Name: a preclinical mouse model of osteosarcoma to define the extracellular vesicle-mediated communication between tumor and mesenchymal stem cells
Uploaded: 2018-05-06T12:30:00
Description: 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

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. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+within+tumour+stroma+promote+breast+cancer+metastasis.">Mesenchymal stem cells within tumour stroma promote breast cancer metastasis.</a> Nature. 449 (7162), 557-563 (2007).</li><li>Jung, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+mesenchymal+stem+cells+into+prostate+tumours+promotes+metastasis.">Recruitment of mesenchymal stem cells into prostate tumours promotes metastasis.</a> Nat Commun. 4, 1795 (2013).</li><li>Shahar, T., 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=Percentage+of+mesenchymal+stem+cells+in+high-grade+glioma+tumor+samples+correlates+with+patient+survival.">Percentage of mesenchymal stem cells in high-grade glioma tumor samples correlates with patient survival.</a> Neuro Oncol. 19 (5), (2016).</li><li>Behnan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruited+brain+tumor-derived+mesenchymal+stem+cells+contribute+to+brain+tumor+progression.">Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression.</a> Stem Cells. 32 (5), 1110-1123 (2014).</li><li>Giallongo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-like+myeloid+derived+suppressor+cells+(G-MDSC)+are+increased+in+multiple+myeloma+and+are+driven+by+dysfunctional+mesenchymal+stem+cells+(MSC).">Granulocyte-like myeloid derived suppressor cells (G-MDSC) are increased in multiple myeloma and are driven by dysfunctional mesenchymal stem cells (MSC).</a> Oncotarget. 7 (52), 85764-85775 (2016).</li><li>Baglio, S. R., 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=Blocking+tumor-educated+MSC+paracrine+activity+halts+osteosarcoma+progression.">Blocking tumor-educated MSC paracrine activity halts osteosarcoma progression.</a> Clin Cancer Res. 23 (14), 3721-3733 (2017).</li><li>Pittenger, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284 (5411), 143-147 (1999).</li><li>Shi, Y., Du, L., Lin, L., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-associated+mesenchymal+stem/stromal+cells:+emerging+therapeutic+targets.">Tumour-associated mesenchymal stem/stromal cells: emerging therapeutic targets.</a> Nat Rev Drug Discov. 16 (1), 35-52 (2017).</li><li>Ridge, S. M., Sullivan, F. J., Glynn, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells:+key+players+in+cancer+progression.">Mesenchymal stem cells: key players in cancer progression.</a> Mol Cancer. 16 (1), 31 (2017).</li><li>Luo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infiltrating+bone+marrow+mesenchymal+stem+cells+increase+prostate+cancer+stem+cell+population+and+metastatic+ability+via+secreting+cytokines+to+suppress+androgen+receptor+signaling.">Infiltrating bone marrow mesenchymal stem cells increase prostate cancer stem cell population and metastatic ability via secreting cytokines to suppress androgen receptor signaling.</a> Oncogene. 33 (21), 2768-2778 (2013).</li><li>Huang, W. -. H., Chang, M. -. C., Tsai, K. -. S., Hung, M. -. C., Chen, H. -. L., Hung, S. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+promote+growth+and+angiogenesis+of+tumors+in+mice.">Mesenchymal stem cells promote growth and angiogenesis of tumors in mice.</a> Oncogene. 32 (37), 4343-4354 (2013).</li><li>Patel, S. A., Meyer, J. R., Greco, S. J., Corcoran, K. E., Bryan, M., Rameshwar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+protect+breast+cancer+cells+through+regulatory+T+cells:+role+of+mesenchymal+stem+cell-derived+TGF-beta.">Mesenchymal stem cells protect breast cancer cells through regulatory T cells: role of mesenchymal stem cell-derived TGF-beta.</a> J Immunol. 184 (10), 5885-5894 (2010).</li><li>Becker, A., Thakur, B. K., Weiss, J. M., Kim, H. S., Peinado, H., Lyden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+in+cancer:+Cell-to-cell+mediators+of+metastasis.">Extracellular vesicles in cancer: Cell-to-cell mediators of metastasis.</a> Cancer Cell. 30 (6), 836-848 (2016).</li><li>Skog, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+microvesicles+transport+RNA+and+protein+that+promote+tumor+growth+and+provide+diagnostic+biomarkers.">Glioblastoma microvesicles transport RNA and protein that promote tumor growth and provide diagnostic biomarkers.</a> Nat Cell Biol. 10 (12), 1470-1476 (2008).</li><li>Peinado, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanoma+exosomes+educate+bone+marrow+progenitor+cells+toward+a+pro-metastatic+phenotype+through+MET.">Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.</a> Nat Med. 18 (6), 883-891 (2012).</li><li>Zomer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+reveals+extracellular+vesicle-mediated+phenocopying+of+metastatic+behavior.">In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior.</a> Cell. 161 (5), 1046-1057 (2015).</li><li>Webber, J., Steadman, R., Mason, M. D., Tabi, Z., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+exosomes+trigger+fibroblast+to+myofibroblast+differentiation.">Cancer exosomes trigger fibroblast to myofibroblast differentiation.</a> Cancer Res. 70 (23), 9621-9630 (2010).</li><li>Webber, J. P., 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=Differentiation+of+tumour-promoting+stromal+myofibroblasts+by+cancer+exosomes.">Differentiation of tumour-promoting stromal myofibroblasts by cancer exosomes.</a> Oncogene. 34 (3), 290-302 (2015).</li><li>Zhou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-secreted+miR-105+destroys+vascular+endothelial+barriers+to+promote+metastasis.">Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis.</a> Cancer Cell. 25 (4), 501-515 (2014).</li><li>Whiteside, T., Anastasopoulou, E., Voutsas, I., Papamichail, M., Perez, S., Nunes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+tumor-mediated+immune+suppression.">Exosomes and tumor-mediated immune suppression.</a> Expert Rev Mol Diagn. 15 (10), 1293-1310 (2016).</li><li>Verweij, F. J., Van Eijndhoven, M. A. J., Middeldorp, J., Pegtel, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+viral+microRNA+exchange+via+exosomes+in+vitro+and+in+vivo.">Analysis of viral microRNA exchange via exosomes in vitro and in vivo.</a> Methods Mol Biol. 1024, 53-68 (2013).</li><li>Baglio, S. R., 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=Human+bone+marrow-+and+adipose-mesenchymal+stem+cells+secrete+exosomes+enriched+in+distinctive+miRNA+and+tRNA+species.">Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species.</a> Stem Cell Res Ther. 6 (1), 127 (2015).</li><li>Naaijkens, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+as+a+fetal+bovine+serum+substitute+improves+human+adipose-derived+stromal+cell+culture+for+future+cardiac+repair+applications.">Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications.</a> Cell Tissue Res. 348 (1), 119-130 (2012).</li><li>Grisendi, 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=Adipose-derived+mesenchymal+stem+cells+as+stable+source+of+tumor+necrosis+factor-related+apoptosis-inducing+ligand+delivery+for+cancer+therapy.">Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy.</a> Cancer Res. 70 (9), 3718-3729 (2010).</li><li>Cosette, J., Abdelwahed, R. B., Donnou-Triffault, S., Saut&#232;s-Fridman, C., Flaud, P., Fisson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence-based+tumor+quantification+method+for+monitoring+tumor+progression+and+treatment+effects+in+mouse+lymphoma+models.">Bioluminescence-based tumor quantification method for monitoring tumor progression and treatment effects in mouse lymphoma models.</a> J Vis Exp. (113), (2016).</li><li>Carbone, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+cervical+dislocation+as+a+humane+euthanasia+method+in+mice.">Assessing cervical dislocation as a humane euthanasia method in mice.</a> J Am Assoc Lab Anim Sci. 51 (3), 352-356 (2012).</li><li>Costa-Silva, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer+exosomes+initiate+pre-metastatic+niche+formation+in+the+liver.">Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.</a> Nat Cell Biol. 17 (6), 816-826 (2015).</li><li>Hoshino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+exosome+integrins+determine+organotropic+metastasis.">Tumour exosome integrins determine organotropic metastasis.</a> Nature. 527 (7578), 329-335 (2015).</li><li>Clayton, A., Mitchell, J. P., Court, J., Mason, M. D., Tabi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+tumor-derived+exosomes+selectively+impair+lymphocyte+responses+to+interleukin-2.">Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2.</a> Cancer Res. 67 (15), 7458-7466 (2007).</li><li>Wieckowski, E. U., Visus, C., Szajnik, M., Szczepanski, M. J., Storkus, W. J., Whiteside, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-derived+microvesicles+promote+regulatory+t+cell+expansion+and+induce+apoptosis+in+tumor-reactive+activated+cd8++T+lymphocytes.">Tumor-derived microvesicles promote regulatory t cell expansion and induce apoptosis in tumor-reactive activated cd8+ T lymphocytes.</a> J Immunol. 183 (6), 3720-3730 (2009).</li><li>Valenti, R., Huber, V., Iero, M., Filipazzi, P., Parmiani, G., Rivoltini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-released+microvesicles+as+vehicles+of+immunosuppression.">Tumor-released microvesicles as vehicles of immunosuppression.</a> Cancer Res. 67 (7), 2912-2915 (2007).</li><li>Costa-Silva, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer+exosomes+initiate+pre-metastatic+niche+formation+in+the+liver.">Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.</a> Nat Cell Biol. 17 (6), 816-826 (2015).</li><li>Lin, L. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+cell-derived+exosomes+endow+mesenchymal+stromal+cells+with+tumour-promotion+capabilities.">Tumour cell-derived exosomes endow mesenchymal stromal cells with tumour-promotion capabilities.</a> Oncogene. 35 (46), 6038-6042 (2016).</li></ol>

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 ...

Research

JoVE Journal

Cancer Research

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

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A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

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The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

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Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

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Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields)

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Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), ...

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

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A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

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Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

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Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...