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 authors have nothing to disclose.

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 ...

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3.1K Views.  La Jolla Institute for Immunology. 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.