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).

Secondo la legge austriaca, e l&#39;Ufficio di animali da laboratorio benessere del servizio sanitario pubblico embrioni aviaria degli Stati Uniti non sono considerati animali vertebrati vivi fino hatching.The NIH Office of Laboratory Animal Welfare ha fornito indicazioni scritte in questo settore (http: // www.grants.nih.gov/grants/olaw/references/ilar91.htm e NIH Pubblicazione n .: 06-4515). 1. Colture Cellulari e lentivirali trasfezione <ol><li> Linee Cultura MM cellule OPM-2, RPMI-8226 e cellule staminali mesenchimali umane da midollo osseo in mezzo RPMI1640, integrato con 10% di siero fetale bovino vitello e 100 UI / ml di penicillina, 100 mg / ml di streptomicina e glutammina 2 mM in presenza 5% di CO 2 a 37 ° C. </li><li> Trasfettare 5 x 10 6 cellule HEK 293FT con mix virale imballaggio (9 mg) DNA e 3 mg pLenti6 / V5 dest eGFP vettore per l&#39;uso di 30 microlitri di reagente liposomiale trasfezione e medie trasfezione (10 ml). Rimuovere medio trasfezione dopo 12 ore eaggiungere 10 ml DMEM con 10% di siero di vitello e 1% di aminoacidi non essenziali (NEAA). </li><li> Dopo 5 giorni, raccogliere surnatanti di cellule HEK293FT dopo centrifugazione di cellule nuoto (1.000 XG, 5min). Determinare titolo virale mediante real time PCR come descritto altrove 28. </li><li> Trasfettare 1 x 10 6 cellule di MM con particelle lentivirali eGFP (1 x 10 5 particelle) in un piatto 24 bene in mezzo di crescita completa. Dopo 3 giorni, iniziare il processo di selezione aggiungendo 2 ug / ml blasticidin al mezzo di coltura. Per il eGFP lentivirus disponibile in commercio, usare 500 ug / ml di neomicina. </li><li> Dopo 2 settimane di selezione, grappoli di eGFP esprimono cellule di MM apparirà; raccogliere le cellule per centrifugazione (1.000 xg, 5 min) e di espandersi come OPM-2 eGFP e RPMI-8226 sottolinee eGFP per esperimenti (sezione 2 e 3). </li></ol> 2. 3D Multiplo modello mieloma Spheroid <ol><li> Tipo I Freddo collagene soluzione e 10 x DMEM on ghiaccio. </li><li> Mescolare 1/10 Volume di 10 x DMEM medio in matrice di collagene; aggiungere NaOH (0,2 N) per neutralizzare la soluzione acida di collagene ad un valore pH di 7,4; immagazzinare il collagene / soluzione a medio sul ghiaccio. </li><li> Mescolare linee cellulari di MM transgeniche (OPM-2 eGFP o RPMI-8226 eGFP; 250.000 per sferoide,) con cellule umane mesenchimali (50.000 cellule / sferoide, vale a dire, il 30 microlitri drop). </li><li> Miscela di cellule Centrifuga in provette da 15 ml (1000 xg, 5min), aggiungere freddo miscela di collagene preparato (1 ml) al pellet e mescolare bene (con 1.000 punta microlitri). </li><li> Subito pipetta 30 microlitri della miscela di collagene / cella (con 100 microlitri punta) in una piastra a 24 pozzetti su parafilm sterile e lasciare miscela di cellule / collagene polimerizzare per 30 minuti a 37 ° C (vedere Figura 1A). </li><li> Sferoidi MM sovrapposizione con mezzo di coltura contenente 1 ml 1, 10 e 100 nM bortezomib (vedi Figura 1B). </li><li> Dopo 72 h di incubazione a 37 ° C, sferoidi documento dafluorescenza stereomicroscopia (vedi Figura 1C). </li><li> Trasferire ogni spheroid dall&#39;uso di pinze con ganasce piatte larghe in un tubo di reazione per la misura di GFP (vedere Figura 1D). </li></ol> 3. 3D mieloma multiplo Xenograft modello in CAM <ol><li> Incubare uova di gallina in un incubatore speciale per uova di volatili a 37 ° C e 70% di umidità per tre giorni. </li><li> Successivamente, uova aperte e trasferimento embrioni sterilizzati con barche etanolo, quadrato, di 10 cm in plastica di peso con la cultura di cellule coperchio piatto e incubare &quot;ex ovo&quot; per altri sei giorni, in modo che la CAM è in grado di sviluppare (vedere Figura 2A). </li><li> Tipo I freddo collagene soluzione e 10 x DMEM su ghiaccio, mescolare 1/10 volume di 10 x terreno DMEM nella matrice di collagene, Aggiungere NaOH (0,2 N) per neutralizzare la soluzione acida di collagene ad un valore pH di 7,4; immagazzinare il collagene / soluzione a medio sul ghiaccio. </li><li> Mescolare linee cellulari di MM transgenici (OPM-2 o eGFPRPMI-8226 eGFP; 250.000 per sferoide,) con cellule mesenchimali umane (50.000 cellule / sferoide). </li><li> Centrifugare le cellule in 15 ml provette (1.000 XG, 5min, per ciascun composto di prova 1 fiala), aggiungere 1 ml di freddo miscela di collagene preparata con la droga (a concentrazione di lavoro desiderato) al pellet e mescolare bene (con 1.000 punta microlitri). </li><li> Inserire collagene gocce (30 ml ciascuno) su parafilm in 6 ben pate per 30 min per permettere la polimerizzazione della matrice extracellulare a 37 ° C. </li><li> Transfer &quot;onplants&quot; dal punto 3.6 con l&#39;uso del forcipe alla superficie trattata del CAM (2 cm da embrione) di 9 giorni di embrioni di pollo (4 onplants per ogni embrione di pollo, si veda la Figura 2B) </li><li> Dopo 5 giorni di crescita in vivo in un incubatore uovo alla 37 ° C e 70% di umidità, xenotrapianti documento per fluorescenza stereomicroscopia (vedere la Figura 2C). Euthanize embrioni di pollo da ipotermia a 4 ° C in frigorifero per 5h. </li><li> Rimuovere xenotrapianti con tessuto CAM soggiacente da una forbice oftalmica e pinze con ganasce piatte larghe. Usali per la misura di GFP (sezione 4, vedi Figura 2D) o per l&#39;analisi immunoistochimica dei vasi sanguigni e / o cellule tumorali invadono (sezione 5). </li></ol> 4. Quantificazione di eGFP proteine ​​mediante ELISA <ol><li> Trasferire ogni sferoide MM o xenotrapianto asportato in 0,5 ml RIPA buffer contenente 200 mcg / ml inibitori della proteasi. </li><li> Omogeneizzare sferoide / xenotrapianto con un omogeneizzatore tessuto sul ghiaccio. </li><li> Eseguire tre congelamento / scongelamento-cicli in azoto liquido e 37 ° C bagnomaria. </li><li> Centrifugare omogenato a 4 ° C per 20 min (12,000 g) e memorizzare supernatanti. </li><li> Diluire i campioni 1:20 in tampone (200 microlitri) del kit ELISA. Misurare GFP livelli da un GFP commerciale ELISA Kit utilizzando anticorpi biotinilati anti-GFP, secondo il protocollo del produttore. </li></ol> 5. ImmunohiAnalisi stochemical dei vasi sanguigni e invadere le cellule tumorali <ol><li> Fissare xenotrapianti asportati con area CAM in paraformaldeide al 4% O / N a 4 ° C. </li><li> Inserire xenotrapianti fissi in cassette incorporamento e trasferirli in una stazione embedding tessuto con una serie alcool crescente graduata (50%, 70%, 80%, 95% di etanolo, xilolo e paraffina; ogni passo 60 min). </li><li> Xenotrapianti Sezione (5 micron) mediante l&#39;uso di un microtomo rotativo da banco. Cuocere sezioni di paraffina sui vetrini O / N a 56 ° C. </li><li> Deparaffinare le sezioni di una serie graduata alcool riduzione acqua bidistillata (xilolo, 95%, 80%, 70%, 50% di etanolo, acqua bidistillata; ciascun passo 10 min). </li><li> Eseguire recupero dell&#39;antigene in un bagno d&#39;acqua (95 ° C, 20 min) con una soluzione di recupero dell&#39;antigene (tampone citrate-; pH 7,0; volume di 100 microlitri). </li><li> Bloccare l&#39;attività perossidasica endogena con 100 microlitri 3% H 2 O 2 / metanolo per 30 min. </li><li> Sezioni di blocco in PBS contenenteSiero bovino fetale al 10% per 45 min (volume 100 ml). </li><li> Stain per 1 ora con 100 microlitri di anticorpo primario (1 ug / ml) diluito in PBS contenente siero fetale di vitello 1% a RT. </li><li> Dopo aver lavato 3 volte in PBS, incubare per 1 ora con l&#39;anticorpo secondario biotinilato (0,1 mg / ml) in PBS contenente siero fetale di vitello 1% a RT. </li><li> Dopo aver lavato 3 volte in PBS effettuare reazione colorata dalla soluzione avidina / biotina-complesso (ABC) e la diaminobenzidina (DAB) substrato secondo le istruzioni del produttore. </li><li> 5.11. Smettere di reazione trasferendo sezioni doppio acqua distillata, di contrasto con ematossilina e montare le sezioni con un mezzo di montaggio sintetico. </li></ol>

<ol>
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Lo sviluppo di nuovi agenti terapeutici per refrattario MM richiede meno tempo e costosi sistemi in vivo per valutare la sensibilità di cellule MM umane ai farmaci. Finora, solo pochi sistemi in vivo sono a disposizione per la valutazione preclinica di nuove terapie anti-mieloma. Tutti loro hanno i loro limiti per lo screening su larga scala di librerie di composti 29. I migliori modelli attuali di cellule di MM umane sono altamente immuni topi con deficit 7,13,30 ed embrioni di tacch...

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

Il mieloma multiplo (MM), una malattia delle cellule del plasma maligne, resta incurabile e nuovi farmaci sono necessari per migliorare la prognosi dei pazienti. A causa della mancanza del microambiente dell&#39;osso e fattori di crescita auto / paracrino cellule MM umane sono difficili da coltivare. Pertanto, non vi è un urgente bisogno di stabilire una corretta in vitro e in sistemi di coltura in vivo per studiare l&#39;azione di nuove terapie sulle cellule umane di MM. Presentiamo qui un modello di crescere cellule di mieloma multiplo umano in un ambiente 3D complesso in vitro e in vivo. Linee cellulari di MM OPM-2 e RPMI-8226 sono state trasfettate per esprimere la GFP transgene e sono stati coltivati ​​in presenza di cellule mesenchimali umane e collagene di tipo I matrice come sferoidi tridimensionali. Inoltre, sferoidi sono stati innestati sulla membrana corioallantoidea (CAM) di embrioni di pollo e la crescita del tumore è stata monitorata mediante microscopia a fluorescenza stereo. Entrambi i modelli consentono lo studio del romanzo dru terapeuticogs in un ambiente 3D complesso e la quantificazione della massa tumorale dopo omogeneizzazione di innesti in un GFP-ELISA specifico transgene. Inoltre, le risposte angiogenici dell&#39;ospite e l&#39;invasione delle cellule tumorali nel tessuto ospite soggiacente possono essere monitorate quotidianamente da un microscopio stereo e analizzati mediante immunoistochimica contro le cellule tumorali umane (Ki-67, CD138, Vimentin) o cellule murali ospiti che coprono i vasi sanguigni (desmin / ASMA). In conclusione, il sistema permette onplant studiare crescita cellulare MM e l&#39;angiogenesi in un ambiente 3D complesso e permette lo screening per composti terapeutici mirati sopravvivenza e la proliferazione delle cellule di MM.

L&#39;analisi in vitro dei composti target in 3D mieloma multiplo test spheroid A causa delle limitazioni di coltura di cellule primarie umane di MM in vitro abbiamo stabilito nuovi modelli di coltura in vitro 3D per linee cellulari MM umane che fanno uso di una matrice extracellulare crescita e cellule mesenchimali umane primarie di supporto dal midollo osseo (Figura 1A, B). Linee cellulari di MM transgenici EGFP consentono la vis...

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

Mieloma multiplo (MM), le cellule umane richiedono il microambiente favorevole cellule mesenchimali e componenti della matrice extracellulare per la sopravvivenza e la proliferazione. Abbiamo stabilito un vivo pollo modello embrione con cellule di mieloma e mesenchimali umane trapiantate per studiare gli effetti dei farmaci anti-tumorali sulla crescita del tumore, l&#39;invasione e l&#39;angiogenesi.

Istituzione di un mieloma multiplo umano Xenograft modello nel pollo per studiare la crescita del tumore, invasione e l&#39;angiogenesi

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

13.0K Views.  Innsbruck Medical University.  Mieloma multiplo (MM), le cellule umane richiedono il microambiente favorevole cellule mesenchimali e componenti della matrice extracellulare per la sopravvivenza e la proliferazione. Abbiamo stabilito un vivo pollo modello embrione con cellule di mieloma e mesenchimali umane trapiantate per studiare gli effetti dei farmaci anti-tumorali sulla crescita del tumore, l'invasione e l'angiogenesi. 

Video: 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.

Un In Vitro Sistema per studio dormienza tumorale e l&#39;interruttore di crescita metastatica

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 ...

Ricerca

Medicina

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-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

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 modello murino del saggio Cornea Pocket Study Angiogenesi

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.

Umano arteria mammaria interna (IMA) Trapianti e Stenting: un modello umano per studiare lo sviluppo della ristenosi in-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.

Neuroendocrini umani cella linee tumorali come un modello tridimensionale per lo studio dei diritti dell&#39;uomo e terapia dei tumori neuroendocrini

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.

Un romanzo ad alta risoluzione<em&gt; In vivo</em&gt; Tecnica di imaging per studiare la risposta dinamica di strutture intracraniche alla crescita del tumore e Terapeutica

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

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.

Distacco della retina Modello in Roditori da iniezione sottoretinico di ialuronato di sodio

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.

A Modified<em&gt; In vitro</em&gt; Invasion test per determinare il ruolo potenziale di ormoni, citochine e / o fattori di crescita nella mediazione Cancer Invasion cellulare

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.

Iniezione ortotopico di cellule di carcinoma mammario nel mammaria Fat Pad di Mice per studiare la crescita del tumore.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensionale (3D) Tumore Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...