Name: in vivo immunogenicity screening of tumor-derived extracellular vesicles by flow cytometry of splenic t cells
Uploaded: 2021-09-23T14:30:00
Description: Watch this Scientific Journal Video about In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells at JoVE.com

This study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Projektnummer 360372040 - SFB 1335 and Projektnummer 395357507 - SFB 1371 (to H.P.), a Mechtild Harf Research Grant from the DKMS Foundation for Giving Life (to H.P.) a Young Investigator Award by the Melanoma Research Alliance (to S.H.), a scholarship by the Else-Kr&#246;ner-Fresenius-Stiftung (to F.S.), a Seed Fund by the Technical University Munich (to S.H.) and a research grant by the Wilhelm Sander Foundation (2021.041.1, to S.H.). H. P. is supported by the EMBO Young Investigator Program.
AUTHOR CONTRIBUTIONS:
F.S., H.P., and S.H. designed the research, analyzed, and interpreted the results. F.S. and S.H. wrote the manuscript. H.P. and S.H. guided the study.

At the onset of experiments, mice were at least 6 weeks of age and were maintained under specific pathogen-free conditions. The present protocol complies with the Institutional ethical standards and prevailing local regulations. Animal studies were approved by the local regulatory agency (Regierung von Oberbayern, Munich, Germany). Possible sex-related biases were not investigated in these studies.
1. Generation and isolation of EVs derived from tumor cells after chemotherapy exposure
<ol>
	...

<ol>
	<li>Kroemer, G., Galluzzi, L., Kepp, O., Zitvogel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenic+cell+death+in+cancer+therapy.">Immunogenic cell death in cancer therapy.</a> Annual Review of Immunology. 31, 51-72 (2013).</li><li>Kepp, O., 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=Consensus+guidelines+for+the+detection+of+immunogenic+cell+death.">Consensus guidelines for the detection of immunogenic cell death.</a> Oncoimmunology. 3 (9), 955691 (2014).</li><li>Fuertes, M. B., 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=Host+type+I+IFN+signals+are+required+for+antitumor+CD8++T+cell+responses+through+CD8{alpha}++dendritic+cells.">Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8{alpha}+ dendritic cells.</a> The Journal of Experimental Medicine. 208 (10), 2005-2016 (2011).</li><li>Sistigu, A., 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=Cancer+cell-autonomous+contribution+of+type+I+interferon+signaling+to+the+efficacy+of+chemotherapy.">Cancer cell-autonomous contribution of type I interferon signaling to the efficacy of chemotherapy.</a> Nature Medicine. 20 (11), 1301-1309 (2014).</li><li>Fridman, W. H., Pages, F., Sautes-Fridman, C., Galon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+contexture+in+human+tumours:+impact+on+clinical+outcome.">The immune contexture in human tumours: impact on clinical outcome.</a> Nature reviews. Cancer. 12 (4), 298-306 (2012).</li><li>Shankaran, V., 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=IFNgamma+and+lymphocytes+prevent+primary+tumour+development+and+shape+tumour+immunogenicity.">IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity.</a> Nature. 410 (6832), 1107-1111 (2001).</li><li>Tesniere, A., 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=Immunogenic+death+of+colon+cancer+cells+treated+with+oxaliplatin.">Immunogenic death of colon cancer cells treated with oxaliplatin.</a> Oncogene. 29 (4), 482-491 (2010).</li><li>van Niel, G., D'Angelo, G., Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shedding+light+on+the+cell+biology+of+extracellular+vesicles.">Shedding light on the cell biology of extracellular vesicles.</a> Nature reviews. Molecular Cell Biology. 19 (4), 213-228 (2018).</li><li>Zomer, A., van Rheenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+extracellular+vesicle+transfer+on+cellular+heterogeneity+in+cancer:+What+are+the+potential+clinical+ramifications.">Implications of extracellular vesicle transfer on cellular heterogeneity in cancer: What are the potential clinical ramifications.</a> Cancer Research. 76 (8), 2071-2075 (2016).</li><li>Whiteside, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+tumor-mediated+immune+suppression.">Exosomes and tumor-mediated immune suppression.</a> The Journal of Clinical Investigation. 126 (4), 1216-1223 (2016).</li><li>Wolfers, 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=Tumor-derived+exosomes+are+a+source+of+shared+tumor+rejection+antigens+for+CTL+cross-priming.">Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming.</a> Nature Medicine. 7 (3), 297-303 (2001).</li><li>Zeelenberg, I. S., 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=Targeting+tumor+antigens+to+secreted+membrane+vesicles+in+vivo+induces+efficient+antitumor+immune+responses.">Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.</a> Cancer Research. 68 (4), 1228-1235 (2008).</li><li>Diamond, J. M., 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=Exosomes+Shuttle+TREX1-Sensitive+IFN-Stimulatory+dsDNA+from+Irradiated+Cancer+Cells+to+DCs.">Exosomes Shuttle TREX1-Sensitive IFN-Stimulatory dsDNA from Irradiated Cancer Cells to DCs.</a> Cancer Immunology Research. 6 (8), 910-920 (2018).</li><li>Kitai, 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=DNA-containing+exosomes+derived+from+cancer+cells+treated+with+topotecan+activate+a+STING-dependent+pathway+and+reinforce+antitumor+immunity.">DNA-containing exosomes derived from cancer cells treated with topotecan activate a STING-dependent pathway and reinforce antitumor immunity.</a> Journal of Immunology. 198 (4), 1649-1659 (2017).</li><li>Heidegger, S., 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=RIG-I+activation+is+critical+for+responsiveness+to+checkpoint+blockade.">RIG-I activation is critical for responsiveness to checkpoint blockade.</a> Science Immunology. 4 (39), 8943 (2019).</li><li>Schadt, 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=Cancer-cell-intrinsic+cGAS+expression+mediates+tumor+immunogenicity.">Cancer-cell-intrinsic cGAS expression mediates tumor immunogenicity.</a> Cell Reports. 29 (5), 1236-1248 (2019).</li><li>Cheng, 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=In+situ+immunization+of+a+TLR9+agonist+virus-like+particle+enhances+anti-PD1+therapy.">In situ immunization of a TLR9 agonist virus-like particle enhances anti-PD1 therapy.</a> Journal for Immunotherapy of Cancer. 8 (2), (2020).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Current Protocols in Immunology. 111, 1-3 (2015).</li><li>Jeyaram, A., Jay, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+and+storage+stability+of+extracellular+vesicles+for+therapeutic+applications.">Preservation and storage stability of extracellular vesicles for therapeutic applications.</a> The AAPS Journal. 20 (1), 1 (2017).</li><li>Thery, C., 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=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Vestad, B., 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=Size+and+concentration+analyses+of+extracellular+vesicles+by+nanoparticle+tracking+analysis:+a+variation+study.">Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study.</a> Journal of Extracellular Vesicles. 6 (1), 1344087 (2017).</li><li>Suarez, 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=A+bead-assisted+flow+cytometry+method+for+the+semi-quantitative+analysis+of+Extracellular+Vesicles.">A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles.</a> Scientific Reports. 7 (1), 11271 (2017).</li><li>Yuana, 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=Cryo-electron+microscopy+of+extracellular+vesicles+in+fresh+plasma.">Cryo-electron microscopy of extracellular vesicles in fresh plasma.</a> Journal of Extracellular Vesicles. 2, (2013).</li><li>Kapasi, Z. F., Murali-Krishna, K., McRae, M. L., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+generation+but+normal+maintenance+of+memory+T+cells+in+old+mice.">Defective generation but normal maintenance of memory T cells in old mice.</a> European Journal of Immunology. 32 (6), 1567-1573 (2002).</li><li>Bloom, M. B., 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=Identification+of+tyrosinase-related+protein+2+as+a+tumor+rejection+antigen+for+the+B16+melanoma.">Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma.</a> The Journal of Experimental Medicine. 185 (3), 453-459 (1997).</li><li>Patel, G. K., 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=Comparative+analysis+of+exosome+isolation+methods+using+culture+supernatant+for+optimum+yield,+purity+and+downstream+applications.">Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications.</a> Scientific Reports. 9 (1), 5335 (2019).</li><li>DeGrendele, H. C., Kosfiszer, M., Estess, P., Siegelman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44+activation+and+associated+primary+adhesion+is+inducible+via+T+cell+receptor+stimulation.">CD44 activation and associated primary adhesion is inducible via T cell receptor stimulation.</a> The Journal of Immunology. 159 (6), 2549-2553 (1997).</li><li>Yang, S., Liu, F., Wang, Q. J., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shedding+of+CD62L+(L-selectin)+regulates+the+acquisition+of+lytic+activity+in+human+tumor+reactive+T+lymphocytes.">The shedding of CD62L (L-selectin) regulates the acquisition of lytic activity in human tumor reactive T lymphocytes.</a> PLoS One. 6 (7), 22530 (2011).</li><li>Bek, S., 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=Targeting+intrinsic+RIG-I+signaling+turns+melanoma+cells+into+type+I+interferon-releasing+cellular+antitumor+vaccines.">Targeting intrinsic RIG-I signaling turns melanoma cells into type I interferon-releasing cellular antitumor vaccines.</a> Oncoimmunology. 8 (4), 1570779 (2019).</li><li>Nabet, B. 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=Exosome+RNA+unshielding+couples+stromal+activation+to+pattern+recognition+receptor+signaling+in+cancer.">Exosome RNA unshielding couples stromal activation to pattern recognition receptor signaling in cancer.</a> Cell. 170 (2), 352-366 (2017).</li><li>Pitt, J. M., Kroemer, G., Zitvogel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles:+masters+of+intercellular+communication+and+potential+clinical+interventions.">Extracellular vesicles: masters of intercellular communication and potential clinical interventions.</a> The Journal of Clinical Investigation. 126 (4), 1139-1143 (2016).</li></ol>

This protocol provides an immunological in vivo assessment of EVs derived from melanoma cells under chemotherapy-induced stress while adapting to EVs emitted from various cancers under various treatments. Immunizing mice with EVs derived from oxaliplatin-treated B16-OVA cells, for instance, expands IFN-&#947;-producing CD8+ T cells in the spleen, which are further stimulated by ex vivo incubation with ovalbumin, indicating a tumor-specific immune response. Thus, detection of immunogenic EVs b...

The immune system plays a pivotal role in the fight against cancer, both when incited by immune checkpoint inhibition and for the efficacy of conventional cancer therapies. Tumor cells succumbing to genotoxic therapies such as the chemotherapeutic agents oxaliplatin and doxorubicin, or ionizing radiation treatment can release antigens and danger-associated molecular patterns (DAMPs) that potentially initiate an adaptive anti-tumor immune response1. The most prominent DAMPs, in the context of immunogenic cell death, include find-me signals such as chemotactic ATP, eat-me signals such as the exposure of calreticulin, that promotes tumor cell uptake by antigen-presenting cells, and the release of HMGB1, that activates pattern recognition receptors, thereby enhancing the cross-presentation of tumor antigens2. Furthermore, type I interferons (IFN-I), induced via tumor-derived immunogenic nucleic acids or other stimuli, are sensed by dendritic cells, enabling them to effectively prime tumor-specific cytotoxic T cells3,4. Clinically, activated and proliferating CD8+ T cells infiltrating the tumor provide an independent prognostic factor for prolonged survival in many cancer patients. Released from such activated T cells, IFN-&#947; mediates direct antiproliferative effects on cancer cells and drives Th1 polarization and cytotoxic T cell differentiation, thereby contributing to effective immunosurveillance against cancer5,6. Oxaliplatin is a bona fide immunogenic cell death inducer, mediating such adaptive immune response against cancer7. However, the plethora of initial immunogenic signals released by tumor cells under therapy-induced stress remains to be fully unveiled. Despite significant advances in cancer immunotherapy, expanding its benefits to a larger portion of patients remains a challenge. A more detailed understanding of immunogenic signals that initiate T cell activation may guide the development of novel therapies.
A heterogeneous group of membrane-enclosed structures, known as extracellular vesicles (EVs), seem to serve as intercellular communication devices. Emitted by virtually all cell types, EVs carry functional proteins, RNA, DNA, and other molecules to a recipient cell or may alter a cell&#39;s functional state just by binding to receptors on the cell surface. Their biologically active cargo varies significantly by the type and functional state of the generating cell8. In cancer immunology, EVs released from tumor cells have been predominantly regarded as adversarial to immunotherapy because they eventually promote invasive growth, preform metastatic niches9, and suppress the immune response10. In contrast, some studies have shown that EVs can transfer tumor antigens to dendritic cells for effective cross-presentation11,12. EVs may provide immunostimulatory nucleic acids if they emerge under therapy-induced stress, facilitating an anti-tumor immune response13,14. Sensing of such RNA and DNA&#160;innate immune ligands in the tumor microenvironment has recently been shown to modulate responsiveness to checkpoint blockade significantly15,16,17. Hence, the immunogenic role of EVs released by tumor cells under different therapy-induced stress needs to be further elucidated. Since EVs constitute a young yet growing field of research, standardization of methods is still ongoing. Therefore, sharing knowledge is essential to improve the research reproducibility on interactions between EVs and cancer immunology. With this in mind, this manuscript describes a simple protocol to assess the immunogenic effect of tumor-derived EVs in vivo.
This assessment is performed by generating tumor-derived EVs, immunizing recipient mice with those EVs, and analyzing splenic T cells via flow cytometry. EV generation is ideally performed by seeding murine tumor cells in an EV-free cell culture medium for a high degree of purity. Cells are treated with a specific cell stress stimulus, such as chemotherapy, to compare the effect of therapy-induced EVs against baseline immunogenicity of respective tumor-derived EVs. The isolation of EVs may well be performed by various techniques that should be selected according to in vivo applicability and local availability. The following protocol describes a precipitation-based assay with a commercial kit for EV purification. Mice are immunized twice with those EVs. Fourteen days after the first injection, T cells are extracted from the spleen and analyzed for IFN-&#947; production via flow cytometry to evaluate a systemic immune response. With this, the potential of tumor-derived EVs, emerging under different therapeutic regimens, to induce anti-tumor T cell responses is assessed relatively easily, quickly, and with high validity13. Therefore, this method is suitable for an immunological screening of EVs derived from cancer cells under various conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-CD3 FITC</td>
			<td>Biolegend</td>
			<td>100204</td>
			<td>Clone 17A2</td>
		</tr>
		<tr>
			<td>Anti-CD4 PacBlue</td>
			<td>Biolegend</td>
			<td>100428</td>
			<td>Clone GK1.5</td>
		</tr>
		<tr>
			<td>Anti-CD8 APC</td>
			<td>Biolegend</td>
			<td>100712</td>
			<td>Clone 53-6.7</td>
		</tr>
		<tr>
			<td>Anti-IFN&#947; PE</td>
			<td>eBioscience</td>
			<td>RM90022</td>
			<td>Clone XMG1.2</td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Biolegend</td>
			<td>420601</td>
			<td>Brefeldin A Solution (1,000x)</td>
		</tr>
		<tr>
			<td>Cell Strainer, 100 &#181;m</td>
			<td>Greiner</td>
			<td>542000</td>
			<td>EASYstrainer 100 &#181;m</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium with D-glucose (4.5 g/L) and L-glutamine (4 mM)</td>
		</tr>
		<tr>
			<td>FBS Good Forte</td>
			<td>PAN BIOTECH</td>
			<td>P40-47500</td>
			<td>Fetal Calf Serum (FCS)</td>
		</tr>
		<tr>
			<td>Fixable Viability Dye eFluor 506</td>
			<td>eBioscience, division of Thermo Fischer Scientific</td>
			<td>65-0866-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation/Permeabilization Concentrate</td>
			<td>eBioscience</td>
			<td>00-5123-43</td>
			<td>Fixation/Permeabilization Concentrate (10x)</td>
		</tr>
		<tr>
			<td>Fixation/Permeabilization Diluent</td>
			<td>eBioscience</td>
			<td>00-5223-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>407952</td>
			<td>From Streptomyces conglobatus - CAS 56092-82-1, &#8805; 97% (HPLC)</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-032</td>
			<td>L-Glutamine (200 mM)</td>
		</tr>
		<tr>
			<td>Ovalbumin</td>
			<td>InvivoGen</td>
			<td>vac-pova</td>
			<td>Ovalbumine with &#60; 1 EU/mg endotoxin - CAS 9006-59-1</td>
		</tr>
		<tr>
			<td>Oxaliplatin</td>
			<td>Pharmacy of MRI hospital</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Phosphate Buffered Saline without calcium chloride and magnesium chloride</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>1514-122</td>
			<td>Mixture of penicillin (10,000 U/mL) and streptomycin (10,000 ug/mL)</td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma-Aldrich</td>
			<td>P1585</td>
			<td>Phorbol 12-myristate 13-acetate, &#8805; 99% (HPLC)</td>
		</tr>
		<tr>
			<td>PVDF filter, 0,22 &#181;m, for syringes</td>
			<td>Merck Millipore</td>
			<td>SLGV033RS</td>
			<td>Millex-GV Filter Unit 0.22 &#181;m Durapore PVDF Membrane</td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Invitrogen</td>
			<td>00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fischer Scientific</td>
			<td>11875</td>
			<td>Roswell Park Memorial Institute 1640 Medium with D-glucose (2.00 g/L) and L-glutamine (300 mg/L), without HEPES</td>
		</tr>
		<tr>
			<td>Syringe, 26 G</td>
			<td>BD Biosciences</td>
			<td>305501</td>
			<td>1 mL Sub-Q Syringes with needle (0.45 mm x 12.7 mm)</td>
		</tr>
		<tr>
			<td>Total Exosome Isolation Reagent</td>
			<td>Invitrogen</td>
			<td>4478359</td>
			<td>For isolation from cell culture media</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Thermo Fischer Scientific</td>
			<td>31350</td>
			<td>&#946;-Mercaptoethanol (50 mM)</td>
		</tr>
	</tbody>
</table>

in vivo immunogenicity screening of tumor-derived extracellular vesicles by flow cytometry of splenic t cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

This protocol is intended to facilitate the straightforward and easily reproducible assessment of the immunogenicity of tumor-derived EVs. Hereby, mice are inoculated with EVs derived from in vitro cultures of tumor cells expressing the model antigen chicken ovalbumin (OVA). The subsequent immune response is analyzed in splenic T cells via flow cytometry.
Figure 1 gives an overview of the practical steps of the entire protocol. Since the work foc...

Watch this Scientific Journal Video about In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells at JoVE.com

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Research

JoVE Journal

Cancer Research

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

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

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

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

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Extraction of Extracellular Vesicles from Whole Tissue

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

Cell Cycle-specific Measurement of ÃƒÅ½Ã‚Â³H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...