Name: Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1α Nanoparticles (Video) | JoVE Journal
Uploaded: 2021-06-28T14:30:00.000+00:00
Duration: 9 min 57 s
Description: Learn to evaluate antitumor activity of IL-1α nanoparticles in a syngeneic mouse model of head and neck squamous cell carcinoma. This protocol enables assessment of efficacy and toxicity for cancer immunotherapy research.

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5 minutes

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>

<ol><li>Dinarello, C. A., Simon, A., vander Meer, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treating+inflammation+by+blocking+interleukin-1+in+a+broad+spectrum+of+diseases%5BTitle%5D)+AND+%22Nature+Reviews+Drug+Discovery%22%5BJournal%5D)">Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases.</a> Nature Reviews Drug Discovery. 11 (8), 633-652 (2012).</li>
<li>de Mooij, C. E. M., Netea, M. G., vander Velden, W., Blijlevens, N. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+interleukin-1+pathway+in+patients+with+hematological+disorders%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Targeting the interleukin-1 pathway in patients with hematological disorders.</a> Blood. 129 (24), 3155-3164 (2017).</li>
<li>Veltri, S., Smith, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin+1+trials+in+cancer+patients%3A+a+review+of+the+toxicity%2C+antitumor+and+hematopoietic+effects%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Interleukin 1 trials in cancer patients: a review of the toxicity, antitumor and hematopoietic effects.</a> Stem Cells. 14 (2), 164-176 (1996).</li>
<li>Grandis, J. R., Chang, M. J., Yu, W. D., Johnson, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antitumor+activity+of+interleukin-1+alpha+and+cisplatin+in+a+murine+model+system%5BTitle%5D)+AND+%22Archives+of+Otolaryngology-+Head+%26+Neck+Surgery%22%5BJournal%5D)">Antitumor activity of interleukin-1 alpha and cisplatin in a murine model system.</a> Archives of Otolaryngology- Head & Neck Surgery. 121 (2), 197-200 (1995).</li>
<li>Curti, B. D., Smith, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin-1+in+the+treatment+of+cancer%5BTitle%5D)+AND+%22Pharmacology+%26+Therapeutics%22%5BJournal%5D)">Interleukin-1 in the treatment of cancer.</a> Pharmacology & Therapeutics. 65 (3), 291-302 (1995).</li>
<li>Smith, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+treatment+with+interleukin-1+alpha+on+platelet+recovery+after+high-dose+carboplatin%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">The effects of treatment with interleukin-1 alpha on platelet recovery after high-dose carboplatin.</a> New England Journal of Medicine. 328 (11), 756-761 (1993).</li>
<li>Senapati, S., Mahanta, A. K., Kumar, S., Maiti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlled+drug+delivery+vehicles+for+cancer+treatment+and+their+performance%5BTitle%5D)+AND+%22Signal+Transduction+and+Targeted+Therapy%22%5BJournal%5D)">Controlled drug delivery vehicles for cancer treatment and their performance.</a> Signal Transduction and Targeted Therapy. 3, 7(2018).</li>
<li>Carbone, A. L., Uhrich, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+synthesis+of+fast-degrading+Poly%28anhydride-esters%29%5BTitle%5D)+AND+%22Macromolecular+Rapid+Communications%22%5BJournal%5D)">Design and synthesis of fast-degrading Poly(anhydride-esters).</a> Macromolecular Rapid Communications. 30 (12), 1021(2009).</li>
<li>Gopferich, A., Tessmar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyanhydride+degradation+and+erosion%5BTitle%5D)+AND+%22Advanced+Drug+Delivery+Reviews%22%5BJournal%5D)">Polyanhydride degradation and erosion.</a> Advanced Drug Delivery Reviews. 54 (7), 911-931 (2002).</li>
<li>Jain, J. P., Chitkara, D., Kumar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyanhydrides+as+localized+drug+delivery+carrier%3A+an+update%5BTitle%5D)+AND+%22Expert+Opinion+on+Drug+Delivery%22%5BJournal%5D)">Polyanhydrides as localized drug delivery carrier: an update.</a> Expert Opinion on Drug Delivery. 5 (8), 889-907 (2008).</li>
<li>Jain, J. P., Modi, S., Domb, A. J., Kumar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+polyanhydrides+as+localized+drug+carriers%5BTitle%5D)+AND+%22Journal+of+Controlled+Release+%3A+Official+Journal+of+the+Controlled+Release+Society%22%5BJournal%5D)">Role of polyanhydrides as localized drug carriers.</a> Journal of Controlled Release : Official Journal of the Controlled Release Society. 103 (3), 541-563 (2005).</li>
<li>Kumar, N., Langer, R. S., Domb, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyanhydrides%3A+an+overview%5BTitle%5D)+AND+%22Advanced+Drug+Delivery+Reviews%22%5BJournal%5D)">Polyanhydrides: an overview.</a> Advanced Drug Delivery Reviews. 54 (7), 889-910 (2002).</li>
<li>Goldman, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+human+cell+engraftment+in+mice+deficient+in+RAG2+and+the+common+cytokine+receptor+gamma+chain%5BTitle%5D)+AND+%22British+Journal+of+Haematology%22%5BJournal%5D)">Enhanced human cell engraftment in mice deficient in RAG2 and the common cytokine receptor gamma chain.</a> British Journal of Haematology. 103 (2), 335-342 (1998).</li>
<li>Seth, A., Park, H. S., Hong, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+perspective+on+in+vivo+molecular+imaging+of+immune+cells%5BTitle%5D)+AND+%22Molecules%22%5BJournal%5D)">Current perspective on in vivo molecular imaging of immune cells.</a> Molecules. 22 (6), (2017).</li>
<li>Espinosa-Cotton, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin-1+alpha+increases+antitumor+efficacy+of+cetuximab+in+head+and+neck+squamous+cell+carcinoma%5BTitle%5D)+AND+%22Journal+for+Immunotherapy+of+Cancer%22%5BJournal%5D)">Interleukin-1 alpha increases antitumor efficacy of cetuximab in head and neck squamous cell carcinoma.</a> Journal for Immunotherapy of Cancer. 7 (1), 79(2019).</li>
<li>Veltri, S., Smith, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin+1+trials+in+cancer+patients%3A+A+review+of+the+toxicity%2C+antitumor+and+hematopoietic+effects%5BTitle%5D)+AND+%22The+Oncologist%22%5BJournal%5D)">Interleukin 1 trials in cancer patients: A review of the toxicity, antitumor and hematopoietic effects.</a> The Oncologist. 1 (4), 190-200 (1996).</li>
</ol>

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><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&#039;s Modified Eagle Medium)</td><td>Thermo Fisher Scientific</td><td>11965092</td><td></td></tr><tr><td>DMEM/Hams F12 (Dulbecco&#039;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&amp;nbsp;Digi-Max&amp;nbsp;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

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-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...

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; 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 ...

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The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

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.

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

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.

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

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

Learn to evaluate antitumor activity of IL-1α nanoparticles in a syngeneic mouse model of head and neck squamous cell carcinoma. This protocol enables assessment of efficacy and toxicity for cancer immunotherapy research.

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

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