Name: experimental melanoma immunotherapy model using tumor vaccination with a hematopoietic cytokine
Uploaded: 2023-02-24T14:30:00
Description: Watch this Scientific Journal Video about Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine at JoVE.com

We thank Dr. Stephen Schoenberger for providing B16-Flt3L cells and the staff of the LJI animal and flow cytometry facilities for excellent support.

All mice used in the study were maintained and housed in the vivarium of the La Jolla Institute for Immunology (LJI) under specific pathogen-free conditions with controlled temperature and humidity. Animal experiments were performed with 8-14 weeks old female C57BL/6 mice according to guidelines and protocols approved by the LJI Animal Care Committee.
1. Preparation of cultured tumor cells for implantation
<ol>
	<li>Culture B16-F10 melanoma cells in Iscove&#39;s Modified Dul...

<ol>
	<li>Zhang, Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+and+advances+in+cancer+immunotherapy:+understanding+the+characteristics+of+tumor-infiltrating+immune+cells+and+their+therapeutic+implications.">The history and advances in cancer immunotherapy: understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications.</a> Cell &amp; Molecular Immunology. 17 (8), 807-821 (2020).</li><li>Banchereau, J., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+the+control+of+immunity.">Dendritic cells and the control of immunity.</a> Nature. 392 (6673), 245-252 (1998).</li><li>Banchereau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunobiology+of+dendritic+cells.">Immunobiology of dendritic cells.</a> Annual Review of Immunology. 18, 767-811 (2000).</li><li>Martinez-Lostao, L., Anel, A., Pardo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+cytotoxic+lymphocytes+kill+cancer+cells.">How do cytotoxic lymphocytes kill cancer cells.</a> Clinical Cancer Research. 21 (22), 5047-5056 (2015).</li><li>Maraskovsky, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+increase+in+the+numbers+of+functionally+mature+dendritic+cells+in+Flt3+ligand-treated+mice:+multiple+dendritic+cell+subpopulations+identified.">Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified.</a> Journal of Experimental Medicine. 184 (5), 1953-1962 (1996).</li><li>Talmadge, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral,+injection+of+adenoviral+Flt3+ligand+has+therapeutic+activity+in+association+with+increased+intratumoral+levels+of+T+cells+but+not+dendritic+cells.">Intratumoral, injection of adenoviral Flt3 ligand has therapeutic activity in association with increased intratumoral levels of T cells but not dendritic cells.</a> Blood. 104 (11), 5280 (2004).</li><li>Curran, M. A., Allison, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+vaccines+expressing+flt3+ligand+synergize+with+ctla-4+blockade+to+reject+preimplanted+tumors.">Tumor vaccines expressing flt3 ligand synergize with ctla-4 blockade to reject preimplanted tumors.</a> American Association for Cancer Research. 69 (19), 7747-7755 (2009).</li><li>Simon, S. R., Ershler, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+influences+on+growth+of+B16+murine+melanoma.">Hormonal influences on growth of B16 murine melanoma.</a> Journal of the National Cancer Institute. 74 (5), 1085-1088 (1985).</li><li>Broz, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+tumor+myeloid+compartment+reveals+rare+activating+antigen-presenting+cells+critical+for+T+cell+immunity.">Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity.</a> Cancer Cell. 26 (6), 938 (2014).</li><li>Salmon, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+and+activation+of+CD103(+)+dendritic+cell+progenitors+at+the+tumor+site+enhances+tumor+responses+to+therapeutic+PD-L1+and+BRAF+inhibition.">Expansion and activation of CD103(+) dendritic cell progenitors at the tumor site enhances tumor responses to therapeutic PD-L1 and BRAF inhibition.</a> Immunity. 44 (4), 924-938 (2016).</li><li>Liu, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leveraging+the+Treg-intrinsic+CTLA4-PKCeta+signaling+pathway+for+cancer+immunotherapy.">Leveraging the Treg-intrinsic CTLA4-PKCeta signaling pathway for cancer immunotherapy.</a> Journal for Immunotherapy Cancer. 9 (9), 002792 (2021).</li><li>Kong, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-eta+controls+CTLA-4-mediated+regulatory+T+cell+function.">Protein kinase C-eta controls CTLA-4-mediated regulatory T cell function.</a> Nature Immunology. 15 (5), 465-472 (2014).</li></ol>

The protocol described here is based on the study by Allison&#39;s group. They demonstrated that combination of B16-Flt3L vaccine with CTLA-4 blockade showed a synergistic effect on survival rate and tumor growth, whereas no reduction of tumor growth was seen in mice receiving the B16-Flt3L vaccine or anti-CTLA-4 antibody treatment alone7. Recent studies have revealed a novel Treg-intrinsic CTLA4-PKC&#951; signaling pathway that plays an important obligatory role in regulating the contact-dependen...

Cancer immunotherapy has been recognized as a promising therapeutic strategy based on its less toxic side effects and more durable responses. Several types of immunotherapies have been developed, including oncolytic virus therapies, cancer vaccines, cytokine therapies, monoclonal antibodies, adoptive cell transfer (CAR-T cells or CAR-NK), and immune checkpoint blockade1.
For cancer vaccines, there are different forms of therapeutic vaccines, such as whole cell-based vaccines, protein or peptide vaccines, and RNA or DNA vaccines. Vaccination relies on the ability of antigen-presenting cells (APCs) to process tumor antigens, including tumor-specific antigens, and present them in an immunogenic form to T cells. Dendritic cells (DCs) have been known to be the most potent APCs and are believed to play an important role in antitumor immunity2,3. These cells take up and process tumor antigens, and then migrate to the draining lymph nodes (dLN) to prime and activate tumor-specific T effector (Teff) cells through engagement of the T-cell receptor (TCR) and costimulatory molecules. This results in differentiation and expansion of tumor-specific cytotoxic T cells (CTL), which infiltrate the tumor and kill tumor cells4. Consequently, activation and maturation of DCs represent attractive strategies to stimulate immunity against tumor antigens.
Flt3L is known to promote the maturation and expansion of functionally mature DCs that express MHC class II, CD11c, DEC205, and CD86 proteins5. Intratumoral, but not intravenous, administration of an adenovirus vector incorporating the Flt3L gene (Adv-Flt3L) has been shown to promote immune therapeutic activity against orthrotopic tumors6. Flt3L has also been used in tumor cell-based vaccines consisting of irradiated B16-F10 cells stably expressing retrovirally transduced Flt3L as a way of enhancing the cross-presentation of tumor antigens by DCs and, thus, increasing antitumor responses. The protocol of B16-Flt3L tumor vaccination described here is based on a study published by Dr. James Allison's group7. In this paper, they reported that a B16-Flt3L vaccine combined with CTLA-4 blockade synergistically induced the rejection of established melanoma, resulting in increased survival.
The goal of this protocol is to provide a preclinical immunotherapy model for melanoma. Here, detailed procedures of how to prepare and implant tumor vaccines, and how to analyze the composition and function of intratumoral immune cells from solid tumor are described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA&#160;</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>10% heat-inactivated FBS</td>
			<td>Omega Scientific</td>
			<td>FB-02&#160;</td>
			<td>Lot# 209018</td>
		</tr>
		<tr>
			<td>30G needle</td>
			<td>BD Biosciences</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well V-shape-bottom plate</td>
			<td>SARSTEDT</td>
			<td>83.3926.500</td>
			<td></td>
		</tr>
		<tr>
			<td>B16 cell line expressing Fms-like tyrosine kinase 3 ligand (B16-Flt3L)</td>
			<td>Gift of Dr. Stephen Schoenberger, LJI&#160;</td>
			<td></td>
			<td>Flt3L cDNAs were cloned into the pMG-Lyt2 retroviral vector, as in refernce 5, Supplemental Figure 1</td>
		</tr>
		<tr>
			<td>B16-F10 cell lines</td>
			<td>ATCC</td>
			<td>CRL-6475</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofix fixation buffer&#160;</td>
			<td>BD Biosciences</td>
			<td>BDB554655</td>
			<td>Cell fixation buffer (4.2% PFA)&#160;</td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm kit&#160;</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Fixation/Permeabilization Solution Kit</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium&#160; (DMEM)&#160;</td>
			<td>Corning</td>
			<td>10013CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic digital caliper</td>
			<td>Fisherbrand</td>
			<td>14-648-17</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo software&#160;</td>
			<td>Tree Star</td>
			<td></td>
			<td>Flow cytometer data analysis</td>
		</tr>
		<tr>
			<td>GolgiStop (protein transport inhibitor)</td>
			<td>BD Biosciences</td>
			<td>554724</td>
			<td>1:1500 dilution</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#8217;s modified Dulbecco&#8217;s medium (IMDM)</td>
			<td>Thermo Fisher</td>
			<td>12440053</td>
			<td></td>
		</tr>
		<tr>
			<td>LSR-II cytometers&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>MEM nonessential amino acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin and streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>GE17-0891-02</td>
			<td>density gradient specific medium</td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma</td>
			<td>P1585</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysing Buffer Hybri-Max liquid</td>
			<td>Sigma</td>
			<td>R7757-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Corning</td>
			<td>10-040-CV</td>
		</tr>
		<tr>
			<td>RS2000 X-ray Irradiator</td>
			<td>Rad Source Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell strainer 40 &#956;m</td>
			<td>Fisherbrand</td>
			<td>22-363-547</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell strainer 70 &#956;m</td>
			<td>Fisherbrand</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>TL Liberase</td>
			<td>Roche</td>
			<td>477530</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua fixable viability kit</td>
			<td>BioLegend</td>
			<td>423101</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mCD45</td>
			<td>BioLegend</td>
			<td>103135</td>
			<td>Clone: 30-F11 
			Fluorophore: BV570 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mCD3&#949;</td>
			<td>BioLegend</td>
			<td>100327</td>
			<td>Clone: 145-2C11 
			Fluorophore: PerCP-Cy5.5 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mCD8</td>
			<td>BioLegend</td>
			<td>100730 
			100724</td>
			<td>Clone: 53-6.7 
			Fluorophore: Alexa Fluor 700, Alexa Fluor 647 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mCD4</td>
			<td>BioLegend</td>
			<td>100414</td>
			<td>Clone: GK1.5 
			Fluorophore: APC-Cy7 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mFoxp3</td>
			<td>Thermo Fisher Scientific</td>
			<td>11577382</td>
			<td>Clone: FJK-16s 
			Fluorophore: FITC 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-m/hGzmB</td>
			<td>BioLegend</td>
			<td>372208</td>
			<td>Clone: QA16A02 
			Fluorophore: PE 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mIFNg</td>
			<td>BioLegend</td>
			<td>505826</td>
			<td>Clone: XMG1.2 
			Fluorophore: PE-Cy7 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mCD19</td>
			<td>BioLegend</td>
			<td>115543</td>
			<td>Clone: 6D5 
			Fluorophore: BV785 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mGr1</td>
			<td>BioLegend</td>
			<td>108423</td>
			<td>Clone: RB6-8C5 
			Fluorophore: APC/Cy7 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mCD11b</td>
			<td>BioLegend</td>
			<td>101223</td>
			<td>Clone: M1/70 
			Fluorophore: Pacific blue 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mF4/80</td>
			<td>BioLegend</td>
			<td>123114</td>
			<td>Clone: BM8 
			Fluorophore: PECy7 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mCD11c</td>
			<td>BioLegend</td>
			<td>117328</td>
			<td>Clone: N418 
			Fluorophore: PerCP Cy5.5 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mMHCII</td>
			<td>BioLegend</td>
			<td>107622</td>
			<td>Clone: M5/114.15.2 
			Fluorophore: AF700 
			Dilution: 1:400</td>
		</tr>
		<tr>
			<td>Anti-mCD103</td>
			<td>BioLegend</td>
			<td>121410</td>
			<td>Clone: 2E7 
			Fluorophore: Alexa Fluor 647 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mCD86</td>
			<td>BioLegend</td>
			<td>105007</td>
			<td>Clone: GL-1 
			Fluorophore: PE 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>FC-blocker (Rat anti-mouse CD16/CD32)</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td>Clone: 2.4G2 
			Dilution: 1:200</td>
		</tr>
	</tbody>
</table>

experimental melanoma immunotherapy model using tumor vaccination with a hematopoietic cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

A visible black dot of the implanted B16-F10 cells is usually observed on the skin surface ~3 days after tumor implantation. Mice are treated with the tumor vaccine 3, 6, and 9 days after the tumor nodule has reached a size of &#8805;2 mm. We observed a significant reduction in tumor growth in vaccinated mice group ~2 weeks after tumor implantation (Figure 1). At the end of the experiment, we isolated the intratumoral immune cells and analyzed their number and cell surface marker expression,...

Watch this Scientific Journal Video about Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine at JoVE.com

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...

This protocol provides with clinical solid tumor immunotherapy model and a research platform to study the relationship between tumor cells and infiltrating immune cells. This cell-based tumor vaccine is convenient, straightforward to use, and can be combined with other therapeutics modalities such as checkpoint blockade, to achieve additive or synergistic effect that can result in more potent and high tumor immunity. Helping to demonstrate the procedure will be Ann Balancio, a research technician from my laboratory.To begin, culture B16-F10 melanoma cells in IMDM, containing 10%heat inactive FBS, 2 millimolar glutamine, 1 millimolar sodium pyruvate, 1 millimolar MEM non-essential amino acids and 100 units per milliliter each of penicillin and streptomycin. Maintain the cell line at 37 degrees Celsius with 5%carbon dioxide. Seed and harvest the B16-F10 cells as described in the text manuscript.Remove the culture medium and wash the flasks once with PBS. Aspirate PBS and add 5 milliliters of 0.25%trypsin-EDTA, followed by harshly tapping the rim of the culture flask. Add 15 milliliters of culture medium to neutralize the trypsin-EDTA and pour the contents of the flask into a 50 milliliters centrifuge tube.Wash the dish surface with 10 milliliters of PBS and pour into the same 50 milliliters centrifuge tube. Centrifuge the cells for 5 minutes at 200 RCF. Discard the supernatant and break the cell pellet by finger tapping the bottom of the tube.Add cold 10 milliliters of PBS and gently pipette the cell suspension. Then, manually count the cells using a hemocytometer. Keep the cells on ice before injection.Intradermally implant 500, 000 cells B16-F10 tumor cells in 50 microliters of cold PBS in the left flank, using a 30 gauge needle. The dose of implanted B16-F10 tumor cells may need to be adjusted in a range of 50, 000 to 500, 000 cells for successful tumor development. After implementation, measure the tumor length and width three times a week using an electronic digital caliper.Calculate the tumor volume and treat the mice with tumor vaccine when tumors have reached a size of two millimeters as described in the text manuscript. Using an x-ray irradiator set at 160 kilovolts and 25 milliamperes, irradiate cells at 150 gray doses of gamma rays. Count and check the cell viability by trypan blue staining before injection.Intradermally inject the mice with 1 million irradiated B16-Flt3L cells in 50 microliters of cold PBS on the same flank as the original tumor implantation. Approximately one centimeter from the site of the primary tumor on days 3, 6, and 9 after the initial cell implantation. Mark the vaccine injection sites with a colored pen to distinguish them from the primary tumor.If 50, 000 B16-F10 cells were initially implanted, it is recommended to perform vaccine treatment on days 8, 11 and 14. Surgically removed the tumor with the skin from each euthanized mouse and put it into a 24 well plate with 1 milliliter of 10%FBS RPMI 1640 medium. Cut the tumors into small pieces in two milliliters of digestion buffer and incubate for 25 minutes at 37 degrees Celsius.Add 10 milliliters of 10%FBS RPMI 1640 medium to stop the digestion. Transfer the cells to a 40 micrometer cell strainer using a 25 milliliter serological pipette. Then use the plunger of a one milliliter syringe to grind the tissues.Centrifuge the cells at 500 RCF for 5 minutes at 4 degrees Celsius. Resuspend the pellet in 5 milliliters of 40%density gradient specific medium in PBS diluted to 1x concentration. Add the cell suspension slowly on top of 5 milliliters of 80%density gradient specific medium containing PBS.Centrifuge to the cells at 325 RCF with a low brake setting for 23 minutes at room temperature. After centrifugation, carefully collect the leukocytes layer at the interface between 40%and 80%density gradient specific medium, and pass it through a 40 micrometer cell strainer. Centrifuge the cells at 500 RCF for 5 minutes at 4 degrees Celsius.Incubate the pellet in two milliliters of red blood cell lysis buffer for five minutes at room temperature. After incubation, add 10 milliliters of 10%FBS RPMI 1640 medium to quench the RBC lysis buffer. Centrifuge the cells at 400 RCF for 5 minutes at 4 degrees Celsius.Resuspend the cells in 0.5 milliliters of 10%FBS RPMI 1640 medium, and count the total number of cells before use for further analysis. Collect the spleen or the draining lymph nodes as controls for the gating strategy of immune cell subsets by flow cytometry analysis. Follow the cell isolation method described in the text manuscript.A visible black dot of the implanted B16-F10 cells was usually observed on the skin surface approximately three days after tumor implantation. Mice were treated with a tumor vaccine three, six and nine days after the tumor nodule had reached or exceeded the size of two millimeters. A significant tumor growth reduction was observed in the vaccinated mice group about two weeks after tumor implantation.Cells collected from the leukocytes layer contained many tumor cells, making it difficult to readily define the lymphocyte population. Therefore, splenocyte were used in parallel, for proper gating of intratumoral immune cell subsets in flow cytometry analysis. The gating strategies of CD103 positive, CD11C positive DC, CD8 positive, CD4 positive, and Treg were plotted along with the compensation matrix.Representative data of acquired accounts and frequency of each population was also provided. Intratumoral CD103 positive, CD11C positive DC from vaccinated mice, displayed a significantly elevated expression of the co-stimulatory ligand CD86. Vaccinated mice also displayed an increase in tumor infiltrating CD8 positive and CD4 positive, Foxp3 negative cells, as well as in CD8 positive granzyme B positive and interferon gamma positive CTLs.Keep a sufficient distant between the primary tumor and the tumor vaccine. This physical separation is critical to avoid potential fusion between the two tumor implants.

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Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

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 ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...