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>
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<ol>
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The authors declare that there is no conflict of interest.

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...

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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 ...

This video shows how to assess the in vivo immunogenicity of tumor-derived extracellular vesicles, or EVs, using flow cytometry. The screening method described herein is relatively easy to perform and can be adapted to various settings. In cancer, identifying conditions of immunogenic extracellular vesicle releases of particular translational relevance.As such, autologous EVs might be used as personalized anti-cancer treatments. Demonstrating the first part of the procedure will be Tatiana Nedelko, a technical assistant from my laboratory. To start the preparation of cell culture media required for extracellular vesicle, or EVs, deplete the bovine EVs in FCS media by ultracentrifugation at 100, 000 times g for 24 hours at 4 degrees Celsius.Discard the pellet after centrifugation. Harvest the murine B16 melanoma cells expressing ovalbumin, or B16-OVA cells, from the culture and wash the cells twice with PBS. Then, seed the cells at a concentration of 400, 000 cells per milliliter in the EV-depleted media.Next, treat the B16-OVA cells with 30 micrograms of oxaliplatin per milliliter and incubate for 24 hours at 37 degrees Celsius with untreated control samples. After 24 hours, collect the supernatant to centrifuge at 400 times g for five minutes at 4 degree Celsius. Discard the pellet before centrifugation again at 2, 000 times g for 30 minutes at 4 degrees Celsius.After discarding the pellet, filter the supernatant through a 220 nanometer PVDF membrane filter into a fresh tube. Then, mix 1 milliliter of supernatant with 0.5 milliliters of a specific commercially-available exosome isolation reagent. Vortex and transfer the mixture into 1.5 milliliter tubes, followed by overnight incubation at 4 degrees Celsius.The next day, centrifuge the mixture at 10, 000 times g for 60 minutes at 4 degrees Celsius. After carefully discarding the supernatant, remove the remaining drops by tapping the 1.5 milliliter tube upside down on a paper towel and by aspiration through a fine tip of pipette without touching the EV pellet at the bottom. Resuspend the pellet in cold PBS by pipetting up and down without scratching the pellet with the tip.Then, pool the EVs from all tubes. If not using immediately, store the EV suspensions at 80 degrees Celsius in siliconized vessels for up to 28 days until application. To immunize each mouse in the treatment group with EVs isolated from 4.0 x 10 to the fifth B16-OVA cells, mix 5 microliters of the EV suspension with 55 microliters of cold PBS.Next, fill syringes mounted with 26 to 30-gauge needles with 60 microliters of the diluted EVs or PBS and then immediately put the syringe on ice. Inoculate the EVs or PBS subcutaneously into the medial aspect of the mice's thigh. Repeat the immunization after seven days.14 days after the first treatment, sacrifice the mice to resect the spleen. Then, mash the resected spleen with a moistened 100-micrometer cell strainer and the plastic plunger of a syringe. Flush the splenic cells into a 50 milliliter tube with 5 to 10 milliliters of the cool complete RPMI.Next, centrifuge the cells at 400 times g for five minutes at 4 degrees Celsius before discarding the supernatant. To remove erythrocytes from the cell suspension, resuspend and incubate the cell pellet with 2 milliliters of the red blood cell lysis buffer for five minutes at room temperature. Then, stop the reaction by adding five to 10 milliliters of complete RPMI, followed by centrifugation as described earlier.After resuspending the cell pellet in complete RPMI, count the cells from every mouse and place the triplicates of 200, 000 splenocytes with 200 microliters of cRPMI into each well of a 96-well plate with a U-shaped bottom. Then, incubate the plates for 48 hours at 37 degrees Celsius. After 48 hours, treat the cell culture with 5 nanograms per milliliter Brefeldin A, 20 nanograms per milliliter PMA, and 1 microgram per milliliter Ionomycin at 37 degrees Celsius for 4 hours.Post incubation, transfer the splenocytes to a 96-well plate with a V-shaped bottom and wash the cells twice with PBS. Resuspend the pelleted splenocytes in the freshly-prepared staining solution and incubate the cell suspension for 30 minutes at 4 degrees Celsius protected from light. For fixation and permeabilization, wash the splenocytes twice in the FACS-buffer and then resuspend the splenocytes in 100 microliters per well of the fixation and permeabilization solution, followed by incubation in the dark as explained earlier.For staining the intracellular interferon gamma, wash the splenocytes in the fixation or permeabilization buffer before resending the cells with the fluorescent antibodies against interferon gamma diluted 1:200 in the buffer. Incubate the splenocytes for 1 to 12 hours at 4 degree Celsius in the dark. After thorough washing and resuspension in FACS-buffer, analyze the activation of cytotoxic T cells according to the gating strategy explained in the manuscript.In the representative image, the gating strategy to analyze the activation of cytotoxic T-cells is displayed. The cells were gated as the single cells, the lymphocyte subset, the viable CD3-positive T cells, and the CD4-negative CD8-positive cytotoxic T-cells. The intracellular accumulation of the interferon gamma was assessed as a surrogate marker for the activation.After the immunization with EVs derived from the tumor cells, the induction of antigen-specific T cell responses in recipient mice was studied. The tumor-derived EVs cultured under genotoxic stress conditions, such as oxaliplatin treatment, induced potent activation of the splenic cytotoxic T cells in contrast to untreated EVs. The vehicle-treated group of animals showed the lowest response.The activation of splenic T cells was determined after the ex vivo restimulation either in the presence or absence of ovalbumin. The production of the interferon gamma was increased when the splenocytes of tumor EV-treated animals were restimulated with the model tumor antigen ovalbumin before the analysis. Please note that reproducible results rely on the constant isolation efficacy of EVs.Thus, ensure that EV pellets are resuspended entirely and promptly to avoid desiccation. With this protocol, we were able to analyze specific pathways within tumor cells that determine the immunogenicity of tumor cell-derived EVs. This may pave the road to harness such immunogenic EVs in cancer treatment.

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Research

JoVE Journal

Cancer Research

2.4K Views.  Technical University of Munich. This video shows how to assess the in vivo immunogenicity of tumor-derived extracellular vesicles, or EVs, using flow cytometry. The screening method described herein is relatively easy to perform and can be adapted to various settings. In cancer, identifying conditions of immunogenic extracellular vesicle releases of particular translational relevance.As such, autologous EVs might be used as personalized anti-cancer treatments. Demonstrating the first part of the procedure will be Tat...