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 Dulbecco&#39;s medium (IMDM) containing 10% heat-inactivated FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1 mM MEM nonessential amino acids, and 100 U/mL each of penicillin and streptomycin. Maintain the cell line at 37 &#176;C under 5% CO2.</li>
	<li>Seed 1.5-2 x 106 B16-F10 cells in a 175T flask and culture for 2 days. Harvest cells when they are 75%-80% confluent.</li>
	<li>Remove the culture medium and wash the flask once with PBS. Aspirate PBS and add 5 mL of 0.25% trypsin-EDTA followed by harshly tapping the rim of the culture flask.</li>
	<li>Add 15 mL of culture medium to neutralize the trypsin-EDTA and pour the contents of the flask into a 50 mL centrifuge tube. Wash the dish surface with 10 mL of PBS and pour into the same 50 mL tube.</li>
	<li>Centrifuge the cells for 5 min at 200 x g. Discard the supernatant and break the cell pellet by finger tapping the bottom of the tube.</li>
	<li>Add cold 10 mL of PBS and gently pipet the cell suspension; then, manually count cells using hemocytometer. Keep the cells on ice before injection.</li>
</ol>
2. Tumor implantation
<ol>
	<li>Gas anesthetize mice with 5% isoflurane at gas flow rate of 1.0 L per min in the fume hood.&#160;Change the flow rate to the maintenance dose of 2% isoflurane&#160;once the mice are fully anesthetized. For this protocol, anesthetization was performed by the veterinarian following&#160;the institutional animal care and use guidelines.</li>
	<li>Shave the hair on the left flank of the mice and sterilize the injection site using alcohol wipes. Intradermally (i.d.) implant B16-F10 tumor cells at 5 x 105 cells in 50 &#181;L of cold PBS in the left flank using a 30 G needle. 
	NOTE: The dose of implanted B16-F10 tumor cells may need to be adjusted in the range of 0.5-5 x 105 cells for successful tumor development.</li>
	<li>After implantation, measure the tumor length and width three times a week using an electronic digital caliper. Calculate the tumor volume (mm3) using the formula: 
	Tumor volume (mm3) = width2 &#215; length &#215; 0.5 
	Treat mice with tumor vaccine when tumors have reached a size of &#8805;2 mm. 
	&#8203;NOTE: Tumors usually can be measured on day 3 after implantation of 5 x 105 tumor cells. Faster B16-F10 tumor growth rate was observed in male C57BL/6, Rag1-/-, or Rag2-/-&#947;c-/-mice. A similar observation was described in other studies8. Keeping the gender of the mice consistent is recommended. However, note that the NIH places emphasis on sex as an important biological variable in biomedical research.</li>
</ol>
3. Vaccine preparation and injection of Flt3L-expressing B16-F10 (B16-Flt3L) cells
<ol>
	<li>Maintain the B16-Flt3L cells in DMEM containing 8% heat-inactivated FBS, 2 mM glutamine, and 100 U/mL each of penicillin and streptomycin at 37 &#176;C under 5% CO2.</li>
	<li>Seed 1 x 106 B16-Flt3L cells in a 175T flask and culture for 2 days. Harvest cells when they are 75%-80% confluent as described in steps 1.3 to 1.6 and suspend in 1 mL of cold PBS.</li>
	<li>Irradiate cells at 150 Gy dose of gamma rays using X-ray Irradiator with 160 kV and 25 mA parameter setting. Count and check the cell viability by trypan blue staining before injection.</li>
	<li>Gas anesthetize mice as described earlier and sterilize the injection site using alcohol wipes. Intradermally inject the mice with 1 x 106 irradiated B16-Flt3L cells in 50 &#181;L of cold PBS on the same flank as the original tumor implantation, ~1 cm away from the site of the primary tumor on days 3, 6, and 9 after the initial cell implantation.</li>
	<li>Mark the vaccine injection sites with a colored pen to distinguish it from the primary tumor. 
	&#8203;NOTE: If 0.5 x 105 B16-F10 cells are initially implanted, it is recommended to perform vaccine treatment on days 8, 11, and 14.</li>
</ol>
4. Intratumoral immune cell isolation
<ol>
	<li>Sacrifice mice using CO2 and cervical dislocation in the fume hood at the end of the experiment (on day 15 after tumor implantation; Figure 1).</li>
	<li>Surgically remove the tumor with the skin from each mouse and put it into 24-well plate with 1 mL 10% FBS/RPMI-1640 medium. Dry the tumors using a paper towel before weighing it.</li>
	<li>Cut the tumors into small pieces. Add 2 mL of digestion buffer (100 &#181;g/mL TL Liberase and 200 &#181;g/mL DNase I in RPMI-1640 medium) and incubate for 25 min at 37 &#176;C.</li>
	<li>Add 10 mL of 10% FBS/RPMI-1640 medium to stop the digestion. Transfer the cells using a 25 mL serological pipette and use the plunger of a 1 mL syringe to grind tissue on a 40 &#181;m cell strainer.</li>
	<li>Centrifuge the cells at 500 x g for 5 min at 4 &#176;C. Resuspend the pellet in 5 mL of 40% density gradient specific medium in PBS, diluted to 1x concentration).</li>
	<li>Add the cell suspension slowly on top of 5 mL of 80% density-gradient-specific medium containing PBS. Centrifuge the cells at 325 x g with low brake setting for 23 min at room temperature (RT).</li>
	<li>After centrifugation, carefully collect the leukocytes layer at the interface between 40% and 80% density-gradient-specific medium and pass it through a 40 &#181;m cell strainer. Centrifuge the cells at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Incubate the pellet in 2 mL of red blood cell (RBC) lysis buffer for 5 min at RT. After incubation, add 10 mL of 10% FBS/RPMI-1640 medium to quench the RBC lysis buffer.</li>
	<li>Centrifuge the cells at 500 x g for 5 min at 4 &#176;C. Resuspend the cells in 0.5 mL of 10% FBS/RPMI-1640 medium and count the total number of cells before use for further analysis. 
	&#8203;NOTE: Collect the spleen or dLN as controls for gating strategy of immune cell subsets by flow cytometry analysis. Follow the cell isolation method as described above with the following changes: Use the plunger of a 1 mL syringe to grind tissue on a 70 &#181;m mesh filter. Wash the tissue with 10% FBS/RPMI-1640 medium to get single-cell suspensions. Lyse the RBC as described.</li>
</ol>
5. Flow cytometry analysis
NOTE: Cells collected from the leukocytes layer contain immune cells and tumor cells. Two independent staining panels are recommended.
<ol>
	<li>Surface staining
	<ol>
		<li>Transfer the cells into a 96-well V-shape-bottom plate. Wash the cells with PBS and stain them with cell viability dye (50-100 &#181;L/well) for 15 min at RT.</li>
		<li>Centrifuge the cells at 500 x g for 5 min at 4 &#176;C. Stain the surface markers with mixed antibodies (detailed dilution is provided in Table of Materials) in FACS buffer (1% FBS and 0.05% NaN3 in PBS) for 30 min on ice (50-100 &#181;L/ well).</li>
		<li>Centrifuge the cells at 500 x g for 5 min and wash them with FACS buffer twice.</li>
		<li>Fix the cells with cell fixation buffer (50-100 &#181;L/ well) for 35 min on ice. Centrifuge the cells at 500 x g for 5 min and wash them twice with FACS buffer.</li>
		<li>Store the samples in FACS buffer (150-200 &#181;L/tube) at 4 &#176;C and protect from light. Acquire the samples on a flow cytometer. For DCs and T cells population, follow the gating strategies provided in Figure 2B and Figure 3A. 
		NOTE: For the staining of myeloid DCs, incubating FC-blocker (Rat anti-mouse CD16/CD32) for 15 min on ice before surface antibodies incubation (step 5.1.2) is recommended.</li>
	</ol>
	</li>
	<li>Cytokine analysis upon ex vivo re-stimulation
	<ol>
		<li>Plate the cells in complete medium (RPMI-1640 supplemented with 10% FBS, 10 mM HEPES, pH 7.2-7.6, 0.1 mM non-essential amino acid, 1 mM sodium pyruvate, 100 U/mL each penicillin and streptomycin, 50 &#956;M 2-mercaptoethanol, and 2 mM L-glutamine) and stimulate with 50 ng/mL of PMA plus 1 &#181;M ionomycin in the presence of protein transport inhibitor for 4 h at 37 &#176;C under 5% CO2.</li>
		<li>Perform surface staining for surface markers as described above in step 5.1.</li>
		<li>Add the permeabilization solution (50-100 &#181;L/well) and incubate for 5 min at RT for intracellular staining. Incubate the cells with antibodies specific for cytokines or nuclear proteins in permeabilization solution (50-100 &#181;L/well) for 60 min on ice or overnight at 4 &#176;C.</li>
		<li>Centrifuge the cells at 500 x g for 5 min and wash them with FACS buffer twice. Store the samples at 4 &#176;C and protect from light. Acquire the samples on a flow cytometer. 
		NOTE: Antibody dilution may have to be adjusted as necessary to accomplish optimal staining.</li>
	</ol>
	</li>
</ol>

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	<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><tbody><tr><td>0.25% trypsin-EDTA&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>BD Biosciences</td><td>BDB554655</td><td>Cell fixation buffer (4.2% PFA)&amp;nbsp;</td></tr><tr><td>Cytofix/Cytoperm kit&amp;nbsp;</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&#039;s Modified Eagle Medium&amp;nbsp; (DMEM)&amp;nbsp;</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&amp;nbsp;</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&amp;rsquo;s modified Dulbecco&amp;rsquo;s medium (IMDM)</td><td>Thermo Fisher</td><td>12440053</td><td></td></tr><tr><td>LSR-II cytometers&amp;nbsp;</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&amp;nbsp;</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Percoll&amp;nbsp;</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><td></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 &amp;mu;m</td><td>Fisherbrand</td><td>22-363-547</td><td></td></tr><tr><td>Sterile cell strainer 70 &amp;mu;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&lt;br/&gt; Fluorophore: BV570&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Anti-mCD3&amp;epsilon;</td><td>BioLegend</td><td>100327</td><td>Clone: 145-2C11&lt;br/&gt; Fluorophore: PerCP-Cy5.5&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Anti-mCD8</td><td>BioLegend</td><td>100730&lt;br/&gt; 100724</td><td>Clone: 53-6.7&lt;br/&gt; Fluorophore: Alexa Fluor 700, Alexa Fluor 647&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Anti-mCD4</td><td>BioLegend</td><td>100414</td><td>Clone: GK1.5&lt;br/&gt; Fluorophore: APC-Cy7&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Anti-mFoxp3</td><td>Thermo Fisher Scientific</td><td>11577382</td><td>Clone: FJK-16s&lt;br/&gt; Fluorophore: FITC&lt;br/&gt; Dilution: 1:100</td></tr><tr><td>Anti-m/hGzmB</td><td>BioLegend</td><td>372208</td><td>Clone: QA16A02&lt;br/&gt; Fluorophore: PE&lt;br/&gt; Dilution: 1:100</td></tr><tr><td>Anti-mIFNg</td><td>BioLegend</td><td>505826</td><td>Clone: XMG1.2&lt;br/&gt; Fluorophore: PE-Cy7&lt;br/&gt; Dilution: 1:100</td></tr><tr><td>Anti-mCD19</td><td>BioLegend</td><td>115543</td><td>Clone: 6D5&lt;br/&gt; Fluorophore: BV785&lt;br/&gt; Dilution: 1:100</td></tr><tr><td>Anti-mGr1</td><td>BioLegend</td><td>108423</td><td>Clone: RB6-8C5&lt;br/&gt; Fluorophore: APC/Cy7&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Anti-mCD11b</td><td>BioLegend</td><td>101223</td><td>Clone: M1/70&lt;br/&gt; Fluorophore: Pacific blue&lt;br/&gt; Dilution: 1:100</td></tr><tr><td>Anti-mF4/80</td><td>BioLegend</td><td>123114</td><td>Clone: BM8&lt;br/&gt; Fluorophore: PECy7&lt;br/&gt; Dilution: 1:100</td></tr><tr><td>Anti-mCD11c</td><td>BioLegend</td><td>117328</td><td>Clone: N418&lt;br/&gt; Fluorophore: PerCP Cy5.5&lt;br/&gt; Dilution: 1:100</td></tr><tr><td>Anti-mMHCII</td><td>BioLegend</td><td>107622</td><td>Clone: M5/114.15.2&lt;br/&gt; Fluorophore: AF700&lt;br/&gt; Dilution: 1:400</td></tr><tr><td>Anti-mCD103</td><td>BioLegend</td><td>121410</td><td>Clone: 2E7&lt;br/&gt; Fluorophore: Alexa Fluor 647&lt;br/&gt; Dilution: 1:200</td></tr><tr><td>Anti-mCD86</td><td>BioLegend</td><td>105007</td><td>Clone: GL-1&lt;br/&gt; Fluorophore: PE&lt;br/&gt; 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&lt;br/&gt; 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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3.3K Views.  La Jolla Institute for Immunology. Dit protocol voorziet in een klinisch solide tumorimmunotherapiemodel en een onderzoeksplatform om de relatie tussen tumorcellen en infiltrerende immuuncellen te bestuderen. Dit op cellen gebaseerde tumorvaccin is handig, eenvoudig te gebruiken en kan worden gecombineerd met andere therapeutische modaliteiten zoals checkpointblokkade, om een additief of synergetisch effect te bereiken dat kan resulteren in krachtigere en hogere tumorimmuniteit. Ann Balancio, een onderzoekstechnicus uit mijn l...

Video: Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Developmental Biology

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 ...

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 vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

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

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Immunology and Infection

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

Patient-Derived Melanoma Organoids: A Frontier in Cancer Immunotherapy

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While traditional two- and three-dimensional models can offer insights into cancer development and progression, these lack crucial aspects that hinder a faithful mimic of native tumors. An alternative model that has gained a lot of attention is the patient-derived organoid. The development of these organoids recapitulates the complex intercellular communication, tumor microenvironment, and histoarchitecture of tumors. This paper describes the protocol for establishing melanoma patient-derived organoid (MPDO) models. To validate these models, we assessed the immune cell composition, including the expression levels of T-cell activation markers, to confirm the cellular heterogeneity of the organoids. Additionally, to describe the potential utility of MPDOs in cellular therapies, we evaluated the cytotoxic capabilities of treating the organoids with &#947;&#948; T-cells. In conclusion, the MPDO models offer promising avenues for understanding tumor complexity, validating therapeutic strategies, and potentially advancing personalized treatment.

Author Spotlight: Recreating Melanoma Complexity with Patient-Derived Organoids for Immunotherapy Evaluation

Here, we present a protocol to generate melanoma patient-derived organoids by culturing disassociated cell suspensions from fresh melanoma tissues. These organoids faithfully recapitulate patient-specific tumors in vitro, offering an innovative approach to exploring tumor immunosuppressive mechanisms, drug screening, drug resistance mechanisms, and cancer surveillance approaches.

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Tumor Implantation and Injection of a Tumor Vaccine in a Mouse Model

Source: Liu, H. Y., et al. <a href="https://app.jove.com/v/64082">Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine.</a> J. Vis. Exp. (2023).
This video demonstrates a cell-based tumor vaccination assay. A mouse, intradermally injected with B16-F10 melanoma cells for tumor formation, is vaccinated with B16-F10 cells secreting Flt3L cytokine near the developed tumor. The secreted Flt3L from the vaccine promotes the maturation and proliferation of dendritic cells that then present tumor antigens to T cells in lymph nodes, triggering a targeted anti-tumor immune response.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

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