We are grateful to Mr. Arkadiusz Slusarczyk and Kentucky Biomedical Research Infrastructure Network (KBRIN, P20GM103436) for acquiring transmission electron microscope images. This work was supported in part by grants from NIH AA018016-01 (J.W.E.), Commonwealth of Kentucky Research Challenge Trust Fund (J.W.E.), NIH CA106599 and CA175003 (C.L.), NIH CA198249 (K.Y.), and Free to Breathe Research Grant (K.Y.).

1. Culturing ES-D3 cells
<ol>
	<li>To generate exosome-free fetal bovine serum (FBS), load FBS into an ultracentrifuge and centrifuge at 100,000 x g for 16 h at 4 &#176;C. Following centrifugation, collect serum supernatant as exosome-free FBS for culturing the murine ESC cell line ES-D3 and acquiring exosome-enriched EVs.</li>
	<li>Before plating the ES-D3 cells, coat 15 cm tissue culture dishes using gelatin (0.1%) at room temperature for 30 min.</li>
	<li>Following a previously described protocol16, culture the ES-D3 cells without feeder layer cells in the gelatin-coated 15 cm tissue culture dishes. The ES-D3 cell culture medium is composed of DMEM, exosome-free FBS (15%), nonessential amino acids (0.1 mM), L-glutamine (2 mM), &#946;-mercaptoethanol (0.1 mM), penicillin (50 units/mL), streptomycin (50 &#181;g/mL), and leukemia inhibitory factor (LIF; 100 units/mL). Culture ES-D3 cells at 37 &#176;C in a 5% CO2 humidified incubator.</li>
	<li>Once the ES-D3 cells reach around 90% confluency in 15 cm tissue culture dishes, remove the medium by aspiration. Wash the cells using trypsin (5 mL; 0.05%). Add trypsin (5 mL) to the dishes and incubate at 37 &#176;C for 5 min. Collect the cells from the dishes.</li>
	<li>Add fresh culture medium (5 mL) to the collected cells to inactivate the trypsin. Centrifuge the cells at 390 x g for 5 min. Resuspend the cells in fresh medium and determine the cell number using a hemocytometer.</li>
	<li>For passaging cells, plate the ES-D3 cells (5 x 106) in a gelatin-coated (0.1%) 15 cm tissue culture dish with fresh medium (15 mL) and culture for 3 days before subculturing the cells.</li>
	<li>To collect the cell culture supernatant for isolation of exosome-enriched EVs, plate the ES-D3 cells (1 x 107) in a gelatin-coated (0.1%) 15 cm tissue culture dish with fresh medium (15 mL) for 3 days prior to collecting cell culture supernatant.</li>
</ol>
2. Generation of GM-CSF expression plasmid
NOTE: Generate the transfection plasmid pEF1&#945;-mGM-CSF-IRES-hrGFP to overexpress GM-CSF in ES-D3 cells. In this plasmid, expression of murine GM-CSF cDNA along with the marker protein humanized Renilla reniformis GFP (hrGFP) is driven by the human polypeptide chain elongation factor 1&#945; (EF1&#945;) promoter17,18.
<ol>
	<li>Generate the vector backbone.
	<ol>
		<li>Digest the plasmid pEF1&#945;-FD3ER-IRES-hrGFP (20 &#181;g of DNA) using the restriction enzyme EcoRI (100 units) at 37 &#176;C for 2 h to generate two DNA fragments: the vector backbone (6.0 kb) and FD3ER insert (2.5 kb).</li>
		<li>Transfer 50% of digested plasmid DNA (10 &#181;g) into one 1.5 mL microcentrifuge tube and treat with alkaline phosphatase (20 units) at 37 &#176;C for 1 h. Resolve both untreated and dephosphorylated DNA using agarose gel electrophoresis (2%).</li>
		<li>Purify the vector backbone DNA fragments (6.0 kb) using a DNA gel extraction kit . While the untreated vector backbone will serve as the empty vector (pEF1&#945;-IRES-hrGFP), the dephosphorylated vector backbone will be used to generate GM-CSF-expressing plasmid (pEF1&#945;-mGM-CSF-IRES-hrGFP).</li>
	</ol>
	</li>
	<li>Generate the GM-CSF cDNA insert.
	<ol>
		<li>Digest the plasmid pMSCV-mGM-CSF-IRES-EGFP19 (20 &#181;g) using the restriction enzyme EcoRI (100 units) at 37 &#176;C for 2 h to produce two DNA fragments: the vector backbone (6.5 kb) and the murine GM-CSF cDNA insert (474 bp).</li>
		<li>Resolve the digested DNA through agarose gel electrophoresis (2%). Purify the murine GM-CSF cDNA fragment (474 bp) using a DNA gel extraction kit.</li>
	</ol>
	</li>
	<li>Generate the expression plasmids.
	<ol>
		<li>Set up 2 ligation reactions (10 &#956;L total volume) using a DNA ligation kit.
		<ol>
			<li>To generate the empty vector pEF1&#945;-IRES-hrGFP, ligate the untreated vector backbone from step 2.1.3 (6.0 kb; 500 ng).</li>
			<li>To generate pEF1&#945;-mGM-CSF-IRES-hrGFP, ligate the following DNA fragments: (1) the dephosphorylated vector backbone from step 2.1.3 (6.0 kb; 500 ng) and (2) the mGM-CSF cDNA fragment generated in step 2.2.2 (474 bp; 200 ng).</li>
		</ol>
		</li>
		<li>Ligate at 25 &#176;C for 5 min. Transform ligated DNA into DH5&#945; competent E. coli cells. Plate the transformed E. coli cells on LB-agar plates containing carbenicillin (50 &#956;g/mL).</li>
		<li>Purify plasmids from single E. coli colonies using a DNA isolation kit. Validate the identities of the plasmids by DNA sequencing.</li>
	</ol>
	</li>
</ol>
3. Generation of ES-D3 cells overexpressing GM-CSF
NOTE: Transfect the ES-D3 cells with the plasmid pEF1&#945;-mGM-CSF-IRES-hrGFP to overexpress GM-CS. Cotransfect the plasmid pBabe-Neo into ES-D3 cells to facilitate selection of stably transfected cells18,20.
<ol>
	<li>Transfect the plasmids into the ES-D3 cells.
	<ol>
		<li>Plate the ES-D3 cells (1.4 x 106) in a gelatin-coated (0.1%) 10 cm tissue culture dish with culture medium (10 mL) for transfection. Culture plated ES-D3 cells at 37 &#176;C for 24 h.</li>
		<li>Prepare two plasmid mixtures in 1.5 mL microcentrifuge tubes: (1) pEF1&#945;-IRES-hrGFP (28 &#181;g, vector control) with pBabe-Neo (4 &#181;g) and (2) pEF1&#945;-mGM-CSF-IRES-hrGFP (28 &#181;g, expressing GM-CSF) with pBabe-Neo (4 &#181;g). Carry out transfection using a transfection kit following the manufacturer&#39;s protocol.</li>
		<li>Add transfection medium (1 mL) and transfection reagent (64 &#181;L) to each tube containing the plasmid mixtures and incubate at room temperature for 5 min.</li>
		<li>Add the transfection mixtures to respective 10 cm dishes of ES-D3 cells. Incubate at 37 &#176;C for 5 h.</li>
		<li>Replace the medium in 10 cm dishes with fresh culture medium (10 mL). Incubate at 37 &#176;C for 24 h.</li>
	</ol>
	</li>
	<li>Generate bulk populations of stably transfected ES-D3 cells.
	<ol>
		<li>Remove the medium from transfected ES-D3 cells. Wash the cells with trypsin (2 mL; 0.05%). Add trypsin (2 mL) and incubate at 37 &#176;C for 5 min. Transfer the cells to a 15 mL centrifuge tube and add 2 mL of fresh culture medium to neutralize trypsin. Centrifuge at 390 x g for 5 min.</li>
		<li>Resuspend the transfected cells in fresh culture medium (10 mL). Evaluate the fluorescence intensity of GFP in transfected ES-D3 cells using fluorescence-activated cell sorting (FACS), following manufacturer&#39;s protocol.</li>
		<li>Transfer the transfected cells into two 10 cm dishes containing fresh culture medium (10 mL). Add neomycin (0.5 mg/mL) to eliminate untransfected cells.</li>
		<li>Continue culturing the transfected cells in culture medium containing neomycin (0.5 mg/mL). When the transfected ES-D3 reach 90% confluency, transfer cells to 15 cm tissue culture dishes again. Repeat the procedure for 2 weeks.</li>
	</ol>
	</li>
	<li>Generate clones of stably transfected ES-D3 cells.
	<ol>
		<li>Once bulk populations of stably transfected ES-D3 cells are generated, collect the cells as before. Determine the cell numbers using a hemocytometer. Centrifuge the cells at 390 x g for 5 min. Resuspend the cells (1 x 107 cells/mL) in fresh culture medium.</li>
		<li>Filter the cells through a sterile 40 &#181;m cell strainer. Purify GFP-positive ES-D3 cells using FACS, following manufacturer&#39;s protocol.</li>
		<li>Plate a single sorted ES-D3 cell into one well of a gelatin-coated (0.1%) 96-well tissue culture plate containing parental ES-D3 cells (1 x 103) in neomycin-free medium (200 &#181;L). Co-culturing transfected ES-D3 cells with their untransfected parental counterparts ensures that stably transfected single ES-D3 cells survive and proliferate as a single clone.</li>
		<li>Culture the cells for 48 h, and then add neomycin (0.5 mg/mL) to 96-well plates to eliminate untransfected parental ES-D3 cells.</li>
		<li>Continue culturing the GFP-positive ES-D3 cells in 96-well tissue culture plates with medium containing neomycin (0.5 mg/mL) for 1 week. Transfer clonal ES-D3 cell lines to gelatin-coated (0.1%) 6 cm tissue culture dishes with culture medium (5 mL) containing neomycin (0.5 mg/mL) for 1 week.</li>
		<li>Determine the intensity of GFP fluorescence in each of transfected ES-D3 cell clones using FACS, following manufacturer&#39;s protocol. Select the ES-D3 clones expressing either GM-CSF or the empty vector with high levels of green fluorescence.</li>
		<li>Determine the amounts of GM-CSF secreted by ES-D3 cells using a murine GM-CSF ELISA kit, following manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>
4. Isolation of exosome-enriched extracellular vesicles
<ol>
	<li>Culture the ES-D3 cells (1 x 107) in 15 cm tissue culture dishes for 72 h at 37 &#176;C. Collect the cell culture supernatant. Store collected supernatant at 4 &#176;C up to 1 week to maintain exosomal integrity.</li>
	<li>Centrifuge the cell culture supernatant at 5,000 x g for 60 min at 4 &#176;C using a centrifuge to sediment large cell fragments.</li>
	<li>Collect the supernatant and centrifuge at 100,000 x g for 90 min at 4 &#176;C using an ultracentrifuge.</li>
	<li>Remove the supernatant. Gently rinse each pellet twice with phosphate-buffered saline (PBS; 1 mL) to remove residual culture supernatant.</li>
	<li>Resuspend each pellet in PBS. Quantify exosome-enriched EVs by their protein content5.
	<ol>
		<li>Measure the protein concentration of exosome-enriched EVs with a bicinchoninic acid (BCA) assay. The expected yield of exosome-enriched EVs from ES-D3 cells is approximately 4 &#181;g protein/mL of cell culture supernatant. Resuspend the exosome-enriched EVs in PBS (protein concentration: ~6 &#181;g/&#181;L). Store the exosome-enriched EVs at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
5. Characterization of exosome-enriched extracellular vesicles by transmission electron microscopy
NOTE: Investigate the composition and the structure of the exosome-enriched EVs isolated from ESCs using transmission electron microscopy (TEM)5.
<ol>
	<li>Fix the exosome-enriched EVs (3-5 &#181;g/&#181;L) with a final concentration of 2% EM grade paraformaldehyde at room temperature for 2 h.</li>
	<li>Load fixed samples (10 &#181;L) onto copper grids with carbon support film. Incubate the samples with copper grids for 1 min, and then drain the grids with filter paper.</li>
	<li>Stain the grids with a staining solution following manufacturer&#39;s protocol.</li>
	<li>Transfer the grids to a piece of filter paper using tweezers. Allow the grids to dry overnight at room temperature.</li>
	<li>Acquire electron microscopy images using a transmission electron microscope (50,000x magnification), following manufacturer&#39;s protocol.</li>
</ol>
6. Evaluation of exosome-enriched extracellular vesicles by western blot analysis
<ol>
	<li>Prepare whole cell extracts.
	<ol>
		<li>Remove the medium from ES-D3 cells cultured in 15 cm dishes. Wash the cells with trypsin (5 mL; 0.05%). Add trypsin (5 mL) to the cells. Incubate at 37 &#176;C for 5 min. Collect the cells and add fresh culture medium (5 mL) to neutralize trypsin. Centrifuge the cells at 390 x g for 5 min. Resuspend the cells in PBS.</li>
		<li>Determine cell numbers using a hemocytometer. Centrifuge the cells again at 390 x g for 5 min. Resuspend the cells in SDS-PAGE loading buffer containing 0.5% SDS (5,000 cells/&#181;L).</li>
		<li>Sonicate the samples for 10 s using a sonicator with 10% amplitude (wattage: 500 W; ultrasonic frequency: 20 kHz). Heat the samples at 100 &#176;C for 5 min.</li>
	</ol>
	</li>
	<li>Prepare the lysates of exosome-enriched EVs.
	<ol>
		<li>Resuspend the exosome-enriched EVs in SDS-PAGE loading buffer containing 0.5% SDS at a concentration of 1.2 &#181;g/&#181;L.</li>
		<li>Sonicate the samples for 10 s using a sonicator with 10% amplitude (wattage: 500 W; ultrasonic frequency: 20 kHz). Heat the samples at 100 &#176;C for 5 min.</li>
	</ol>
	</li>
	<li>Detect proteins by Western blot.
	<ol>
		<li>Load whole cell extracts (10 &#181;L; 5,000 cells/&#181;L) and exosome-enriched EV lysates (10 &#181;L; 1.2 &#181;g/&#181;L) into each well of a Bis-Tris PAGE gel (4-20%). Transfer proteins onto polyvinylidene fluoride (PVDF) membranes.</li>
		<li>Incubate membranes with appropriate primary and secondary antibodies. Dilute antibodies (at the concentrations indicated below) in blotting buffer containing PBS, Tween-20 (0.2%), and nonfat dry milk (10% w/v).
		<ol>
			<li>Use the following primary antibodies: anti-Annexin V (200 ng/mL), anti-CD81 (50 ng/mL), anti-Flotillin-1 (200 ng/mL), anti-cytochrome c (100 ng/mL), anti-protein disulfide isomerase (200 ng/mL), anti-GAPDH (33 ng/mL), and anti-Oxphos COX IV-subunit IV (600 ng/mL).</li>
			<li>Use the following secondary antibodies: peroxidase-conjugated goat anti-rabbit IgG (20 ng/mL) and peroxidase-conjugated goat anti-mouse IgG (20 ng/mL).</li>
		</ol>
		</li>
		<li>Detect proteins using an enhanced chemiluminescence detection kit.</li>
	</ol>
	</li>
</ol>
7. Determining GM-CSF concentrations in exosome-enriched extracellular vesicles by ELISA
NOTE: Evaluate the amounts of GM-CSF in exosome-enriched EVs by ELISA using a kit for murine GM-CSF, following manufacturer&#39;s protocol with some modifications.
<ol>
	<li>Coat the ELISA plate with capture antibody. Treat exosome-enriched EVs (0.6 &#181;g) in PBS alone or PBS + 0.05% Tween-20 (100 &#181;L) at room temperature for 30 min. Add treated samples to the coated ELISA plate and incubate at room temperature for 1 h. Wash the plate with PBS alone or PBS + 0.05% Tween-20.</li>
	<li>Add detection antibody to the samples. Incubate at room temperature for 1 h. Wash the plate with PBS alone or PBS + 0.05% Tween-20. Add Avidin-HRP to the samples. Incubate at room temperature for 30 min. Wash the plate with PBS alone or PBS + 0.05% Tween-20.</li>
	<li>Determine the concentrations of GM-CSF in exosome-enriched EVs by measuring the absorbance at 450 nm on a microplate reader.</li>
</ol>

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G., 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=Effects+of+Feeder+Cell+Types+on+Culture+of+Mouse+Embryonic+Stem+Cell+In+Vitro.">Effects of Feeder Cell Types on Culture of Mouse Embryonic Stem Cell In Vitro.</a> Development and Reproduction. 19 (3), 119-126 (2015).</li><li>Lin, S., Talbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+culturing+mouse+and+human+embryonic+stem+cells.">Methods for culturing mouse and human embryonic stem cells.</a> Methods in Molecular Biology. 690, 31-56 (2011).</li><li>Yaddanapudi, 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=Exosomes+from+GM-CSF+expressing+embryonic+stem+cells+are+an+effective+prophylactic+vaccine+for+cancer+prevention.">Exosomes from GM-CSF expressing embryonic stem cells are an effective prophylactic vaccine for cancer prevention.</a> OncoImmunology. 8 (3), 1561119 (2019).</li></ol>

Kavitha Yaddanapudi, Chi Li, and John W. Eaton submitted a US patent application "Compositions comprising engineered embryonic stem cell-derived exosomes and methods of use thereof."

This study shows a highly efficient method of producing exosome-enriched EVs carrying the immune-stimulatory protein GM-CSF, which can be employed to study the immune-modulatory effects of exosome-enriched EVs. Several studies suggest that exosomes exhibit immune-regulatory and anti-tumor functions22. Thus, exosomes from ESCs expressing GM-CSF might also possess biological activities that regulate the immune response. In this protocol, exogenous murine GM-CSF was stably overexpressed in murine ES-...

ESCs are derived from the blastocyst stage of a preimplantation embryo1. As pluripotent stem cells, ESCs have the capability to self-renew and differentiate into any type of embryonic cell. Due to their remarkable developmental potential and long-term proliferative capacity, ESCs are extremely valuable for biomedical research1. Current research efforts have largely focused on the therapeutic potential of ESCs for a variety of major pathological disorders, including diabetes, heart disease, and neurodegenerative diseases2,3,4.
Mammalian cells, including ESCs, are known to release vesicles with variable sizes to the extracellular environment, and these EVs possess many physiological and pathological functions due to their role in intercellular communication5. Among different subtypes of EVs, exosomes are small membrane vesicles released from various cell types into the extracellular space upon fusion of intermediate endocytic compartments, multivesicular bodies (MVBs), with the plasma membrane6. Exosomes have been reported to mediate intercellular communication and are critically involved in many (patho)physiological processes7,8. Exosomes inherit some biological functions from their own parental cells, because exosomes contain biological materials acquired from the cytosol, including proteins and nucleic acids. Thus, the associated antigens or factors stimulating the immune response specific for a given disease are encapsulated in the exosomes from particular types of cells9. This paved the way for clinical trials exploring tumor-derived exosomes as an anti-cancer vaccine10.
GM-CSF is a cytokine secreted by different types of immune cells11. Emerging evidence demonstrates that GM-CSF activates and regulates the immune system and plays an essential role in the antigen-presenting process12. For instance, a clinical report suggests that GM-CSF stimulates the immune response to tumors as a vaccine adjuvant13. Several GM-CSF-based cancer immunotherapy strategies to exploit the potent immune-stimulatory activity of GM-CSF have been investigated in clinical trials14. Among these, a cancer vaccine composed of irradiated GM-CSF-secreting tumor cells has shown some promise in advanced melanoma patients by inducing cellular and humoral antitumor responses and subsequent necrosis in metastasized tumors15.
Because the exosomes derived from ESCs possess similar biological activities as the original ESCs, maybe GM-CSF-carrying exosomes from ESCs could function as cell-free vesicles to regulate the immune response. In this paper, a detailed method to produce high-quality exosome-enriched EVs from ESCs expressing GM-CSF is described. These exosome-enriched EVs have the potential to serve as immune-regulatory vesicles to modulate the immune response.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alkaline phosphate, Calf Intestinal</td>
			<td>New England Biolabs</td>
			<td>M0290S</td>
			<td>Dephosphorylating DNA plasmid</td>
		</tr>
		<tr>
			<td>anti-Annexin V mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone H-3, sc-74438</td>
			<td>Western blot, RRID:AB_1118989</td>
		</tr>
		<tr>
			<td>anti-CD81 mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone B-11, sc-166029</td>
			<td>Western blot, RRID:AB_2275892</td>
		</tr>
		<tr>
			<td>anti-cytochrome c mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone A-8, sc-13156</td>
			<td>Western blot, RRID:AB_627385</td>
		</tr>
		<tr>
			<td>anti-Flotillin-1 mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone C-2; sc-74566</td>
			<td>Western blot, RRID:AB_2106563</td>
		</tr>
		<tr>
			<td>anti-GAPDH pAb</td>
			<td>Rockland</td>
			<td>600-401-A33S</td>
			<td>Western blot, RRID:AB_11182910</td>
		</tr>
		<tr>
			<td>anti-mouse IgG, goat, peroxidase-conjugated</td>
			<td>Thermo Fisher</td>
			<td>31430</td>
			<td>Western blot, RRID:AB_228307</td>
		</tr>
		<tr>
			<td>anti-Oxphos COX IV-subunit IV mAb</td>
			<td>Thermo Fisher</td>
			<td>clone 20E8C12 A21348</td>
			<td>Western blot, RRID:AB_221509</td>
		</tr>
		<tr>
			<td>anti-protein disulfide isomerase (PDI) pAb</td>
			<td>Enzo</td>
			<td>ADI-SPA-890</td>
			<td>Western blot, RRID:AB_10616242</td>
		</tr>
		<tr>
			<td>anti-rabbit IgG, goat, peroxidase-conjugated</td>
			<td>Thermo Fisher</td>
			<td>31460</td>
			<td>Western blot, RRID:AB_228341</td>
		</tr>
		<tr>
			<td>BCA (bicinchoninic acid) assay</td>
			<td>Thermo Fisher</td>
			<td>23223</td>
			<td>Determining protein concentrations</td>
		</tr>
		<tr>
			<td>Bis-Tris PAGE Gel, ExpressPlus, 4-20%</td>
			<td>Genscript</td>
			<td>M42015</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Carbenicillin, Disodium Salt</td>
			<td>Thermo Fisher</td>
			<td>10177012</td>
			<td>Selecting E. coli colonies</td>
		</tr>
		<tr>
			<td>Centrifuge, Avanti J-26 XPI</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Low speed centrifugation</td>
		</tr>
		<tr>
			<td>Centrifuge rotor, JA-10</td>
			<td>Beckman Coulter</td>
			<td>09U1597</td>
			<td>Low speed centrifugation</td>
		</tr>
		<tr>
			<td>Centrifuge bottle, Nalgene PPCO</td>
			<td>Thermo Fisher</td>
			<td>3120-0500PK</td>
			<td>Low speed centrifugation</td>
		</tr>
		<tr>
			<td>Cu grids with carbon support film</td>
			<td>Electron Microscopy Sciences</td>
			<td>FF200-Cu</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
		<tr>
			<td>EcoRI</td>
			<td>New England Biolabs</td>
			<td>R0101</td>
			<td>Digesting DNA plasmid</td>
		</tr>
		<tr>
			<td>Enhanced chemiluminescence detection system</td>
			<td>Thermo Fisher</td>
			<td>32106</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>FACScalibur flow cytometer</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>Examining GFP levels of ES-D3 cells</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ATCC</td>
			<td>SCRR-30-2020</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Cell Strainers; Mesh Size: 40&#956;m</td>
			<td>Thermo Fisher</td>
			<td>22-363-547</td>
			<td>Filtering ES-D3 cells for FACS sorting</td>
		</tr>
		<tr>
			<td>Gelatin (0.1%)</td>
			<td>Thermo Fisher</td>
			<td>ES006B</td>
			<td>Culturing ES-D3 cells</td>
		</tr>
		<tr>
			<td>GM-CSF ELISA kit</td>
			<td>Thermo Fisher</td>
			<td>88733422</td>
			<td>Determining GM-CSF concentrations</td>
		</tr>
		<tr>
			<td>KnockOut Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Thermo Fisher</td>
			<td>10-829-018</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor</td>
			<td>Thermo Fisher</td>
			<td>ESG1106</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>VWR</td>
			<td>VWRL0131-0100</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 transfection reagent</td>
			<td>Thermo Fisher</td>
			<td>11668019</td>
			<td>Transfecting ES-D3 cells</td>
		</tr>
		<tr>
			<td>Microplate reader, PowerWave XS</td>
			<td>BioTek</td>
			<td></td>
			<td>Determining GM-CSF concentrations</td>
		</tr>
		<tr>
			<td>MoFlo XDP high-speed cell sorter</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Isolating single ES-D3 cell clones</td>
		</tr>
		<tr>
			<td>NEB 5-alpha Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2988J</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>Neomycin</td>
			<td>Thermo Fisher</td>
			<td>10-131-035</td>
			<td>Selecting ES-D3 clones</td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Thermo Fisher</td>
			<td>SH3023801</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Non-fat dry milk</td>
			<td>Thermo Fisher</td>
			<td>NC9022655</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher</td>
			<td>31985062</td>
			<td>Transfecting ES-D3 cells</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>VWR</td>
			<td>sc45000-652</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Plasmid pEF1a-FD3ER-IRES-hrGFP</td>
			<td>Addgene</td>
			<td>37270</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>PVDF membranes</td>
			<td>Millipore EMD</td>
			<td>IPVH00010</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit (50)</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>Quick Ligation Kit</td>
			<td>New England Biolabs</td>
			<td>M2200S</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>Hitachi</td>
			<td>HT7700</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR</td>
			<td>45000-660</td>
			<td>Culturing ES-D3 cells</td>
		</tr>
		<tr>
			<td>Ultracentrifuge, OptimaTM L-100 XP</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>High speed centrifugation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor, 45Ti</td>
			<td>Beckman Coulter</td>
			<td>09U4454</td>
			<td>High speed centrifugation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge polycarbonate bottle</td>
			<td>Beckman Coulter</td>
			<td>355622</td>
			<td>High speed centrifugation</td>
		</tr>
		<tr>
			<td>UranyLess staining solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>22409</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
	</tbody>
</table>

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 types, ESCs release small membrane vesicles, such as exosomes, to the extracellular environment. Exosomes serve as essential mediators of intercellular communication and play a basic role in many (patho)physiological processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) functions as a cytokine to modulate the immune response. The presence of GM-CSF in exosomes has the potential to boost their immune-regulatory function. Here, GM-CSF was stably overexpressed in the murine ESC cell line ES-D3. A protocol was developed to isolate high-quality exosome-enriched extracellular vesicles (EVs) from ES-D3 cells overexpressing GM-CSF. Isolated exosome-enriched EVs were characterized by a variety of experimental approaches. Importantly, significant amounts of GM-CSF were found to be present in exosome-enriched EVs. Overall, GM-CSF-bearing exosome-enriched EVs from ESCs might function as cell-free vesicles to exert their immune-regulatory activities.

GM-CSF is overexpressed in murine ESCs. 
To stably overexpress GM-CSF in ES-D3 cells, murine GM-CSF cDNA was cloned into a transfection vector to generate the expression vector pEF1&#945;-mGM-CSF-IRES-hrGFP (Figure 1A). GM-CSF was overexpressed in ES-D3 cells by transfection, and about 20% of transiently transfected ES-D3 cells were GFP-positive. Cell clones stably overexpressing GM-CSF or the empty vector control were a...

Watch this Scientific Journal Video about Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells at JoVE.com

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

This study describes a method to isolate exosome-enriched extracellular vesicles carrying immune-stimulatory granulocyte macrophage colony-stimulating factors 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 ...

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Cancer Research

4.1K Views.  University of Louisville. This study describes a method to isolate exosome-enriched extracellular vesicles carrying immune-stimulatory granulocyte macrophage colony-stimulating factors from embryonic stem cells.

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

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

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 prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

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

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

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.

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.

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