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).

실험 동물 복지의 hatching.The NIH 사무실이이 지역 (HTTP 작성 지침을 제공 할 때까지 오스트리아 법, 미국 공중 보건 서비스 조류 배아의 실험 동물 복지 사무소에 따르면 라이브 척추 동물로 간주되지 않습니다 // www.grants.nih.gov/grants/olaw/references/ilar91.htm 및 NIH 출판 번호 : 06-4515). 1. 세포 배양 및 렌티 바이러스 형질 <ol><li> 문화 MM 세포주 OPM-2, RPMI-8226 및 RPMI1640 배지에서 골수로부터 인간 중간 ?...

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nonobese+diabetic/severe+combined+immunodeficient+(NOD/SCID)+mouse+model+of+childhood+acute+lymphoblastic+leukemia+reveals+intrinsic+differences+in+biologic+characteristics+at+diagnosis+and+relapse.">The nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of childhood acute lymphoblastic leukemia reveals intrinsic differences in biologic characteristics at diagnosis and relapse.</a> Blood. 99 (11), 4100-4108 (2002).</li><li>Feuring-Buske, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+engraftment+of+human+acute+myeloid+leukemia+progenitor+cells+in+beta+2-microglobulin-deficient+NOD/SCID+mice+and+in+NOD/SCID+mice+transgenic+for+human+growth+factors.">Improved engraftment of human acute myeloid leukemia progenitor cells in beta 2-microglobulin-deficient NOD/SCID mice and in NOD/SCID mice transgenic for human growth factors.</a> Leukemia. 17 (4), 760-763 (2003).</li><li>Pearce, D. 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내화성 MM에 대한 새로운 치료제의 개발은 약물에 대한 인간 MM 세포 민감도를 평가하는 데 시간이 덜 소요되고 비용이 생체 시스템을 필요로한다. 지금까지, 단 몇 생체 시스템은 새로운 항 골수종 치료의 임상 평가에 사용할 수 있습니다. 그들 모두는 화합물 라이브러리 (29)의 대규모 스크리닝에 대한 자신의 한계를 가지고있다. 인간의 MM 셀에 대한 가장 좋은 현재의 모...

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

다발성 골수종 (MM), 악성 형질 세포 질환, 난치성 유지 및 신규 약물은 환자의 예후를 개선하기 위해 요구된다. 인해 골 미세 자동 / 분비 성장 인자의 부족으로 인간 MM 세포는 배양하기 어렵다. 따라서, 인간의 MM 세포에 새로운 치료제의 작용을 연구하기 위해 시험 관내 및 생체 내 배양 시스템에서 적절한 확립이 절실히 요구되고있다. 여기서 우리는 시험 관내 및 생체 내에서 복잡한 3D 환경에서 인간 다발성 골수종 세포를 성장 모델을 제시한다. MM 세포주 OPM-2 및 RPMI-8226를 형질 전환 유전자 GFP를 발현하는 형질 감염시키고 인간 중간 엽 세포와 콜라겐의 존재 하에서 배양 하였다 타입 I 입체 타원체로 매트릭스합니다. 더하여, 구 상체는 닭 배아 융모 막 (CAM)에 그래프트 된 종양 성장은 스테레오 형광 현미경에 의해 모니터 하였다. 두 모델 모두 새로운 치료 DRU의 연구를 허용복잡한 3D 환경 및 트랜스 특정 GFP-ELISA에서의 균질화 이식 후 종양 세포 질량의 정량화에 GS. 또한, 직하 숙주 조직으로 호스트 및 종양 세포의 침윤 혈관 신생 반응을 입체 현미경으로 매일 모니터링 및 인간 종양 세포 (기-67, CD138, 멘틴) 또는 호스트 벽화 세포 덮는 혈관에 대해 면역 조직 화학 염색에 의해 분석 될 수있다 (최근 desmin / ASMA). 결론적 onplant 시스템은 복잡한 3 차원 환경에서 MM 세포 성장 및 혈관 신생을 연구 할 수 있으며 MM 세포의 생존과 증식을 치료 표적 신규 화합물에 대한 스크리닝을 가능하게한다.

3D 다발성 골수종 타원체 내 분석 대상 화합물의 시험 관내 분석에서 때문에 체외에서 일차 인간 MM 세포 배양의 한계로는 골수에서 세포 성장 매트릭스 및지지 차 인간 중간 엽 세포 이용하게 인간 MM 세포 라인에 대한 새로운 3D 체외 배양 모델을 확립 (도 1a를, B). EGFP 유전자 MM 셀 라인은 회전 타원체의 3D 성장 후 시각화 및 MM 종?...

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

인간 다발성 골수종 (MM) 세포는 간엽 세포의 생존과 증식을위한 세포 외 매트릭스 성분의지지 미세 환경을 필요로한다. 우리는 종양의 성장, 침윤 및 혈관 신생에 항암제의 효과를 연구하기 위해 접목 인간 골수종 및 중간 엽 세포와 생체 내 닭 배아 모델을 설립했다.

닭에서 인간의 다발성 골수종 이종 이식 모델의 설립은 종양의 성장, 침략과 혈관 신생을 공부하기

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

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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 ...

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

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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 ...

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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

소설 고해상도<em&gt; 생체 내</em종양의 성장과 치료학에 두개 내 구조물의 동적 응답을 공부하기&gt; 이미징 기술

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

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<em&gt; OVO에서</em육종을위한 Xenograftmodel으로&gt; CAM-분석

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

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수정<em&gt; 시험관</em&gt; 침공 분석은 호르몬, 중재 암 세포 침략의 사이토 카인 및 / 또는 성장 인자의 잠재적 인 역할을 확인하는

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.

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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

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

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

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The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...