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).

根据奥地利法律，美国公共卫生服务禽流胚胎实验室动物福利办公室不被视为活的脊椎动物，直到实验动物福利hatching.The NIH办公室在这方面（HTTP提供了书面指导:// www.grants.nih.gov/grants/olaw/references/ilar91.htm和NIH出版号:06-4515）。 1.细胞培养和慢病毒转染<ol><li>培养的MM细胞系OPM-2，RPMI-8226，并从骨髓在RPMI1640培养基中的人类间质干细胞，补充有在存在10％牛胎儿小牛血清和100IU / ml青霉素，100微克/毫升链霉素和2mM谷氨酰胺的5％的CO 2在37℃。 </li><li>转染5×10 6的HEK 293FT细胞病毒包装结构（9微克）的DNA和3微克pLenti6 / V5 dest中eGFP的向量通过使用30微升脂质体转染试剂转染培养基（10毫升）中。 12小时后取出转染中，加入10 mL的DMEM培养基含10％小牛血清和1％非必需氨基酸（NEAA）。 </li><li> 5天后，游泳细胞（1000 XG，5分钟），离心后收集HEK293FT细胞的上清液。通过实时PCR测定病毒滴度为28别处描述。 </li><li>转染1×10 6个MM细胞与eGFP的慢病毒颗粒（1×10 5个颗粒）在24孔板中的完全生长培养基。 3天后，加入2微克/毫升瘟到培养基开始选择过程。对于市售的eGFP慢病毒，使用500微克/ ml新霉素的。 </li><li> 2周选择后，eGFP的簇表达MM细胞会出现;通过离心（1,000×g离心，5分钟）收集细胞，并扩大其作为OPM-2 的eGFP和RPMI-8226 eGFP的亚系进行实验（第2和3）。 </li></ol> 2. 3D-多发性骨髓瘤球体模型<ol><li>寒意胶原I型解决方案和10×DMEMØñ冰。 </li><li>混合1/10体积10​​×DMEM培养基进入胶原基质;添加NaOH水溶液（0.2 N），以中和酸性的胶原溶液以7.4的pH值;店胶原/冰介质溶液。 </li><li>转基因混合MM细胞系（OPM-2或的eGFP RPMI-8226 绿色荧光蛋白 ; 250,000球体，）与人类间质细胞（50,000个细胞/球体， 即 30微升滴）。 </li><li>在15ml试管（1000×g离心，5分钟）离心细胞混合物，冷制备胶原混合物（1mL）溶液加入到细胞沉淀拌匀（1,000微升尖）。 </li><li>立即吸取30微升胶原/细胞混合物（用100μl尖端）在一个24孔板上无菌石蜡薄膜，并允许细胞/胶原混合物在37℃（ 见图1A）聚合30分钟。 </li><li>覆盖的MM球体用含有1,10和100nM的硼替佐米1mL中培养基（参见图1B）。 </li><li>之后，在37℃72小时温育的，文件球状体由荧光立体显微镜（ 见图1C）。 </li><li>通过使用镊子具有宽扁钳口在反应管转移的每个球体的GFP的测量（ 见图1D）。 </li></ol> 3. 3D多发性骨髓瘤异种移植模型中的CAM <ol><li>孵化鸡蛋中为鸟卵的特别培养箱中在37℃和70％湿度下三天。 </li><li>此后，打开蛋和转移胚胎灭菌用乙醇，方形，10厘米的塑料称量船与细胞培养板盖和孵化" 前卵 "用于进一步六天，使得CAM是能够开发（参见图2A）。 </li><li>寒意胶原I型溶液和10×DMEM在冰上，混合1/10体积10​​×DMEM培养基到胶原基质，添加氢氧化钠（0.2 N），以中和酸性的胶原溶液以7.4的pH值;店胶原/冰介质溶液。 </li><li>混合转基因MM细胞系（OPM-2 的eGFP或RPMI-8226 绿色荧光蛋白 ; 250,000球体，）与人类间质细胞（50,000个细胞/球体）。 </li><li>离心细胞在15ml试管（1000×g离心，5分钟;对于每个试验化合物1小瓶），药物添加1mL中冷制备胶原混合物（在所需的工作浓度）的细胞沉淀并充分混合（1,000微升尖）。 </li><li>放置胶原滴在6孔颈部30分钟上的封口膜（各30微升），以允许细胞外基质的聚合在37℃。 </li><li>转移"onplants"来自步骤3.6与使用镊子到CAM的未处理表面（2厘米的距离胚胎）的9日龄鸡胚（4- onplants每个鸡胚，参见图2B） </li><li>经过5天的体内生长在蛋孵化器在37℃和70％的湿度，文件异种移植物通过荧光立体显微镜（ 见图2C）。受低温的冰箱5小时安乐死鸡胚胎在4℃。 </li><li>由眼科剪刀取出移植与下层组织CAM和镊子与宽阔平坦的下颌。它们用于GFP的测量（第4节， 见图2D）或血管和/或侵入的肿瘤细胞（第5）免疫组化分析。 </li></ol>通过ELISA 4.绿色荧光蛋白定量的蛋白质<ol><li>传送每个MM球体或切除异种移植入0.5毫升RIPA缓冲液含有200微克/毫升蛋白酶抑制剂。 </li><li>均质球体/异种移植物在冰上组织匀浆器。 </li><li>执行三个冷冻/解冻周期在液氮和37℃水浴中。 </li><li>离心机匀浆在4℃下20分钟（12,000克）和存储上清液。 </li><li>稀释样品中的ELISA试剂盒的测定缓冲液（200微升）1:20。测量GFP的水平通过使用生物素化抗GFP抗体商业GFP ELISA试剂盒，根据制造商的协议。 </li></ol> 5. Immunohi对血管和入侵肿瘤细胞stochemical分析<ol><li>切除修复与移植CAM面积在4％多聚甲醛O / N在4℃。 </li><li>放置固定异种移植到嵌入盒，并将它们与增加的梯度醇系（50％，70％，80％，95％的乙醇，二甲苯和石蜡;每个步骤60分钟）转移至组织包埋站。 </li><li>部异种移植物（5微米）通过使用台式旋转切片机。烘烤在56℃下在载玻片O / N石蜡切片。 </li><li> Deparaffinize部分由递减梯度酒精串联双蒸水（混合二甲苯，95％，80％，70％，50％的乙醇，双蒸水;每个步骤10分钟）。 </li><li>在水浴（95℃，20分钟）与抗原修复液进行抗原修复（柠檬酸盐缓冲液; pH值7.0;体积100微升）。 </li><li>阻断内源性过氧化物酶活性用100μl3％的H 2 O 2 /甲醇30分钟。 </li><li>在含PBS块段10％胎牛血清45分钟（体积100微升）。 </li><li>染色1小时，用100μl初级抗体（1微克/毫升）稀释在含有1％胎牛血清在RT PBS中。 </li><li>洗涤3次在PBS中后，孵育1与生物素化第二抗体（0.1微克/毫升）在含有1％胎牛血清在RT PBS中。 </li><li>洗涤3次在PBS中后由抗生物素蛋白/生物素复合物（ABC）和二氨基联苯胺（DAB）底物溶液，根据制造商的说明进行显色反应。 </li><li> 5.11。停止反应通过转移部分，以双蒸水，染液用苏木精和安装部分用合成封固剂。 </li></ol>

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(85), (2014).</li><li>Schuler, J., Ewerth, D., Waldschmidt, J., Wasch, R., Engelhardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+multiple+myeloma:+a+critical+appraisal.">Preclinical models of multiple myeloma: a critical appraisal.</a> Expert.Opin.Biol.Ther. 13, S111-S123 (2013).</li><li>Kirshner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+three-dimensional+model+for+evaluating+the+impact+of+therapy+on+multiple+myeloma.">A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma.</a> Blood. 112 (7), 2935-2945 (2008).</li><li>Jilani, S. 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=Selective+binding+of+lectins+to+embryonic+chicken+vasculature.">Selective binding of lectins to embryonic chicken vasculature.</a> J.Histochem.Cytochem. 51 (5), 597-604 (2003).</li></ol>

作者有没有竞争经济利益

的新的治疗剂用于难治性MM的发展需要较少耗时且昂贵的体内系统评估人类MM细胞的对药物的敏感性。迄今为止，只有少数的体内系统可用于新的抗骨髓瘤治疗的临床前评价。他们都有自己的化合物库29的大规模筛选的限制。 目前最好的模型为人类MM细胞是高度免疫缺陷小鼠7,13,30和火鸡胚胎29。既SCID小鼠和禽流胚胎异种移植模型可用于研究的MM的生物学和?...

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><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&amp;nbsp;</td><td>PromoCell</td><td>PC-C-12974</td><td></td></tr><tr><td>HEK293FT cells&amp;nbsp;</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&amp;nbsp; 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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp; 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>SPF-white hen eggs</td><td>Charles River</td><td></td><td>Fertilized&amp;nbsp; white Leghorn&amp;nbsp; chicken eggs</td></tr><tr><td>Plastic weighing boats</td><td>neoLab</td><td>Art.Nr. 1-1125</td><td>for ex-ovo culture</td></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>Roche</td><td>11 836 170 001</td><td></td></tr><tr><td>Complete Mini EDTA-free</td><td></td><td></td><td></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&amp;nbsp; 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>SuperFrost Microscope Slides</td><td>R. Langenbrinck&amp;nbsp;</td><td>Art.-Nr.</td><td></td></tr><tr><td>Labor- u. Medizintechnik</td><td>03-0060</td><td></td><td></td></tr><tr><td>DakoCytomation Wash Buffer 10x</td><td>DakoCytomation</td><td>Code-Nr.</td><td></td></tr><tr><td>S 3006</td><td></td><td></td><td></td></tr><tr><td>Target Retrieval Solution (10x)&amp;nbsp; pH 6,1</td><td>DAKO</td><td>Code-Nr.</td><td></td></tr><tr><td>S 1699</td><td></td><td></td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</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>&amp;nbsp;M0760</td><td></td></tr><tr><td>m-a-hu Ki67&amp;nbsp; clone MIB-1&amp;nbsp;&amp;nbsp;</td><td>DAKO</td><td>&amp;nbsp;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&amp;rsquo;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>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&amp;nbsp;</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&amp;nbsp;</td><td>Olympus, SZX10</td><td></td><td></td></tr><tr><td>Ultra Turrax&amp;nbsp;</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

多发性骨髓瘤（MM），恶性浆细胞疾病，无法治愈的和新的药物都需要改善患者的预后。由于缺乏骨微环境和自动/旁分泌生长因子的人的MM细胞不易培养。因此，目前迫切需要建立适当的体外和体内培养系统来研究对人MM细胞新的治疗的作用。这里，我们提出一个模型，以在体外和体内生长的人类多发性骨髓瘤细胞在一个复杂的三维环境。 MM细胞系OPM-2和RPMI-8226被转染以表达该转基因GFP和培养在人类间质细胞和胶原的存在I型基质为三维球状体。此外，球状体接枝在鸡胚绒毛尿囊膜（CAM）和肿瘤生长通过立体声荧光显微镜监测。这两种型号允许新的治疗DRU研究GS在一个复杂的三维环境和肿瘤细胞块移植物在转基因特异性GFP-ELISA均化后进行量化处理。此外，主机和肿瘤细胞的侵袭进入下卧宿主组织的血管生成反应，可以每日通过立体显微镜监测和针对人肿瘤细胞（Ki-67的，CD138，波形蛋白）或宿主壁细胞覆盖血管免疫染色分析（结蛋白/ ASMA）。 总之，onplant系统允许研究在一个复杂的三维环境MM细胞生长和血管生成，使筛选靶向生存MM细胞的增殖和新的治疗的化合物。

在目标化合物的3D多发性骨髓瘤球体测定体外分析 由于在体外原代人MM细胞培养的限制，我们建立了新的三维体外培养模型为人类MM细胞系，利用细胞外生长基质和支持原代人骨髓间充质细胞从骨髓的（ 图1A，B）。 EGFP转基因MM细胞系允许可视化和MM肿瘤块的量化的三维生长球状体之后。两个MM细胞系OPM-2 的eGFP和RPMI-8...

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

人多发性骨髓瘤（MM）细胞需要间质细胞和细胞外基质成分为存活和增殖的支持性微环境。我们建立了与植入人类骨髓瘤和间质细胞体内鸡胚模型来研究癌症药物对肿瘤的生长，侵袭和血管生成作用。

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

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 次观看.  Innsbruck Medical University. 人多发性骨髓瘤（MM）细胞需要间质细胞和细胞外基质成分为存活和增殖的支持性微环境。我们建立了与植入人类骨髓瘤和间质细胞体内鸡胚模型来研究癌症药物对肿瘤的生长，侵袭和血管生成作用。 

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

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

癌症转移，使用禽流感胚胎模型的定量分析

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

科研

医学

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

癌症研究

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

比较在小鼠肾脏和鸡胆膜上建立的转移性透明细胞肾细胞癌模型

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

利用活体中鸡胆膜研究妇科和泌尿癌

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

微创建立小鼠原位膀胱移植瘤的

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

<meta charset="utf8"></head><body>骨肉瘤 PDX 小鼠模型的方案：研究生长和耐药性

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

<meta charset="utf8"></head><body>作者聚焦：在免疫缺陷小鼠中复制人骨肉瘤进展用于癌症研究

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

该<em&gt;卵内</em&gt;鸡胚尿囊膜（CAM）含量为肝癌的有效异种移植模型

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

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此视频中的章节

癌症转移，使用禽流感胚胎模型的定量分析

建立胃癌患者衍生异种移植模型和原细胞系

比较在小鼠肾脏和鸡胆膜上建立的转移性透明细胞肾细胞癌模型

利用活体中鸡胆膜研究妇科和泌尿癌

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

微创建立小鼠原位膀胱移植瘤的

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

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