这项工作得到了上海科学技术委员会 (15411951800, 15410723200) 的资助。作者感谢复旦大学病原微生物系玉梅教授和赵教授的技术支持。

该方法是根据复旦大学华山医院研究伦理学委员会批准的指导方针和规定进行的。每个步骤都遵循相应的标准安全措施。有关示意性演示文稿, 请参阅图 1。
1. 细胞培养和质粒结构
<ol><li>例行维持人脐静脉内皮细胞 (HUVEC) 在 Dulbecco 的改良鹰的培养基 (DMEM) 补充10% 胎牛血清 (血清), 100 单位/毫升青霉素, 100 µg/毫升链霉素在加湿 5% CO2孵化器摄氏37摄氏度。</li>
	<li>摘要 PLKO 1 质粒, ApaI 和 EcoRI 合成 shRNA 片段。确保 VHL shRNA 向量的寡核苷酸序列 CCGGGATCTGGAAGACCACCCAAATCTCGAGA TTTGGGTGGTCTTCCAGATCTTTTTG14。</li>
	<li>然后, 混合20µΜ系统与前5µL 的寡核苷酸, 5 µL 的反向寡聚, 5 µL 10x 的纳布缓冲器和35µL ddH2O 一起 (总容量为50µL)。将混合物加热98摄氏度4分钟, 在几个小时内慢慢冷却到室温。</li>
	<li>结扎退火的寡核苷酸和 PLKO 1 质粒与 T4 连接酶在4°c 过夜。16小时后, 添加5µL 结扎组合成25µL 主管 DH5α细胞。然后, 筛选成功结扎的质粒, 并通过测序验证插入片段。</li>
</ol>2. 慢病毒北疆包装和感染
<ol><li>总计, 混合10µg 的 PLKO 1-shVHL 或 PLKO 1-shScramble 向量, 7.5 µg 的包装质粒 psPAX2, 2.5 µg 的信封质粒 pMD2 500 µL 的 DMEM 培养基没有胎牛血清 (血清) 25 分钟。</li>
	<li>添加 7 mLDMEM, 而不给上述解决方案。</li>
	<li>培养293FT 细胞在 DMEM 没有在10厘米直径组织培养皿中的血清。使用单元格计数器确保293FT 单元格的数量为 1 x 107 。</li>
	<li>将上述溶液的1毫升添加到293FT 细胞中以进行转染。 注意:293FT 细胞系来源于293F 细胞系, 稳定表达 SV40 大 T 抗原。因此, 它是一个适合慢病毒载体生产的主机。</li>
	<li>在6小时后将培养基与含有10% 的 DMEM 的培养基替换。</li>
	<li>转染后用吸管在48小时收集介质。 注意:病毒存在于含有10% 血清的 DMEM 中。死细胞漂浮在培养基上。使用0.45 µm 无菌膜过滤死细胞和杂质。一般而言, 没有必要检查感染的多重性。这是建立一个稳定的细胞线。在最后一步, 所有没有成功感染的活细胞将死于嘌呤霉素。shScramble 组和 HUVEC 细胞为对照组。确保所有 HUVEC 细胞与所使用的嘌呤霉素浓度 72 h 一起死亡。每个井都有相同数量的细胞。</li>
	<li>将慢病毒载体介质中的 HUVEC 细胞培养为72小时。然后, 将嘌呤霉素添加到 HUVEC 细胞的培养基中, 浓度为2µg/毫升, 24 小时. 使用 HUVEC 细胞而不接受任何治疗作为对照组。确保所有控制单元在此时死亡。 注意:活细胞代表 VHL 击倒细胞和 shSramble 细胞的稳定细胞系。在96井板中, 镀层密度为 5000/井。</li>
</ol>3. 内皮细胞球体的生成
<ol><li>血管内皮细胞细胞和并用重悬在 DMEM 培养基中胰蛋白酶处理10% 的血清。在10厘米的培养皿中有适量的细胞, 加入1毫升的 typsin EDTA。</li>
	<li>将3D 圆底96井板中的细胞种子, 在细胞密度为 1 x 103单元格/好。用一个能容纳0.50 µL 球体的井来悬浮球体。按照制造商的说明, 使用单元格计数器系统计数单元格。</li>
	<li>连续孵化37°c 和 5% CO2的细胞为 72 h。在这些条件下, 确保悬浮细胞自动形成细胞球体。36小时后替换培养基中的一半。</li>
</ol>4.体外血管生成试验
<ol><li>解冻的凝胶溶液在4°c 和稀释它与减少的血清培养基在稀释率为1:5。</li>
	<li>用微吸 DMEM 培养基中的球体。洗涤后的球体与减少血清培养基的5毫升, 暂停球体轻轻地和谨慎地在稀释凝胶。确保没有气泡。</li>
	<li>在15井板中嵌入300µL 混合液。孵化后在37°c 和 5% CO2为 1 h, 增加400µL 减少的血清培养基对每个井。在水井周围填充无菌水, 以产生湿润的环境, 以阻止蒸发。</li>
	<li>尔后, 文化板材在37°c 在 5% CO2在100% 湿气为 1 h。</li>
	<li>小心地吸入旧培养基, 用600µL 减少血清培养基, 并将每一个井中的1% 个内皮细胞生长补充剂替换。</li>
	<li>在12小时或24小时以后, 在倒置的光显微镜下拍摄图像 (40 *)。</li>
</ol>5. 数据分析
<ol><li>将图像上传到在线图像分析平台上, 以获取发芽长度数据 (材料表)。</li>
	<li>在网页上, 通过电子邮件创建帐户。</li>
	<li>然后单击 "上载" 按钮上载图像。</li>
	<li>单击 "下载" 按钮下载结果。 注意:该软件将生成处理后的图像和数据。数据包含五个部分: 累计发芽长度、平均发芽长度、芽长的标准偏差、发芽面积和球体面积。在处理过的图像中, 每个参数都用不同的颜色勾勒出来, 以简化处理后的图像中的识别: 红色代表芽的骨架, 黄芽的数量, 蓝色的芽结构, 白色芽端, 和橙色球体。</li>
</ol>

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作者没有什么可透露的。

近年来, 血管内皮细胞15的研究刺激了多领域的维管束生物学研究。本文以内皮球发芽技术为实验模型, 研究了 VHL 基因在操作内皮细胞中的功能丧失对血管形成的作用, 以确定新的候选分子血管生成叶。根据我们的知识, 这是第一个检查内生改良内皮细胞血管生成效应的报告。
组织 (包括肿瘤组织) 新生血管是一个复杂的过程, 发生在发育过程中的通过?...

小脑 (哈佛商学院) 是良性血管肿瘤, 是完全在人类中枢神经系统 (CNS) 发现。他们发展在患者与冯冯·希佩尔·林道-林道 (VHL) 疾病或零星的损伤。VHL-哈佛商学院是难以治愈的外科治疗, 由于频繁复发和多病变, 造成这种遗传紊乱1。尽管 VHL 抑癌基因的失活已被认为是 VHL-哈佛商学院肿瘤发生的根本原因, 但哈佛商学院的细胞学起源 (包括新生血管) 和进化过程仍然很有争议2。因此, 更好地了解 HB-新生血管的生物机制, 可以为 VHL-哈佛商学院最有前途的抗血管策略提供有益的见解。
最近的研究表明, HB-新生血管类似于胚胎血管发生 3, 4, 5.典型的血管内皮生长因子 (VEGF) 介导的血管生成是由血管内皮引起的, 由 VHL 功能丧失所驱动, 导致增殖和新生血管形成, 这已被挑战6。在 1965年, Cancilla 和齐默尔曼使用电子显微镜发现, 哈佛商学院起源于内皮细胞7。后来发现基质细胞来源于 vasoformative 元素8。在 1982年, Jurco et 等发现基质细胞是内皮源9。因此, 我们假设, 人血管内皮细胞是血红蛋白的原细胞-新生血管的10。虽然最好使用从 VHL 患者手术中提取的 hb 细胞中的主要培养物, 但我们以前的研究表明, 来自 hb 的主要培养基不稳定, 细胞系无法建立3。此外, 3D 环境中的主要文化无法识别乙肝新生血管的细胞学来源, 因为它们包括 hb-血管成分的祖细胞10,11。因此, 作为一种原始和经典的内皮细胞模型, 人血管内皮细胞 (HUVEC) 可以作为哈佛商学院的替代细胞模型。
球形发芽试验是组织工程中的一个新模型12,13。本文开发了一种基于3D 胶原的共培养系统体外, 其目的是评价哈佛商学院是否存在经典的肿瘤血管生成, 以及它在哈佛商学院的作用。

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			<td height="40" style="height:40px;width:265px;">human umbilical vein endothelial cell</td>
			<td style="width:147px;">Fudan IBS Cell Center</td>
			<td style="width:343px;">FDCC-HXN180</td>
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			<td height="40" style="height:40px;">dulbecco&#8217;s modified eagle&#8217;s medium&#160;</td>
			<td>Gibco</td>
			<td>11995040</td>
			<td style="width:761px;"></td>
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			<td height="40" style="height:40px;">fetal bovine serum</td>
			<td>Gibco</td>
			<td>&#160;26400044</td>
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			<td height="40" style="height:40px;">PLKO.1-puro vector</td>
			<td>Addgene</td>
			<td>#8453</td>
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			<td height="40" style="height:40px;">packing plasmid psPAX2&#160;</td>
			<td>Addgene</td>
			<td>#12260</td>
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			<td height="40" style="height:40px;">envelope plasmid pMD2.G</td>
			<td>Addgene</td>
			<td>#12259</td>
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			<td height="40" style="height:40px;">3D round-bottom 96-well plates</td>
			<td>S-Bio</td>
			<td>MS-9096M</td>
			<td style="width:761px;"></td>
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			<td height="40" style="height:40px;">matrigel</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td style="width:761px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Opti-MEM medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td style="width:761px;">reduced serum medium&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">15-well plate</td>
			<td>Ibidi</td>
			<td>81501</td>
			<td>Air bubbles in the gel can be reduced by equilibrating the &#956;&#8211;Slide angiogenesis before usage inside the incubator overnight</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">endothelial cell growth supplements</td>
			<td>Sciencell</td>
			<td>#1052</td>
			<td style="width:761px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">10-cm culture dish</td>
			<td>Corning</td>
			<td>Scipu000813</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Puromycin</td>
			<td>Gibco</td>
			<td>A1113802</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">typsin-EDTA</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Automated Cell Counter System &#160;</td>
			<td>BioTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Image Analysis software&#160;</td>
			<td>Winmasis</td>
			<td style="width:343px;"></td>
			<td>http://mywim.wimasis.com&#160;</td>
		</tr>
	</tbody>
</table>

a comprehensive procedure to evaluate the in vitro performance of the putative hemangioblastoma neovascularization using the spheroid sprouting assay

冯·希佩尔·林道-林道 (VHL) 抑癌基因的失活在人类中枢神经系统 (CNS) 的小脑 (HBs) 的发展中起着至关重要的作用。然而, 哈佛商学院 (包括新生血管) 的细胞学起源和进化过程仍然存在争议, VHL-哈佛商学院的抗血管生成是基于典型的 HB 血管生成, 在临床试验中产生了令人失望的结果。抗血管治疗成功的临床翻译的一个主要障碍是对血管肿瘤的新生血管缺乏透彻的了解。在本文中, 我们提出了一个综合的程序来评价在体外是否存在经典的肿瘤血管生成在哈佛商学院, 以及它在哈佛商学院的作用。通过这一程序, 研究人员可以准确地了解乙肝新生血管的复杂性, 并确定其在哈佛商学院的这种常见的血管生成功能。这些协议可用于评估最有前景的肿瘤抗血管治疗, 它具有很高的翻译潜力, 无论是肿瘤治疗或协助优化抗血管生成治疗的哈佛商学院在未来的翻译。结果突出了血红蛋白新生血管的复杂性, 认为这种共同的形态血管生成只是血红蛋白新生血管的补充机制。

原图像采用倒置光镜。控件组和 VHL 组的典型图像显示在图 2A1和图 2A2中。对照组的发芽长度短于 VHL 组。
上传图像后, 在线平台直接提供分析结果。输出由两部分组成: 处理后的图像和相应的分析结果。控制组和 VHL 基因静默组的处理图像标记为不同的颜色 ...

Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay at JoVE.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

用球形发芽法评价 Hemangioblastoma 新生血管的体外性能的综合程序

本文提出了一个综合的方法来评价在体外是否存在经典的肿瘤血管生成在小脑 (哈佛商学院) 及其在哈佛商学院的作用。研究结果突出了血红蛋白-新生血管的复杂性, 认为这种共同的血管生成形式仅仅是血红蛋白-新生血管的补充机制。

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

a-comprehensive-procedure-to-evaluate-vitro-performance-putative

Research

JoVE Journal

Cancer Research

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

7.9K 次观看.  Fudan University. 本文提出了一个综合的方法来评价在体外是否存在经典的肿瘤血管生成在小脑 (哈佛商学院) 及其在哈佛商学院的作用。研究结果突出了血红蛋白-新生血管的复杂性, 认为这种共同的血管生成形式仅仅是血红蛋白-新生血管的补充机制。

视频: A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

科研

癌症研究

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

综合程序，以评估<em&gt;在体内</em&gt;癌症纳米医学的性能

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

蒸发减少培养条件的提高多细胞球体形成的微孔板重现

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

用点法分析细胞板的迁移

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

应用 Fraumeni 综合征诱导多潜能干细胞建模骨肉瘤

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

通过颅内注射和磁共振成像对大脑转移进行建模

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...