Name: high-sensitivity detection of micrometastases generated by gfp lentivirus-transduced organoids cultured from a patient-derived colon tumor
Uploaded: 2018-06-14T13:30:00
Description: Watch this Scientific Journal Video about High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor at JoVE.com

这项工作得到了 Juntendo 大学青年调查员奖 (2013、2014和 2015) 对 Y.O.、联合项目奖 (2013 和 2014) k.m. 的支持, 并资助了教育部、文化部、体育、科学和技术, 日本 (16K15625 张佑启和16K15598 到 M.G.)。我们特别感谢肛肠外科和分子病理学部的所有成员进行有益的讨论和技术支持。我们还感谢博行 Konno 博士 (滨松大学 Schoolof 医学) 和北岛博士 (国际卫生和福利大学) 在对小鼠进行原位植入手术过程中给予慷慨的技术指导。吉孝河马 (千叶癌症中心) 为执行肿瘤 organoid 文化提供技术咨询。

该病人提供书面知情同意, 该项目被 Juntendo 大学医学院研究伦理学委员会批准。老鼠实验也被 Juntendo 医学院动物研究伦理学委员会批准。
1. Immunodeficient 小鼠 CRC PDXs 的建立
在图 1A中概述了建立 CRC PDXs (步骤 1) 的实验程序。
<ol><li>在患者手术切除肿瘤后立即准备主 CRC 组织。</li>
	<li>在50毫升锥形管中, 用30毫升的冰冷无菌磷酸盐缓冲...

<ol>
	<li>Siegel, R., Desantis, C., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorectal+cancer+statistics,+2014.">Colorectal cancer statistics, 2014.</a> CA Cancer J Clin. 64 (2), 104-117 (2014).</li><li>Hidalgo, 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=Patient-derived+xenograft+models:+an+emerging+platform+for+translational+cancer+research.">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer Discov. 4 (9), 998-1013 (2014).</li><li>Aparicio, S., Hidalgo, M., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+the+utility+of+patient-derived+xenograft+mouse+models.">Examining the utility of patient-derived xenograft mouse models.</a> Nat Rev Cancer. 15 (5), 311-316 (2015).</li><li>Puig, I., 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+personalized+preclinical+model+to+evaluate+the+metastatic+potential+of+patient-derived+colon+cancer+initiating+cells.">A personalized preclinical model to evaluate the metastatic potential of patient-derived colon cancer initiating cells.</a> Clin Cancer Res. 19 (24), 6787-6801 (2013).</li><li>van de Wetering, 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=Prospective+derivation+of+a+living+organoid+biobank+of+colorectal+cancer+patients.">Prospective derivation of a living organoid biobank of colorectal cancer patients.</a> Cell. 161 (4), 933-945 (2015).</li><li>Fujii, 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=A+Colorectal+tumor+organoid+library+demonstrates+progressive+loss+of+niche+factor+requirements+during+tumorigenesis.">A Colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis.</a> Cell Stem Cell. 18 (6), 827-838 (2016).</li><li>Fumagalli, A., 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=Genetic+dissection+of+colorectal+cancer+progression+by+orthotopic+transplantation+of+engineered+cancer+organoids.">Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids.</a> Proc Natl Acad Sci U S A. 114 (12), E2357-E2364 (2017).</li><li>O'Rourke, K. P., 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=Transplantation+of+engineered+organoids+enables+rapid+generation+of+metastatic+mouse+models+of+colorectal+cancer.">Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer.</a> Nat Biotechnol. 35 (6), 577-582 (2017).</li><li>Roper, 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=In+vivo+genome+editing+and+organoid+transplantation+models+of+colorectal+cancer+and+metastasis.">In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.</a> Nat Biotechnol. 35 (6), 569-576 (2017).</li><li>Kuo, T. H., 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=Liver+colonization+competence+governs+colon+cancer+metastasis.">Liver colonization competence governs colon cancer metastasis.</a> Proc Natl Acad Sci U S A. 92 (26), 12085-12089 (1995).</li><li>Onuma, K., 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=Genetic+reconstitution+of+tumorigenesis+in+primary+intestinal+cells.">Genetic reconstitution of tumorigenesis in primary intestinal cells.</a> Proc Natl Acad Sci U S A. 110 (27), 11127-11132 (2013).</li><li>Onder, T. T., 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=Loss+of+E-cadherin+promotes+metastasis+via+multiple+downstream+transcriptional+pathways.">Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways.</a> Cancer Res. 68 (10), 3645-3654 (2008).</li><li>Cespedes, M. V., 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=Orthotopic+microinjection+of+human+colon+cancer+cells+in+nude+mice+induces+tumor+foci+in+all+clinically+relevant+metastatic+sites.">Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites.</a> Am J Pathol. 170 (3), 1077-1085 (2007).</li><li>Fujii, E., 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=Characterization+of+EBV-related+lymphoproliferative+lesions+arising+in+donor+lymphocytes+of+transplanted+human+tumor+tissues+in+the+NOG+mouse.">Characterization of EBV-related lymphoproliferative lesions arising in donor lymphocytes of transplanted human tumor tissues in the NOG mouse.</a> Exp Anim. 63 (3), 289-296 (2014).</li></ol>

虽然 CRC PDX 模型已被广泛应用于研究原发性肿瘤的生长, 但该模型是否也适用于肿瘤转移的调查尚未充分阐明。自发性转移也几乎无法检测到的肝脏和肺部的各种报告结肠 PDX 模型4,10。为了检测高灵敏度的淋巴结, 我们开发了一种协议, 传感 GFP 慢病毒载体粒子进入 PDX 衍生 CRC organoids 之前, 他们的原位和静脉注射的受体小鼠。值得注意的是, 我们能够证明...

大肠癌 (CRC) 是1全球癌症死亡的第二大原因。对晚期疾病患者常规疗法的反应不足, 表明试图从根本上治疗 CRCs 是无效的。为了制定更有效的治疗方法, 建立了模拟 CRCs 特征的各种癌前小鼠模型。各种 CRC 细胞系因其方便易操作而被广泛用于产生肿瘤移植。然而, 癌症细胞系的长期培养往往会导致在特定的文化条件下, 在特殊的细胞群中有相当大的增殖, 从而导致临床前药物不可靠的结果和关键的限制。发展。
未经体外培养, 患者源性肿瘤移植 (PDXs) 也被植入到人类 CRC 组织的动物模型中, 经手术解剖的患者2,3,4。PDXs 被广泛认为是综述的主要病理学特征和基因改变最初存在于肿瘤的病人, 从他们的来源。此外, 在3D 条件下建立了由肿瘤细胞簇组成的患者源性肿瘤 organoids, 并对原发肿瘤5、6的生物学特性进行了严密的模拟。这些肿瘤 organoids 也被应用于高通量的药物筛选, 从而允许设计5的个性化疗法。然而, 明显的异种 CRC 种群被认为存在于肿瘤肿块中。特殊的 CRC 种群在体内和体外一系列的 PDX 和肿瘤 organoids 中有选择性地增殖和扩张。这也可能使受影响的 CRCs 的整体基因表达谱和免疫/遗传状态发生变化, 从而导致与父母 CRC 的最小相似性。
患者衍生的 CRC organoids 和从癌人结肠工程中提取的致癌突变的组合, 也被用来研究肿瘤侵袭转移的人肿瘤细胞的特征。6,7,8,9。然而, 由于患者源性 CRC 原位植入 immunodeficient 小鼠的自发性转移发生率极低, 因此难以研究侵袭转移级联的多步法过程, 包括局部入侵, intravasation, 运输在血液, 渗出和殖民化遥远的器官4,10。由患者衍生的 CRC organoids 形成的≤2毫米肿瘤细胞沉积所代表的微转移常常被忽略在实验性小鼠模型中受影响的远端器官切片的组织病理学分析中。自发淋巴结的可视化在体内也很少, 因为在注射到受体小鼠之前, 很难有效地将荧光标记引入肿瘤 organoid 细胞。在这项研究中, 我们开发了一个协议, 以有效地传感器 GFP 慢病毒北疆进入 PDX 衍生的 CRC organoid 细胞在3D 的文化注入受体小鼠之前, 并允许高度敏感的检测, 使用立体荧光显微镜, 其不同器官的定植形成淋巴结。

<table>
	<tbody>
		<tr>
			<td>NOD/Shi-scid IL2R&#947; null (NOG) mice</td>
			<td>The Central Institute for Experimental Animals,Kanagawa, Japan</td>
			<td></td>
			<td>Breed 6-week-old male mice under germ-free and specific pathogen-free conditions</td>
		</tr>
		<tr>
			<td>wound clips 
			2&#215;10mm</td>
			<td>Natsume manufacturing, Japan</td>
			<td>#C-21-S</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>Hamilton syringe 
			needle size:22 gauge</td>
			<td>Tokyo Science, Japan</td>
			<td></td>
			<td>Disinfect with 70% alcohol and sterile PBS.</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>BMBio</td>
			<td>#92006</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>BMBio</td>
			<td>#92412</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml conical tube</td>
			<td>Sumitono Bakelite</td>
			<td>MS-57150</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml conical tube</td>
			<td>Sumitomo Bakelite</td>
			<td>MS-57500</td>
			<td></td>
		</tr>
		<tr>
			<td>microtube</td>
			<td>Eppendorf</td>
			<td>#0030120086</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Erma</td>
			<td>#03-202-1</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m cell strainer</td>
			<td>Corning</td>
			<td>#352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel basement membrane matrix</td>
			<td>Corning</td>
			<td>#354234</td>
			<td>Store aliqupts at -20&#176;C. 
			Place on ice until use</td>
		</tr>
		<tr>
			<td>Collagenase type 1</td>
			<td>Sigma</td>
			<td>#C1030</td>
			<td>150 mg/ml collagenase type1 in 1&#215;PBS. Store aliqupts at -20&#176;C for up to 1 year</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovate Cell Technologies</td>
			<td>#5V2623A</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr>
			<td>DMEM/F-12 with GlutaMAX&#8482;</td>
			<td>Gibco</td>
			<td>#10565018</td>
			<td>Store at 4&#176;C. Warm at 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Cell banker 1plus</td>
			<td>ZENOAQ</td>
			<td>#628</td>
			<td>Store at 4&#176;C. Use within 1 month</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Gibco</td>
			<td>#15140122</td>
			<td>Store at 4&#176;C. Use within 1 month</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Gibco</td>
			<td>#15140122</td>
			<td>Store at 4&#176;C. Use within 1 month</td>
		</tr>
		<tr>
			<td>hEGF</td>
			<td>PEPROTECH</td>
			<td>#AF-100-15</td>
			<td>Store at -20&#176;C. Add to medium on same day as use</td>
		</tr>
		<tr>
			<td>Y27632, a ROCK inhibitor</td>
			<td>Wako</td>
			<td>#253-00591</td>
			<td>Store at -20&#176;C. Add to medium on 
			same day as use</td>
		</tr>
		<tr>
			<td>Culture medium</td>
			<td>Gibco</td>
			<td></td>
			<td>DMEM/F-12 with GlutaMAX&#8482; supplement supplemented with 5% FBS, 100 U/ml penicillin and 100 &#181;g/ml streptomycin. 
			Store at 4&#176;C. Use within 1 month.</td>
		</tr>
		<tr>
			<td>CRC organoid culture medium 
			with 1% or 5% FCS</td>
			<td></td>
			<td></td>
			<td>DMEM/F-12 with GlutaMAX&#8482; supplement (Gibco #10565018) supplemented with 1% or 5% FCS, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, 2 ng/ml hEGF and 10 &#181;M Y27632, a ROCK inhibitor. 
			Store at 4&#176;C. Use within 1 month.</td>
		</tr>
		<tr>
			<td>the FuGENE 6 transfection regent</td>
			<td>Roche</td>
			<td>11814 443001</td>
			<td></td>
		</tr>
		<tr>
			<td>Minisart 0.45 &#181;m filter</td>
			<td>Sartorius stedim</td>
			<td>17598-K</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml polypropylene centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>326819</td>
			<td></td>
		</tr>
		<tr>
			<td>PRRL-GFP vector</td>
			<td>Gift from Dr. Robert A. Weinberg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-VSV-G</td>
			<td>Gift from Dr. Robert A. Weinberg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-dR8.2 dvpr</td>
			<td>Gift from Dr. Robert A. Weinberg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>the SW55Ti swinging bucket rotor</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>a Zeiss Axioplan 2 stereo-fluorescence microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

high-sensitivity detection of micrometastases generated by gfp lentivirus-transduced organoids cultured from a patient-derived colon tumor

尽管目前人类大肠癌 (crc) 治疗的进展, 但很少有根治性疗法对晚期的 CRC 有效。为克服这一临床挑战, 采用长期建立的人肿瘤细胞系和许多转基因小鼠模型, 以肿瘤为基础的模型, 开发了移植瘤小鼠模型。它们部分模仿人类肿瘤的特点, 但往往无法概括人类恶性肿瘤的关键方面, 包括侵袭和转移。因此, 人们长期以来一直在等待替代模式, 更好地代表人类 CRC 的恶性进展。
我们在这里显示由病人衍生的肿瘤移植 (PDXs) 通过皮下植入的小 CRC 片段手术解剖的患者。结肠 PDXs 发育, 病理类似于患者的 CRC。然而, 在 PDX 模型中, 在受影响的远端器官的常规剖面中, 很少有自发淋巴结可检测到。为了便于检测转移到远处的器官, 我们从培养的结肠 PDXs 中提取出肿瘤 organoid 细胞, 并在注射至高度 immunodeficient 点头/石-免疫慢病毒北疆前感染了 GFP(酒) 老鼠。原位注射 PDX 衍生的 CRC organoid 细胞持续形成原发肿瘤阳性的 GFP 在接受小鼠。此外, 在这些小鼠的肺部, 通过荧光显微术, 自发地开发出表达 GFP 的淋巴结微转移菌落。此外, 脾注射液的 CRC organoids 经常产生肝定植。结合起来, 这些发现表明 GFP 标记的 PDX 衍生 CRC organoid 细胞在一个多步骤的过程中被视觉检测, 称为侵袭转移级联。所述的协议包括建立 PDXs 的人 crc 和3D 培养的相应的 CRC organoid 细胞转基因由 GFP 慢病毒载体粒子。

经诊断为中度分化的原发性大肠腺癌经手术切除, 76 岁女性, TNM 分级, 术后化疗后。主要的 CRC 细胞免疫组化染色阳性的癌胚抗原, Ki-67, pan 角蛋白和 e-钙黏素。切除的肿瘤的片断也被皮下植入酒小鼠, 以生成结肠 PDX 模型。然后从这些小鼠皮下发育的结肠 PDX 提取 CRC organoid 细胞进行组织培养。在人工细胞外基质的一系列通道中, CRC organoids 能够持续形成多细胞簇。由于肿瘤 or...

Watch this Scientific Journal Video about High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor at JoVE.com

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

为了允许高度敏感的检测传播的人大肠癌 (CRC) 细胞的殖民组织, 我们在此显示了一个协议, 以有效地转导绿色荧光蛋白 (GFP) 慢病毒载体粒子到 PDX 衍生 CRC organoid 细胞在注射到受体小鼠之前, 用立体荧光显微镜观察。

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可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

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Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

患者源性肿瘤外植体作为预测患者耐药性的"实时"临床前平台

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

使用患者源性肿瘤类器官的高通量 体外 检测

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

分析EGFR突变型NSCLC患者衍生类器官靶向治疗的敏感性

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

高级别浆液性卵巢癌患者来源类器官的生成和培养

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...