Name: establishment of a human multiple myeloma xenograft model in the chicken to study tumor growth, invasion and angiogenesis
Uploaded: 2015-05-01T13:00:00
Description: 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

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).

وفقا للقانون النمساوي، ومكتب مختبر رعاية الحيوان في خدمة الصحة العامة أجنة الطيور الولايات المتحدة لا تعتبر الحيوانات الفقارية الحية حتى قدم مكتب NIH hatching.The مختبر رعاية الحيوان الإرشادات المكتوبة في هذا المجال (HTTP: // www.grants.nih.gov/grants/olaw/references/ilar91.htm وNIH النشر رقم: 06-4515). <p class="jove_title...

<ol>
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تطوير عوامل علاجية جديدة لصهر MM تتطلب أقل استهلاكا للوقت ومكلفة نظم في الجسم الحي لتقييم حساسية الخلايا MM الإنسان للمخدرات. حتى الآن، تتوفر لتقييم ما قبل السريرية من العلاجات المضادة للالمايلوما الجديدة سوى عدد قليل من النظم في الجسم الحي. كل منهم حدودها لفحص نطاق و?...

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

المايلوما المتعددة (MM)، وهو مرض الخلية البلازما خبيث، لا يزال غير قابل للشفاء ويطلب أدوية جديدة لتحسين تشخيص المرضى. ونظرا لعدم وجود المكروية العظام وعوامل النمو لصناعة السيارات / نظير الصماوي الخلايا MM الإنسان يصعب زراعتها. ولذلك، هناك حاجة ملحة لإنشاء السليم في التجارب المختبرية ونظم الاستزراع الجسم الحي لدراسة عمل علاجات جديدة على الخلايا MM الإنسان. هنا نقدم نموذجا لزراعة خلايا المايلوما المتعددة الإنسان في بيئة 3D المعقدة في المختبر والمجراة. و transfected خطوط الخلايا MM OPM-2 وRPMI-8226 للتعبير عن GFP التحوير وزرعها في وجود خلايا اللحمة المتوسطة الإنسان والكولاجين النوع الأول المصفوفة كما الكروية ثلاثية الأبعاد. بالإضافة إلى ذلك، والمطعمة الكروية على غشاء مشيمائي (CAM) من أجنة الدجاج ومراقبة نمو الورم عن طريق مضان ستيريو المجهر. كلا النموذجين تسمح دراسة درو علاجية جديدةع في بيئة 3D المعقدة وتقدير من كتلة الخلايا السرطانية بعد التجانس الطعوم في التحوير محددة GFP-ELISA. وعلاوة على ذلك، يمكن رصد ردود عائية المضيف وغزو الخلايا السرطانية في الأنسجة المضيف تحتاني يوميا المجهر ستيريو وتحليلها بواسطة تلطيخ المناعى ضد الخلايا البشرية الورم (كي-67، CD138، Vimentin) أو الخلايا الجدارية المضيفة التي تغطي الأوعية الدموية (desmin / ASMA). في الختام، ونظام onplant يسمح دراسة MM نمو الخلايا والأوعية الدموية في بيئة 3D معقدة ويمكن الكشف عن مركبات علاجية جديدة تستهدف بقاء وتكاثر الخلايا MM.

في تحليل المختبر من المركبات المستهدفة في 3D المايلوما المتعددة المقايسات كروي يرجع ذلك إلى الحد من زراعة الخلايا MM الإنسان الأساسية في المختبر أنشأنا نماذج جديدة 3D الثقافة في المختبر لخطوط الخلايا MM...

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

المايلوما المتعددة (MM) الخلايا البشرية تتطلب المكروية داعمة للخلايا اللحمة المتوسطة ومكونات المصفوفة خارج الخلية من أجل البقاء والانتشار. أنشأنا في الجسم الحي الدجاج نموذج جنين مع خلايا الورم النقوي والوسيطة الإنسان المغروسة لدراسة آثار عقاقير مضادة للسرطان على نمو الورم والغزو والأوعية الدموية.

إنشاء طعم أجنبي نموذج المايلوما المتعددة الإنسان في الدجاج لدراسة نمو الورم والغزو والأوعية الدموية

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

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Medicine

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

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

و في المختبر النظام الدراسي لاستئصال ورم من الخمول وتبديل للنمو النقيلي

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 ...

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

متعدد الفوتون التصوير من غزو خلايا الورم في الفأر نموذج مثلي الفم سرطان الخلايا الحرشفية

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

نموذج الفأر من القرنية الفحص جيب للدراسة الأوعية الدموية

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

الإنسان الخلية السرطانية الهرموني العصبي خطوط بمثابة نموذج ثلاثي الأبعاد لدراسة الإنسان علاج ورم الغدد الصم العصبية

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

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

رواية عالية الدقة<em&gt; في الجسم الحي</em&gt; تقنية التصوير لدراسة الاستجابة الديناميكية للمنشآت داخل القحف لنمو الورم والتداوي

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

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

ال<em&gt; في البيضة</em&gt; CAM-مقايسة باعتبارها Xenograftmodel عن الورم اللحمي

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

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

والتعديل<em&gt; في المختبر</em&gt; الغزو الفحص لتحديد الدور المحتمل للالهرمونات، السيتوكينات و / أو عوامل النمو في التوسط السرطان غزو الخلايا

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.

حقن مثلي من خلايا سرطان الثدي في الثديية الدهون وسادة من الفئران لدراسة نمو الأورام.

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

Three-Dimensional 3D Tumor Spheroid Invasion Assay

ثلاثي الأبعاد (3D) ورم الجسم الشبه الكروي الغزو الفحص

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