, transfer the cell suspension through a 70 &#181;m filter into a 50 mL conical tube. Centrifuge at 277 x g for 5 min at 25 &#176;C to remove the media. Wash and resuspend the cells in 1-2 mL of cold PBS.</li>
</ol>
6. FACS staining of single-cell suspension
<ol>
	<li>Use 0.4% trypan blue staining to count the viable cells in a hemocytometer. Calculate the volume required to get 2-3 million cells and transfer them to the respective FACS tube.</li>
	<li>Use viability dye to gate on live cells; otherwise, nonspecific binding of the antibody with dead cells may occur that can yield a false-positive result. 
	NOTE: Zombie dye was used for live and dead cell staining in this experiment. Several fixable and non-fixable dyes (e.g., propidium iodide) are commercially available.</li>
	<li>Centrifuge (277 x g for 5 min at 25 &#176;C) the counted cells. Decant the supernatant and resuspend the cells into 300 &#181;L of PBS. Add 0.5-1 &#181;L of zombie dye/tube. Incubate at room temperature for 20 min in the dark.</li>
	<li>Centrifuge (277 x g for 5 min at 25 &#176;C) and wash the cells with 1-2 mL of FACS buffer and resuspend them into 200 &#181;L of FACS buffer.</li>
	<li>Add Fc-receptor blocker (2 &#181;L per 200 &#181;L or according to the manufacturer&#39;s protocol) and incubate the cells for 10 min.</li>
	<li>Centrifuge (277 x g for 5 min at 25 &#176;C) and wash the cells with 1-2 mL of FACS buffer. Add the antibody cocktail and incubate for 30 min at 4 &#176;C in the dark.</li>
	<li>After the incubation, centrifuge (277 x g for 5 min at 25 &#176;C) and wash the cells with 1-2 mL FACS buffer. Add 300-400 &#181;L of 2% paraformaldehyde solution and resuspend for fixation. Store the cells at this step for a couple of days at 4 &#176;C or analyze immediately by multicolor flow cytometer.</li>
</ol>

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The authors have nothing to disclose.

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...

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Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#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 ...

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1.9K Views.  University of Iowa. A standard protocol is described to study the antitumor activity and associated toxicity of IL-1α in a syngeneic mouse model of HNSCC.

Video: Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1α Nanoparticles

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% of patients. Surgery has been widely used in association with radiofrequency, cryotherapy, or microwaves to expand the number of treatments performed with a curative intent. Nevertheless, several limitations have been documented when using these techniques (i.e., a traumatic puncture of the parenchyma, a limited size of lesions, and an inability to monitor the treatment in real-time).High-intensity focused ultrasound (HIFU) technology can achieve precise ablations of biological tissues without incisions or radiation. Current HIFU devices are based on an extracorporeal approach with limited access to the liver. We have developed a HIFU device designed for intraoperative use. The use of a toroidal transducer enables an ablation rate (10 cm3&#183;min-1) higher than any other treatment and is independent of perfusion.
The feasibility, safety, and accuracy of intraoperative HIFU ablation were evaluated during an ablate-and-resect prospective study. This clinical phase I and IIa study was performed in patients undergoing hepatectomy for liver metastases. The HIFU treatment was performed on healthy tissue scheduled for resection.Liver metastases measuring less than 20 mm will be targeted during phase IIb (currently ongoing). This set-up allows the real-time evaluation of HIFU ablation while protecting participating patients from any adverse effects related to this new technique.
Fifteen patients were included in phase I&#8211;IIa and 30 HIFU ablations were safely created within 40 s and with a precision of 1&#8211;2 mm. The average dimensions of the HIFU ablations were 27.5 x 21.0 mm2, corresponding to a volume of approximately 7.5 cm3. The aim of the ongoing phase IIb is to ablate metastases of less than 20 mm in diameter with a 5 mm margin.

Here, we present an ablate-and-resect prospective study to evaluate the feasibility, safety, and accuracy of intraoperative high-intensity focused ultrasound ablation in patients undergoing hepatectomy for liver metastases.

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application. The mouse represents the traditional animal model for preclinical testing; however, mice are relatively expensive to keep and have long experimental cycles due to the limited progeny from each mother. The zebrafish has emerged as a powerful model system for developmental and biomedical research, including cancer research. In particular, due to its optical transparency and rapid development, zebrafish embryos are well suited for real-time in vivo monitoring of the behavior of cancer cells and their interactions with their microenvironment. This method was developed to sequentially introduce human cancer cells and functionalized nanoparticles in transparent Casper zebrafish embryos and monitor in vivo recognition and targeting of the cancer cells by nanoparticles in real time. This optimized protocol shows that fluorescently labeled nanoparticles, which are functionalized with folate groups, can specifically recognize and target metastatic human cervical epithelial cancer cells labeled with a different fluorochrome. The recognition and targeting process can occur as early as 30 min postinjection of the nanoparticles tested. The whole experiment only requires the breeding of a few pairs of adult fish and takes less than 4 days to complete. Moreover, zebrafish embryos lack a functional adaptive immune system, allowing the engraftment of a wide range of human cancer cells. Hence, the utility of the protocol described here enables the testing of nanoparticles on various types of human cancer cells, facilitating the selection of optimal nanoparticles in each specific cancer context for future testing in mammals and the clinic.

Described here is a method for utilizing zebrafish embryos to study the ability of functionalized nanoparticles to target human cancer cells in vivo. This method allows for the evaluation and selection of optimal nanoparticles for future testing in large animals and in clinical trials.

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.