S.R. Baglio was supported by a fellowship by Associazione Italiana per la Ricerca sul Cancro (AIRC) co-funded by the European Union,. In addition, this project has received funding from the European Union&#8217;s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 660200 (to S.R. Baglio).&#160;

Human adipose tissues for mesenchymal stem cell isolation were obtained from the department of Plastic Surgery of the Tergooi Hospital (Hilversum, Netherlands) after approval by the Institutional Ethical Committee and written informed consent. GFP-positive adipose MSCs were obtained from the Department of Medical and Surgical Sciences for Children and Adults (University of Modena and Reggio Emilia).
Animal experiments were performed in accordance with the Dutch law on animal experimentation, a...

<ol>
	<li>Hanahan, D., Weinberg, 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=Hallmarks+of+cancer:+The+next+generation.">Hallmarks of cancer: The next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Karnoub, A. 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Tumor-secreted extracellular vesicles (EVs) can alter the physiology of local and distant mesenchymal cells to generate a tumor-supportive environment. Here we describe the generation of a preclinical mouse model of osteosarcoma that allows dissection of the EV-mediated interactions between tumor cells and mesenchymal stem cells (MSCs) in vivo. We show that systemic injection of human tumor EV-educated MSCs in mice bearing osteosarcoma xenografts strongly promotes cancer growth and metastasis formation by activa...

The tumor microenvironment actively participates in most, if not all, aspects of tumorigenesis and cancer progression, including metastasis formation and the development of resistance to therapeutics1. This stresses the need for preclinical orthotopic cancer mouse models that allow dissection of the complex tumor-stroma interactions occurring in the tumor niche.
Among the many cellular components of the tumor microenvironment, mesenchymal stem cells (MSCs) strongly contribute to cancer progression in multiple cancer types such as breast cancer, prostate cancer, brain tumors, multiple myeloma, and osteosarcoma2,3,4,5,6,7. MSCs are multipotent stem cells that reside in various adult and fetal tissues, including bone marrow, adipose tissue, placenta, umbilical cord blood, and others8,9. In response to cancer-generated inflammatory signals, MSCs migrate towards tumor sites, incorporate into the tumor microenvironment and ultimately differentiate into cancer-supporting cells10. These cancer-associated MSCs provide essential factors (i.e., growth factors, chemokines, cytokines, and immunosuppressive mediators) for tumor progression acting both on tumor cells and on the surrounding stroma2,3,11,12,13. While the tumor-promoting effects of cancer-associated MSCs have been investigated in numerous cancer models, the mechanisms by which tumor cells reprogram MSCs to shape a cancer-promoting niche are poorly understood. Here we describe the generation of an orthotopic xenograft model that specifically allows the study of the pro-tumorigenic interaction between bone cancer cells and MSCs via extracellular vesicles (EVs).
EVs are crucial mediators of intercellular communication between tumor and stromal cells14. EVs carry functional biomolecules of the cell of origin, including proteins, lipids, and regulatory RNAs. Once released in the extracellular space, these vesicles can be taken up by surrounding cells or carried to distant sites via the blood or the lymphatic circulation, and can profoundly influence target cell behavior.15,16,17 For instance, uptake of cancer EVs by stromal fibroblasts may result in myofibroblast differentiation supporting angiogenesis and accelerating tumor growth in vivo18,19, internalization by endothelial cells can stimulate tumor angiogenesis and increase vascular permeability16,20, and interaction with immune cells might lead to suppression of the antitumor immune response21.
We recently demonstrated, using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, that tumor cells release high amounts of EVs that prompt MSCs to acquire a pro-tumorigenic and pro-metastatic phenotype. This effect is due to a dramatic change in the MSC cytokine expression profile (referred to as &#34;MSC education&#34;), and can be prevented by the administration of a therapeutic interleukin-6 receptor (IL-6R) antibody7. Our work demonstrated that cancer EVs are crucial modulators of MSC behavior, thus providing a rationale for microenvironment-targeted approaches to halt osteosarcoma progression. Herein, we describe a step-by-step protocol to investigate the EV-mediated tumor-MSC interaction in vivo. This model is intended to: 1) specifically define the cancer EV-induced alterations of MSC behavior in the tumor microenvironment, 2) evaluate how this interaction contributes to bone tumor growth and metastasis formation, and 3) study whether interfering with the EV-mediated crosstalk in vivo prevents cancer progression.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Equipment</td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Ultra Centrifuge</td>
			<td >Beckman</td>
			<td >Optima L-90K</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Rotor SW32Ti</td>
			<td >Beckman</td>
			<td >369650</td>
			<td colspan="4">Referred to in the manuscript as ultra-swinging bucket rotor</td>
			<td></td>
		</tr>
		<tr >
			<td >Transmission electron microscope</td>
			<td >Zeiss</td>
			<td >EM109</td>
			<td>Or similar TEM</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Digital camera</td>
			<td >Nikon</td>
			<td >DMX 1200F</td>
			<td>Or similar camera</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Imaging software TEM&#160;</td>
			<td >Nikon</td>
			<td >ACT-1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescence microscope</td>
			<td >Zeiss</td>
			<td >Imager.D2</td>
			<td colspan="2">Or similar Fluorescence microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Imaging software FM</td>
			<td>Zeiss</td>
			<td >ZEN Blue</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Incubator</td>
			<td >Nuaire</td>
			<td >4750E</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Hettick</td>
			<td >ROTANTA 460R</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >-80 Freezer</td>
			<td >Thermo electro corporation</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >FACS</td>
			<td >BD</td>
			<td >BD FACScalibur</td>
			<td>Or similar flow cytometer</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Drill</td>
			<td >Ferm</td>
			<td >FCT-300</td>
			<td>With 0.8 mm drill</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >HSS micro twist drills, 0.8 mm</td>
			<td >Proxxon</td>
			<td >28 852</td>
			<td>0.8 mm drill</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >IVIS camera</td>
			<td >Xenogen</td>
			<td >Ivis Lumina</td>
			<td colspan="5">Referred to in the manuscript as bioluminescence camera. Xenogen is now part of Perkin Elmer</td>
		</tr>
		<tr >
			<td >Living image software2.60</td>
			<td >Xenogen / Igor Por</td>
			<td >n.a</td>
			<td colspan="2">Xenogen is now part of Perkin Elmer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >10 &#181;L Syringe</td>
			<td >Hamilton</td>
			<td >Neuros Model 1701 RN</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Needle: Hamilton RN Needle for Syringe, 26 Gauge, Pointstyle AS, custom length 2 cm</td>
			<td >Hamilton</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Caliper</td>
			<td >Mitutoyo</td>
			<td >G08004463</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Autoclave</td>
			<td >Astell</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Heat Lamp</td>
			<td >Philips</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Culture media</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >Hyclone</td>
			<td >RYG35912</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Platelet Lysate</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >IMDM medium</td>
			<td >Lonza</td>
			<td >BE12-722F</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >alpha-MEM medium</td>
			<td >Lonza</td>
			<td >BE02-002F</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >DMEM medium</td>
			<td >Lonza</td>
			<td>BE12-614F</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >pen/strep/glutamine</td>
			<td >GIBCO</td>
			<td >10378-016</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >heparin</td>
			<td >LEO</td>
			<td >012866-08</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin/EDTA (10x)</td>
			<td >GIBCO</td>
			<td >15400-054</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Cells</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >adipose deriverd MSCs</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >GFP-positive MSCs</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >human fibroblasts</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >143B cells</td>
			<td >ATCC</td>
			<td >CRL-8303</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >FLUC-143B cells</td>
			<td >ATCC</td>
			<td >CRL-8303</td>
			<td>Transduced</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Disposables</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Culture flasks 175 cm2</td>
			<td >CELLSTAR</td>
			<td >660175</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >50 mL tubes</td>
			<td >Greiner bio-one</td>
			<td >210261</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Freeze tubes</td>
			<td >Thermoscientific</td>
			<td >377224</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Ultra-Clear tubes</td>
			<td >Beckman</td>
			<td >344058</td>
			<td colspan="4">Referred to in the manuscript as ultra-centrifuge tubes</td>
			<td></td>
		</tr>
		<tr >
			<td >0,22 &#181;m filter</td>
			<td >Millex</td>
			<td >SLGV033RS</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >200 mesh Formvar-carbon-coated nickel grids</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >0.5 mL insulin syringes with 29G Needle</td>
			<td >Terumo</td>
			<td >U-100&#160;</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td >Sigma - Aldrich</td>
			<td >P7612</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Filter paper&#160;</td>
			<td >Thermo fisher Scientific</td>
			<td >50363215</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Reagents / kits</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >paraformaldehyde</td>
			<td >Alfa Aeser</td>
			<td >43368.9M</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >Braun</td>
			<td >220/12257974/110</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >glutaraldehyde</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >16300</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >uranyl oxalate</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >22510</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >urany acetate</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >22400</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >methyl cellulose</td>
			<td >EMS (Electron Microscopy Sciences)</td>
			<td >1560</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >PKH67</td>
			<td >Sigma</td>
			<td >mini67-1kt</td>
			<td colspan="2">Referred to in the manuscript as GFLD</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >BSA</td>
			<td >Sigma</td>
			<td >A8412</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >CBA - human inflammatory cytokine kit</td>
			<td >BD</td>
			<td >551811</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Formaldehyde 37%</td>
			<td >VWR</td>
			<td >104003100</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Carbon Steel surgical blades</td>
			<td >Swann-Morton</td>
			<td >206</td>
			<td colspan="3">Referred to in the manuscript as surgical knife</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >anti-human vimentin antibody</td>
			<td >Santa Cruz</td>
			<td >sc-6260</td>
			<td>Clone V9</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Antibody diluent</td>
			<td >DAKO</td>
			<td >S0809</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >HRP-labeled anti mouse IgG antibody</td>
			<td >Life Technologies</td>
			<td >32230</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >DAB-kit</td>
			<td >DAKO</td>
			<td >K500711</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >hematoxyllin</td>
			<td >Sigma</td>
			<td >GHS232</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >EDTA-buffer</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Citrate buffer</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >rabbit polyclonal anti-GFP antibody</td>
			<td >Abcam</td>
			<td >n.a.</td>
			<td>Ab290</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI&#160;</td>
			<td >Life Technologies</td>
			<td >D1306</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Paracetamol, 120 mg / 5 ml syrup</td>
			<td >Bayer</td>
			<td >n.a.</td>
			<td colspan="2">Sinaspril, paracetamol solution for kids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane 1000 mg/g</td>
			<td >Vumc pharmacy</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >buprenofine hydrochloride, 0.3 mg/ml</td>
			<td >Indivior UK Limited</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >lidocaine-HCL 2%</td>
			<td >Vumc pharmacy</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >70% ethanol</td>
			<td >VWR</td>
			<td >93003.1006</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Tissue glue</td>
			<td >Derma+Flex, formulated medical cyanoacrylate</td>
			<td >Vygon</td>
			<td>LB604060</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Eyedrops: Vidisec Carbogel, 2 mg/ml</td>
			<td >Bausch+Lomb</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >D-luciferin, potassium salt</td>
			<td >Gold Biotechnology</td>
			<td >LUCK-1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Glass slides</td>
			<td >Thermo scientific</td>
			<td >630-0954</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Stainless steel loops&#160;</td>
			<td >n.a.</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Mice experiments</td>
			<td ></td>
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Mice, Hsd:Athymic Nude-Foxn1nu,&#160; female, 6 weeks at arrival, bacterial status conform FELASA</td>
			<td >ENVIGO</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Paper-pulp smart home (cage enrichment)</td>
			<td >Bio Services</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Alpha-dri bedding material</td>
			<td >Shepperd Speciality Papers</td>
			<td >n.a.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse food: Teklad global 18% protein rodent diet</td>
			<td >ENVIGO</td>
			<td >2918-11416M</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Sutures</td>
			<td >Ethicon</td>
			<td >V926H</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Scissors</td>
			<td >Sigma-Aldrich</td>
			<td >S3146-1EA</td>
			<td>(or similar)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Tweezers</td>
			<td >Sigma-Aldrich</td>
			<td >F4142-1EA</td>
			<td>(or similar)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In this study, we explored the ability of osteosarcoma-secreted EVs to educate MSCs towards a pro-tumorigenic and pro-metastatic phenotype. We show that osteosarcoma cells release exosome-like EVs that are internalized by MSCs. We measured the alteration of the MSC cytokine expression profile induced by cancer EVs, and evaluated the effect of the EV-educated MSCs on tumor growth and metastasis formation. The general representation of the study design is illustrated in <strong class="xfig"...

Watch this Scientific Journal Video about A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells at JoVE.com

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

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 ...

The overall goal of this experimental procedure is to develop a preclinical mouse model to study the role of extracellular vesicle-mediated communication between tumor and mesenchymal stem cells. This method can help answer several key questions in the cancer biology field, as to how tumor and mesenchymal stem cells communicate by means of extracellular vesicles, and whether this supports cancer progression. The main advantage of this technique is that it allows to study the role of mesenchymal stem cell adaptation by cancer EVs, on the progression and metastasis formation in vivo.The implication of this procedure extends to our therapy of cancer, because we can use GSA to interfere with EV communication and androgen signaling to inhibit cancer progression. Besides providing insights into the EV-mediated communication in osteosarcoma, this method can be also applied to many other cancer types. Indeed, bone marrow derived from the mesenchymal stem cells are readily recruited to more sites where they can differentiate into cancer-supporting cells.Begin by seeding eight 175 square centimeter flasks with 3 million 143B cells each. Culture the cells using EV-depleted medium for 36 to 48 hours. When the cells are 80 to 90%confluent, collect the supernatants from the eight flasks for EV isolation.Next, use differential centrifugation to remove all of the cells and cell debris from the supernatant. This consists of repeated centrifugations at four degrees Celsius, with maximum acceleration and deceleration. After each centrifugation step, collect the supernatant, and leave behind one milliliter of the supernatant in the centrifugation tube.Now, load a pre-cleared supernatant into ultracentrifuge tubes at 38.5 milliliters per tube. Then, isolate the EVs using an ultracentrifuge at 70, 000g for one hour, with a slow break and at four degrees Celsius. After the ultracentrifugation, carefully remove the supernatant, leaving behind about one milliliter.Then, resuspend the EV-containing pellets in the remaining volume. Pool all the suspensions. And wash the pooled suspensions with PBS.Fill the tube to 38.5 milliliters. Then, repeat the ultracentrifugation cycle, but use no break. The rotor should stop spinning after about an hour.Next, carefully remove the supernatant without disturbing the pellet, and leave 100 to 200 microliters of solution. Finally, resuspend the EV pellet, and adjust the final volume to 200 microliters with PBS. The EVs can then be characterized by TEM, labeled with dyes for uptake experiments, or used to educate mesenchymal stem cells for in vivo experiments.It is crucial that the primary MSCs are at the right cell density before exposure to cancer EVs. The cell should be around 70%confluent to achieve optimal cell EV ratio, and to also prevent overgrowth during education periods. This experiment uses acclimated adult Athymic Nude-Foxn1nu mice.One day before the surgical procedure, provide paracetamol via the drinking water. On the day of the surgery, prepare 143B cells at 200, 000 per microliter in PBS. 20 minutes before the surgery, provide the animal with buprenorfine.Just before anesthesia, load a 10 microliter syringe with the concentrated 143B cells. After confirming proper anesthesia and applying eye ointment, position the animal on its back with its rear legs towards the operator. Next, secure an anesthesia mask to the animal.Then, flex the left knee and wipe the skin with three alternating scrubs of 70%ethanol and povidone-iodine, and then apply 2%lidocaine as a local analgesic. Now, make a small incision into the skin just below the knee to expose the tibia. Then, using a 0.8 millimiter microtwist drill, make a pinhole in the tibia, approximately 2 millimeters below the knee.Avoid drilling through both tibia cortices. Proceed by slowly injecting one microliter of cells into the hole over about five seconds. Then, close the hole with a droplet of tissue glue to prevent backflow of the suspension.Complete the surgery by closing the skin with sutures and one drop of tissue glue. Allow the animal to recover with heating, and treat it with paracetamol for 24 hours. Two days later, use a 0.5 millimeter insulin syringe to inject the MSCs into the tail vein.Correct administration during a tail vein injection can be visually confirmed. If a white area appears, or if you encounter resistance, please inject again at an area above the previous injection site. Then, twice per week, follow the tumor growth using bioluminescence.Inject the mice with D-luciferin via an insulin syringe, and 10 minutes later, measure the photon flux generated from the tumor cells using an imaging system. When an animal's tumor diameter becomes greater than 15 millimeters, or its weight loss exceeds 15%euthanize the animal and collect all the relevant tissues. 10 minutes before euthanization, inject D-luciferin, as done prior to imaging.After euthanasia, collect the lungs, liver, spleen, and kidneys, using sterilized scissors and tweezers. Wash the blood from the organs using PBS, and arrange them on a Petri dish for imaging. Using a bioluminescence imaging system, document both sides of the organs.Once images are taken, immediately fix the organs for future histological analysis. To process the photo documents, simply apply manual thresholding, and count the number of nodules in each photo. Be certain to use the same threshold for each photo, and to document the tissues from every direction, especially the lungs.After decalcifying the mouse tibia, proceed with dehydration and paraffin embedding. Then, use a microtome to prepare six-micron paraffin sections, and collect the sections on glass slides. Once the slides have dried, store them overnight at room temperature.The next day, perform a heat-mediated antigen retrieval, using citrate buffer. Then, stain the tissues with anti-GFP antibody at one to 900, and follow with a DAPI counterstain. Finally, document the stained tissue sections using fluorescence microscopy.The purity of isolated EVs was confirmed by electron microscopy. The preparations contained vesicles between 40 and 100 nanometers in diameter. To assess whether cancer EVs interact with MSCs, the EVs were labeled with a green fluorescent lipophilic linker dye, and incubated overnight with MSCs.Efficient EV uptake by MSCs was thus confirmed. An immunomodulatory property of EVs on MSCs was confirmed using a multiplex B-based cytokine immunoassay. Tumor EVs caused increased IL-8 and IL-6 production in MSCs, compared with human fibroblast control EVs.Next, in an orthotopic xenograft mouse model of osteosarcoma, mice treated with EV-educated MSCs were shown to have accelerated tumor growth. Four days after systemic injection of GFP-expressing MSCs, GFP-expressing cells were detected in the bone marrow and in the tumor tissue, demonstrating MSC homing to the tumor site. Ex vivo imaging of various organs highlighted lung nodule formation.Ultimately, mice receiving EV-educated MSCs had more lung metastases. After watching this video, you should have a good understanding of how to educate stromal cells with cancer EVs ex vivo, and study the effects of educated stromal cells in vivo using preclinical mouse models. Once mastered, this procedure can be performed within four weeks, provided that enough cancer EVs are available.While attempting this procedure, it is important to plan ahead and get a reproducible purification of cancer extracellular vesicles, as well as to use low-passage proliferating mesenchymal stem cells.

a-preclinical-mouse-model-osteosarcoma-to-define-extracellular

Research

JoVE Journal

Cancer Research

10.0K vistas.  VU Medical Center. The overall goal of this experimental procedure is to develop a preclinical mouse model to study the role of extracellular vesicle-mediated communication between tumor and mesenchymal stem cells. This method can help answer several key questions in the cancer biology field, as to how tumor and mesenchymal stem cells communicate by means of extracellular vesicles, and whether this supports cancer progression. The main advantage of this technique is that it allows to study the role of mesenchym...