The authors want to thank Ms. Cornelia Heis for her excellent technical assistance in immunohistochemistry and preparation of chicken embryos. This work was supported by the Austrian Science Fund (FWF Grant No. P19552) and the European Union (EU FP7 project Optatio No: 278570).

Selon le droit autrichien, et le Bureau de la protection des animaux de laboratoire des embryons d&#39;oiseaux de services de santé publique des États-Unis ne sont pas considérés comme des animaux vertébrés vivants jusqu&#39;à ce hatching.The NIH Bureau de la protection des animaux de laboratoire a fourni des directives écrites dans ce domaine (http: // www.grants.nih.gov/grants/olaw/references/ilar91.htm et NIH Publication No .: 06-4515). 1. Culture cellulaire et transfection Lentiviral <ol><li> Lignes Culture de cellules MM OPM-2, le milieu RPMI-8226 et les cellules souches mésenchymateuses humaines de la moelle osseuse dans un milieu de RPMI 1640, complété avec 10% de sérum bovin de veau foetal et 100 UI / ml de pénicilline, 100 ug / ml de streptomycine et 2 mM de glutamine en présence 5% de CO 2 à 37 ° C. </li><li> Transfecter 5 x 10 6 cellules HEK 293FT avec mélange viral d&#39;emballage (9 ug) et 3 ug d&#39;ADN pLenti6 / V5 dest eGFP vecteur par l&#39;utilisation de 30 pi de réactif de transfection liposomale et le milieu de transfection (10 ml). Retirer milieu de transfection après 12 h etajouter 10 ml de milieu DMEM avec 10% de sérum bovin de veau et 1% d&#39;acides aminés non essentiels (NEAA). </li><li> Après 5 jours, de recueillir les surnageants de cellules HEK293FT après centrifugation des cellules de natation (1000 xg, 5 min). Déterminer le titre viral par PCR en temps réel comme décrit ailleurs 28. </li><li> Transfecter 1 x 10 6 cellules MM avec des particules lentivirales eGFP (1 x 10 5 particules) dans une plaque à 24 puits dans du milieu de croissance complet. Après 3 jours, commencer le processus de sélection par l&#39;ajout de 2 ug / ml de blasticidine dans le milieu de culture. Pour la eGFP lentivirus disponible dans le commerce, utiliser 500 pg / ml de néomycine. </li><li> Après 2 semaines de sélection, des grappes de cellules exprimant eGFP MM apparaîtront; recueillir des cellules par centrifugation (1000 xg, 5 min) et développez comme OPM-2 eGFP et RPMI-8226 lignées eGFP pour des expériences (article 2 et 3). </li></ol> 2. 3D-Multiple Myeloma Modèle Spheroid <ol><li> Solution-I de type Chill collagène et 10 x DMEM on glace. </li><li> Mélanger 1/10 volume de 10 x milieu DMEM en matrice de collagène; ajouter du NaOH (0,2 N) pour neutraliser la solution acide de collagène à une valeur de pH de 7,4; magasin collagène / solution à moyen sur la glace. </li><li> Mélanger les lignées cellulaires transgéniques de MM (OPM-2 eGFP ou RPMI-8226 eGFP; 250,000 par sphéroïde) avec des cellules mésenchymateuses humaines (50 000 cellules / sphéroïde, c.-à-goutte 30 ul). </li><li> Mélange de cellules Centrifugeuse à tubes de 15 ml (1000 xg, 5 min), ajouter le mélange préparé à froid de collagène (1 ml) au culot cellulaire et bien mélanger (avec 1.000 pointe de pi). </li><li> La pipette juste 30 pi du mélange collagène / cellule (avec 100 pointe pi) dans une plaque à 24 puits sur un film de paraffine stérile et laisser reposer le mélange cellules / collagène de polymériser pendant 30 min à 37 ° C (voir la figure 1A). </li><li> Sphéroïdes de MM superposition avec un milieu de culture 1 ml contenant 1, 10 et 100 nM bortézomib (voir la figure 1B). </li><li> Après 72 h d&#39;incubation à 37 ° C, par le document sphéroïdesfluorescence stéréomicroscopie (voir figure 1C). </li><li> Transférer chaque sphéroïde par l&#39;utilisation de forceps avec de larges mâchoires plates dans un tube de réaction pour la mesure de la GFP (voir figure 1D). </li></ol> 3. 3D myélome multiple modèle de xénogreffe de la CAM <ol><li> Incuber des œufs de poule dans un incubateur spécial pour les œufs aviaires à 37 ° C et 70% d&#39;humidité pendant trois jours. </li><li> Par la suite, les œufs ouvertes et d&#39;embryons de transfert à stérilisés avec des bateaux éthanol, carrée, de 10 cm en plastique pesant avec la culture cellulaire couvercle de la plaque et incuber &quot;ex ovo&quot; pour plus de six jours de sorte que le CAM est capable de développer (voir la figure 2A). </li><li> Solution-I de type collagène et de refroidissement 10 x DMEM sur de la glace, mélanger 1/10 volume de 10 x dans du milieu DMEM matrice de collagène, Ajouter NaOH (0,2 N) pour neutraliser la solution acide de collagène à une valeur de pH de 7,4; magasin collagène / solution à moyen sur la glace. </li><li> Mélanger les lignées cellulaires de MM transgénique (OPM-2 ou eGFPRPMI-8226 eGFP; 250.000 par sphéroïde) avec des cellules mésenchymateuses humaines (50.000 cellules / sphéroïde). </li><li> Centrifuger les cellules à tubes de 15 ml (1 000 xg, 5 min; pour chaque composé de 1 flacon de test), ajouter 1 ml mélange de collagène préparé à froid avec la drogue (à une concentration de travail désirée) au culot cellulaire et bien mélanger (avec 1.000 pointe de pi). </li><li> Placez collagène diminue (30 pi chacun) sur parafilm dans un 6 bien pate pendant 30 min pour permettre la polymérisation de la matrice extracellulaire à 37 ° C. </li><li> Transfert &quot;onplants&quot; de l&#39;étape 3.6 avec l&#39;utilisation de forceps à la surface non traitée de la CAM (2 cm de distance de l&#39;embryon) de 9 jours embryons de poulet (4 onplants pour chaque embryon de poulet, voir la figure 2B) </li><li> Après 5 jours de croissance in vivo dans un incubateur à 37 ° C et 70% d&#39;humidité, des xénogreffes de documents par fluorescence stéréomicroscopie (voir la figure 2C). Euthanasier des embryons de poulet par hypothermie à 4 ° C dans le réfrigérateur pendant 5h. </li><li> Retirer xénogreffes avec tissu CAM sous-jacent par un ciseaux et des pinces ophtalmique avec de larges mâchoires plates. Utilisez-les pour la mesure de la GFP (article 4, voir figure 2D) ou pour l&#39;analyse immunohistochimique des vaisseaux sanguins et / ou des cellules tumorales envahissantes (section 5). </li></ol> 4. Quantification de la protéine eGFP par ELISA <ol><li> Transférer chaque sphéroïde MM ou xénogreffe excisé dans 0,5 ml de tampon RIPA contenant 200 ug / ml des inhibiteurs de protéase. </li><li> Homogénéiser sphéroïde / xénogreffe avec un homogénéiseur de tissu sur la glace. </li><li> Effectuer trois congélation / décongélation-cycles dans l&#39;azote liquide et 37 ° C bain d&#39;eau. </li><li> Centrifugeuse homogénat à 4 ° C pendant 20 min (12 000 g) et les surnageants de magasin. </li><li> Diluer les échantillons 1:20 dans du tampon d&#39;essai (200 ul) de la trousse ELISA. Mesurer GFP-niveaux par un ELISA Kit commercial GFP en utilisant des anticorps anti-GFP biotinylés, selon le protocole du fabricant. </li></ol> 5. Immunohistochemical Analyse des vaisseaux sanguins et des cellules tumorales envahissantes <ol><li> Fixer xénogreffes excisés avec zone de CAM dans 4% de paraformaldehyde O / N à 4 ° C. </li><li> Passer xénogreffes fixes dans des cassettes d&#39;enrobage et de les transférer dans une station d&#39;enrobage de tissu avec un gradient croissant série d&#39;alcool (50%, 70%, 80%, 95% d&#39;éthanol, xylène et la paraffine; chaque étape 60 min). </li><li> Section xénogreffes (5 um) par l&#39;utilisation d&#39;un microtome rotatif paillasse. Cuire sections de paraffine sur des lames de verre O / N à 56 ° C. </li><li> Déparaffiner sections par une série décroissante de l&#39;alcool graduée à l&#39;eau bidistillée (xylène, 95%, 80%, 70%, 50% d&#39;éthanol, de l&#39;eau bidistillée; chaque étape 10 min). </li><li> Effectuer la récupération de l&#39;antigène dans un bain d&#39;eau (95 ° C, 20 min) avec une solution d&#39;extraction d&#39;antigène (tampon citrate, pH 7,0, volume de 100 ul). </li><li> Bloquer l&#39;activité de la peroxydase endogène avec 100 ul de 3% de H 2 O 2 / méthanol pendant 30 minutes. </li><li> sections de blocs dans PBS contenant10% de sérum de veau foetal pendant 45 minutes (volume 100 pi). </li><li> Teinture pendant 1 heure avec 100 pi d&#39;anticorps primaire (1 pg / ml) dilué dans du PBS contenant 1% de sérum de veau foetal à température ambiante. </li><li> Après lavage trois fois dans du PBS, incuber pendant 1 h avec un anticorps secondaire biotinylé (0,1 pg / ml) dans du PBS contenant 1% de sérum de veau foetal à température ambiante. </li><li> Après lavage trois fois dans du PBS effectuer réaction colorée par la solution d&#39;avidine / (DAB) complexe biotine-substrat (ABC) et la diaminobenzidine selon les instructions du fabricant. </li><li> 5.11. Arrêter la réaction en transférant les sections de l&#39;eau bidistillée, contre-coloration à l&#39;hématoxyline et à monter des sections avec un milieu de montage synthétique. </li></ol>

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Le développement de nouveaux agents thérapeutiques pour MM réfractaire nécessite moins de temps et coûteux systèmes in vivo pour évaluer la sensibilité des cellules MM humains aux médicaments. Jusqu&#39;à présent, seulement peu de systèmes in vivo sont disponibles pour l&#39;évaluation préclinique de nouveaux traitements anti-myélome. Tous ont leurs limites pour le criblage à grande échelle de bibliothèques de composés 29. Les meilleurs modèles actuels pour les cellules de MM ...

Multiple myeloma (MM) is characterized by proliferation of malignant plasma cells in the bone marrow, bone lesions and immunodeficiency 1. Although new treatment options such as proteasome inhibitors (bortezomib) and immune modulatory drugs (pomalidomide and lenalidomide) are available, MM still remains an incurable malignancy with a grim prognosis 2. The bad prognosis might be explained by the extraordinary heterogeneity of MM cell clones that contributes to variable responses to therapy, in particular under long time treatment and selection pressure of MM clones 3.
Preclinical testing of new drugs and their combinations in vitro and in vivo is a critical and time-consuming step for future drug development. Thus, useful in-vivo models of MM are required to gain a better understanding of the biology of the disease and to enable the discovery of new drugs. Actually, the best xenotransplantation models for hematological malignancies and therapeutics are immune-deficient mice, such as the severe-combined immunodeficient (SCID) mice 4-7, the non-obese diabetic/SCID (NOD/SCID) mice 8,9 or the &#946;-microglobulin-knockout NOD/SCID mice 10,11.
Although murine models of human MM in some aspects can resemble the phenotype of human disease, immune-deficient mice are inbred, therefore simulate only one individual response to a drug and costs are very high. Due to immunosuppression animals require special maintenance conditions and the engraftment of human MM in mice requires 6 weeks to 2 months 9,12, unless cells are grafted directly to the bone marrow using a technically demanding procedure with lower rates of animal survival 7,13. Therefore, new methods using stem-cell based organoid models 14, tissue engineering 15 or sophisticated 3D cell culture models 16 have been established. They will compete in the near future with classical animal experiments for preclinical drug testing, but cannot replace systemic toxicity tests in living organisms.
The chicken embryo has been demonstrated before to be a suitable organism for xenotransplantation of human cells and tissues due to lack of adaptive immune response until hatching 17-19. Moreover, each chicken embryo reflects an individual reaction to applied drugs or tumor cells due to genetic diversity within the chicken population. The chorioallantoic membrane (CAM) is a well-established system to study tumor-dependent angiogenesis 20-22. When solid tumors are grafted to the CAM, they display many characteristics of cancers in vivo, including proliferation, invasion, angiogenesis and metastasis 23-27. 
Based on the previous experience of our group with CAM xenograft models20,26,27, a human MM model was established that combines the advantage of a human 3D culture system with the model of ex ovo developing chicken embryos. This MM model system allows real time monitoring of MM growth progression, quantification of cell mass and preclinical drug testing.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RPMI-8226 cells</td>
			<td>DSMZ</td>
			<td>ACC 9</td>
			<td>STR profiled</td>
		</tr>
		<tr>
			<td>OPM-2 cells</td>
			<td>DSMZ</td>
			<td>ACC 50</td>
			<td>STR profiled</td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells&#160;</td>
			<td>PromoCell</td>
			<td>PC-C-12974</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293FT cells&#160;</td>
			<td>Invitrogen</td>
			<td>R700-07</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 Medium</td>
			<td>Sigma Aldrich</td>
			<td>R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160; HyClone</td>
			<td>ThermoScientific</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glut- Pen- Strep solution</td>
			<td>Sigma</td>
			<td>G6784</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Medium</td>
			<td>Gibco</td>
			<td>31966</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Sigma Life Sciences</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection Medium/Opti-MEM&#160;</td>
			<td>Gibco</td>
			<td>51985</td>
			<td></td>
		</tr>
		<tr>
			<td>eGFP lentiviral particles</td>
			<td>GeneCopoeia</td>
			<td>LPP-EGFP-LV105</td>
			<td>Ready to use viral particles</td>
		</tr>
		<tr>
			<td>pLenti6/V5Dest6 eGFP vector</td>
			<td>Invitrogen</td>
			<td>PN 35-1271</td>
			<td>from authors</td>
		</tr>
		<tr>
			<td>ViralpowerTM packaging mix&#160;</td>
			<td>Invitrogen</td>
			<td>P/N 35-1275</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagent/ Lipofectamin 2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Invitrogen</td>
			<td>R210-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin</td>
			<td>Biochrom</td>
			<td>A2912</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen-Type1&#160; Rat Tail</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>Life Technologies</td>
			<td>Art.Nr. 10338582</td>
			<td></td>
		</tr>
		<tr>
			<td>plitidepsin</td>
			<td>Pharmamar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bortezomib</td>
			<td>LKT Lab., Inc.</td>
			<td>B5871</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">SPF-white hen eggs</td>
			<td rowspan="3">Charles River</td>
			<td rowspan="3"></td>
			<td rowspan="3">Fertilized&#160; white Leghorn&#160; chicken eggs</td>
		</tr>
		<tr>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Plastic weighing boats</td>
			<td rowspan="2">neoLab</td>
			<td rowspan="2">Art.Nr. 1-1125</td>
			<td rowspan="2">for ex-ovo culture</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Petridish square (Lids)</td>
			<td>Simport</td>
			<td>D210-16</td>
			<td>for ex-ovo culture</td>
		</tr>
		<tr>
			<td>RIPA Buffer (10x)</td>
			<td>Cell Signaling</td>
			<td>#9806</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Tablets</td>
			<td rowspan="2">Roche</td>
			<td rowspan="2">11 836 170 001</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Complete Mini EDTA-free</td>
		</tr>
		<tr>
			<td>GFP ELISA</td>
			<td>Cell Biolabs, Inc.</td>
			<td>AKR-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Histocette II</td>
			<td>Simport</td>
			<td>M493-6</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160; 37%</td>
			<td>Roth</td>
			<td>7398.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Lonza</td>
			<td>BE17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolut</td>
			<td>Normapur</td>
			<td>20,821,321</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti-Histol</td>
			<td>Roth</td>
			<td>Art.Nr.6640.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Sigma</td>
			<td>A6330</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">SuperFrost Microscope Slides</td>
			<td>R. Langenbrinck&#160;</td>
			<td>Art.-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Labor- u. Medizintechnik</td>
			<td>03-0060</td>
		</tr>
		<tr>
			<td rowspan="2">DakoCytomation Wash Buffer 10x</td>
			<td rowspan="2">DakoCytomation</td>
			<td>Code-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>S 3006</td>
		</tr>
		<tr>
			<td rowspan="2">Target Retrieval Solution (10x)&#160; pH 6,1</td>
			<td rowspan="2">DAKO</td>
			<td>Code-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>S 1699</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu ASMA clone 1A4</td>
			<td>DAKO</td>
			<td>M0851</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu CD138 clone MI15</td>
			<td>DAKO</td>
			<td>M7228</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Vimentin clone V9</td>
			<td>DAKO</td>
			<td>M0725</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Desmin clone D33</td>
			<td>DAKO</td>
			<td>&#160;M0760</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Ki67&#160; clone MIB-1&#160;&#160;</td>
			<td>DAKO</td>
			<td>&#160;M7240</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated goat- anti-mouse IgG</td>
			<td>Vector Laboratories Inc.</td>
			<td>BA-9200</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain Elite ABC Kit</td>
			<td>Vector Laboratories Inc.</td>
			<td># PK-6100</td>
			<td></td>
		</tr>
		<tr>
			<td>FAST DAB Tablet Set.</td>
			<td>Sigma Biochemicals</td>
			<td># D4293</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#8217;s haemalaun solution</td>
			<td>Merck</td>
			<td>1,092,490,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti Histokitt</td>
			<td>Roth</td>
			<td>Art.Nr.6638.2</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bench top rotary microtome</td>
			<td>Thermo Electron, Shandon Finesse ME+</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue embedding station</td>
			<td>Leica, TP1020</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg-Incubator</td>
			<td>Grumbach&#160;</td>
			<td>BSS160</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo fluorescence microscope equipped with an connected with a digital camera (Olympus E410) and flexible cold light&#160;</td>
			<td>Olympus, SZX10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Turrax&#160;</td>
			<td>IKA T10</td>
			<td></td>
			<td>Homogenizer</td>
		</tr>
	</tbody>
</table>

establishment of a human multiple myeloma xenograft model in the chicken to study tumor growth, invasion and angiogenesis

Le myélome multiple (MM), une maladie des cellules plasmatiques malignes, reste incurable et de nouveaux médicaments sont nécessaires pour améliorer le pronostic des patients. En raison de l&#39;absence du microenvironnement osseux et des facteurs de croissance auto / paracrine des cellules MM humaines sont difficiles à cultiver. Par conséquent, il ya un besoin urgent d&#39;établir une bonne in vitro et dans les systèmes de culture in vivo pour étudier l&#39;action de nouveaux produits thérapeutiques sur les cellules de MM humaines. Ici, nous présentons un modèle à cultiver des cellules du myélome multiple humain dans un environnement 3D complexe in vitro et in vivo. Des lignées cellulaires MM OPM-2 et le milieu RPMI-8226 ont été transfectées pour exprimer le transgène GFP et ont été cultivées en présence de cellules mésenchymateuses humaines et de collagène de type I Matrice sphéroïdes tridimensionnels. En outre, les sphéroïdes ont été greffés sur la membrane chorio-allantoïde (CAM) d&#39;embryons de poulet et la croissance tumorale a été contrôlée par microscopie à fluorescence stéréo. Les deux modèles permettent l&#39;étude de roman dru thérapeutiquegs dans un environnement 3D complexe et la quantification de la masse des cellules tumorales après des greffes homogénéisation dans un transgène GFP spécifique au test ELISA. En outre, les réponses angiogéniques de l&#39;hôte et l&#39;invasion des cellules tumorales dans le tissu de l&#39;hôte sous-jacent peuvent être surveillées quotidiennement par un microscope stéréoscopique et analysés par coloration immunohistochimique à l&#39;encontre des cellules humaines tumorales (Ki-67, CD138, Vimentin) ou des cellules murales hôtes couvrant vaisseaux sanguins (desmine / ASMA). En conclusion, le système onplant permet d&#39;étudier la croissance des cellules MM et l&#39;angiogenèse dans un environnement 3D complexe et permet le criblage de composés thérapeutiques ciblant la survie et la prolifération des cellules MM.

L&#39;analyse in vitro des composés cibles en 3D myélome multiple dosages de sphéroïdes En raison de la limitation de la culture de cellules primaires de MM humaines in vitro, nous avons établi les nouveaux modèles de culture in vitro en 3D pour les lignées cellulaires MM humaines faisant usage d&#39;une matrice de croissance extracellulaire et les cellules de soutien mésenchymateuses humaines primaires de la moelle osseuse (Figure ...

Watch this Scientific Journal Video about Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis at JoVE.com

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Les cellules humaines myélome multiple (MM) nécessitent le micro-environnement favorable des cellules mésenchymateuses et des composants de la matrice extracellulaire pour la survie et la prolifération. Nous avons établi un modèle in vivo d&#39;embryons de poulet avec des cellules de myélome et mésenchymateuses humaines greffées à étudier les effets des médicaments contre le cancer sur la croissance tumorale, l&#39;invasion et l&#39;angiogenèse.

Mise en place d&#39;un modèle de xénogreffe humain de myélome multiple dans le poulet pour étudier la croissance tumorale, l&#39;invasion et l&#39;angiogenèse

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

establishment-human-multiple-myeloma-xenograft-model-chicken-to-study

Research

JoVE Journal

Medicine

12.9K vues.  Innsbruck Medical University.  Les cellules humaines myélome multiple (MM) nécessitent le micro-environnement favorable des cellules mésenchymateuses et des composants de la matrice extracellulaire pour la survie et la prolifération. Nous avons établi un modèle in vivo d'embryons de poulet avec des cellules de myélome et mésenchymateuses humaines greffées à étudier les effets des médicaments contre le cancer sur la croissance tumorale, l'invasion et l'angiogenèse. 

Vidéo: Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Une In Vitro Système d&#39;étude dormance tumorale et le commutateur à la croissance métastatique

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Recherche

Médecine

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-photon imagerie de l&#39;invasion des cellules tumorales dans un modèle de souris orthotopique du carcinome épidermoïde oral

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

Un modèle murin de l&#39;Assay Pocket cornée pour l&#39;étude de l&#39;angiogenèse

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

Human artère mammaire interne (IMA) à la transplantation et stenting: un modèle humain pour étudier le développement de la resténose intra-stent

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

L&#39;homme neuroendocriniennes lignées cellulaires tumorales comme un modèle tridimensionnel pour l&#39;étude de la thérapie des tumeurs neuro-endocrines humaines

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel haute résolution<em&gt; In vivo</em&gt; Technique d&#39;imagerie pour étudier la réponse dynamique des structures intracrâniennes à la croissance tumorale et de thérapeutique

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Le<em&gt; En ovo</em&gt; CAM-test comme un Xenograftmodel pour le sarcome

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Décollement de la rétine modèle chez les rongeurs par injection sous-rétinienne de hyaluronate de sodium

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Une modification<em&gt; In vitro</em&gt; Invasion Test pour déterminer le rôle potentiel des hormones, des cytokines et / ou facteurs de croissance dans le cancer Médiation invasion cellulaire

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.