Name: evaluating the angiogenetic properties of ovarian cancer stem-like cells using the three-dimensional co-culture system, nico-1
Uploaded: 2020-12-05T13:30:00.000+00:00
Duration: 7 min 59 s
Description: Watch this Scientific Journal Video about Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1 at JoVE.com

这项工作得到了日本文部科学省的科学研究C补助金（K.N.拨款号19K09834）的支持。

所有程序均根据人类福利伦理委员会批准的协议执行。所有患者都为其样本的研究用途提供了书面知情同意书，本研究的组织收集和使用得到了帝京大学人类基因组基因分析研究伦理委员会的批准。
1. 在 2 级生物安全柜中分离和培养卵巢癌和腹水患者的卵巢癌干细胞 （OCSC）
<ol><li>从通过穿刺获得的人卵巢癌腹水中分离癌症干细胞。从患者身上收集至少 100-250 mL 的腹水，以摄取足够数量的癌症干细胞。此外，通过流式细胞术评估癌症干细胞标志物（即 EpCAM、钙视黄素、CD133、CD44、CD45、ALDH1 和 Oct4）和卵巢癌标志物（pAX-8、WT-1）的表达谱。<ol><li>在腹水抽吸后24小时内在室温下以300× g 离心人卵巢癌腹水10分钟。</li>
		<li>除去上清液，加入 2 mL OCSC 培养基和 8 mL 30% 组氨酸盐/磷酸盐缓冲盐水 （PBS，pH 7.4） 溶液。</li>
		<li>制备 OCSC 培养基:StemPro hESC 补充剂、含有 L-谷氨酰胺的 DMEM/F-12（GlutaMAX 培养基）、25% BSA、100 μM 2-巯基乙醇、8 ng/mL FGF BASIC、10 μM 胰岛素和 20 μM Y-27632。</li>
		<li>小心地将 2 mL OCSC 培养基覆盖到 15 mL 管中的步骤 1.1.2 中的细胞溶液中，并在室温下在水平吊篮转子中以 450 x g 离心 20 分钟，无需制动。</li>
		<li>通过移液管小心地将OCSC层（在相间不受干扰）转移到新的15 mL管中。</li>
		<li>用高达 15 mL 的 PBS 填充。在室温下以300× g 离心5分钟，除去上清液。</li>
		<li>将细胞沉淀重悬于OCSC培养基中，并接种在超低附着培养皿上;培养物应保持在37°C的5%CO2中。</li>
		<li>每三天更换一次培养基。小心地将培养皿静置约1分钟，弃去部分上清液并加入新培养基。</li>
	</ol></li>
	<li>CSC的通过<ol><li>将 OCSC 收集在 15 mL 管中，并在室温下以 200 x g 离心 5 分钟。</li>
		<li>除去上清液，填充PBS，并在室温下以200× g 离心5分钟。</li>
		<li>除去上清液，加入1mL由蛋白水解和胶原溶解酶（例如AccuMax）组成的细胞分离溶液，并在37°C下孵育10分钟。</li>
		<li>通过移液充分混合并在37°C孵育5分钟。确保细胞处于单个悬浮液中。</li>
		<li>通过移液充分混合，填充PBS，并在室温下以300 x g 离心5分钟。</li>
		<li>除去上清液并将细胞沉淀重悬于OCSC培养基中，随后接种在超低附着培养皿上，并在37°C的5%CO2中维持。</li>
	</ol></li>
</ol>2. HUEhT-1内皮细胞培养
<ol><li>HUEhT-1细胞的传代<ol><li>从HUEhT-1培养皿中取出培养基并用PBS洗涤细胞。</li>
		<li>加入 1 mL 的 0.025% 胰蛋白酶，并在室温下孵育 3 分钟。</li>
		<li>加入 5 mL 内皮细胞生长培养基 2，将细胞收集在 15 mL 管中，并在室温下以 200 x g 离心 5 分钟。</li>
		<li>除去上清液，将细胞沉淀重悬于HUEhT-1培养基中，并将细胞接种在胶原蛋白包被的培养皿上，然后在37°C的5%CO2中维持。</li>
		<li>每三天更换一次培养基。</li>
	</ol></li>
</ol>3. 使用 HUEhT-1 细胞制备用于管形成测定的 NICO-1 共培养板
<ol><li>组装NICO-1并涂有基于细胞外基质的水凝胶（基质胶基质）。<ol><li>按照制造商的说明组装 NICO-1 的一侧并保持在冰上。</li>
		<li>用 300 μL 冷 PBS 覆盖 NICO-1 表面，然后取出缓冲液。</li>
		<li>加入 300 μL 冷却的基于细胞外基质的水凝胶，并在 37 °C 下孵育 60 分钟。</li>
		<li>为了平衡，用100%乙醇浸入过滤器，然后用PBS洗涤13mm ICCP过滤器（0.6μm）1分钟。</li>
		<li>组装 NICO-1，包括主体部件 A（右腔）和 B（左腔）以及 O 形圈和平衡过滤器。</li>
	</ol></li>
</ol>4. 将 HUEhT-1 细胞和 CSC 接种到 NICO-1 系统上
<ol><li>通过胰蛋白酶消化细胞单层并将细胞重悬于含有2%胎牛血清的内皮细胞生长培养基中来制备HUEhT-1细胞悬液。</li>
	<li>向每个基于细胞外基质的水凝胶包被孔中加入 1.2 mL 细胞悬液（1.5 x 105 个 细胞）。</li>
	<li>将 1.5 mL 培养五天的 OCSC 加入另一个孔中。</li>
	<li>在37°C的5%CO2中孵育NICO-1;可以在显微镜下观察管的形成，并通过通过分支数测量基于细胞外基质的水凝胶上的网络形成。</li>
</ol>

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作者没有什么可透露的。

所提出的协议描述了如何在体外环境中模拟OCSC的肿瘤微环境。该方法的主要组成部分构成了使用NICO-1系统（一种间接Transwell共培养系统）获得的高度可重复的共培养模型。许多目前可用的共培养模型检查了直接细胞-细胞接触对共培养细胞群的影响12，13，14，15，16，17，18。可用于检查共培养效果的最简单模型可以通过两种细胞类型的直接混合来繁殖，并且可以通过改变每种细胞类型的接种密度和亚群的相对接种比例来检查异型和同型相互作用的程度19。然而，由于每个细胞的精确隐形，很难通过显微镜独立地直接确定OCSC对共培养的任何观察到的效果的相对贡献;因此，这些研究通常伴随着条件培养基实验。例如，分离的共培养系统可用于研究旁分泌信号对肿瘤微环境的影响19。相比之下，在本文的方法中，我们描述了一种模型，该模型允许同时评估细胞 - 细胞接触和旁分泌信号传导的影响，其模拟天然肿瘤微环境的结构。
血管生成是卵巢癌的标志，并在其进展中起关键作用，其涉及癌细胞，内皮细胞和周围肿瘤微环境之间的相互作用20。贝伐珠单抗是一种关键的分子靶向药物，已被广泛接受用于晚期卵巢癌的联合化疗21。具体而言，贝伐珠单抗构成针对血管内皮生长因子（VEGF）的单克隆抗体。VEGF通过促进新生血管形成和增强血管通透性，有助于晚期卵巢癌或腹膜癌腹水的发展和恶性腹水的形成22。因此，VEGF抑制已被证明可以抑制腹水的产生和转移部位的大量肿瘤生长。因此，有必要在肿瘤微环境水平上有效靶向VEGF刺激的血管内皮细胞进行进一步研究。然而，可能难以有效地利用临床前模型，例如小鼠模型，进行这些研究。例如，据报道，贝伐珠单抗对人VEGF的亲和力很高，而小鼠蛋白的亲和力较低23。这限制了贝伐珠单抗在实验中的应用，这些实验旨在进一步研究其抗VEGF机制，如使用小鼠模型确定的那样，在肿瘤微环境中形成新生血管。因此，需要一个具有良好控制结构的适当模型来概括体内肿瘤微环境的组成部分。在这种情况下，我们注意到我们的体外共培养模型系统允许在患者来源的OCSC和内皮细胞的培养过程中实时跟踪细胞行为，并允许研究这些单个细胞亚群以响应共培养。
更好地了解OCSC的作用及其血管生态位可以为制定OCSC的治疗策略提供新的见解。如代表性实验所示，该模型的优势在于它提供了一个似乎与临床环境部分一致的研究平台，能够开发和测试更具针对性和更有效的新药。需要进一步的研究，直接临床复制我们的结果，涉及更多患者来源的OCSC。

卵巢癌是全球女性第八大常见恶性肿瘤，每年约有 300，000 例新诊断，估计有 180，000 例死亡1。在初步诊断时，卵巢癌通常出现严重症状，约75%的患者已经处于III-IV期。因此，卵巢癌的5年生存率为<30%，死亡率在妇科癌症中最高2，卵巢癌的治疗效率高度依赖于减瘤手术的成功完成、化疗耐药和初始治疗后复发等临床因素。
卵巢癌组织是分层组织的，并非所有肿瘤成分都能够同样产生后代。唯一能够自我更新并产生异质性肿瘤细胞群的细胞被认为代表癌症干细胞（CSC）3。CSC自我更新和肿瘤起始伴随着促进血管生成以重塑其肿瘤微环境，以维持支持性生态位。然而，以前的模型不能用于体外分析，因为培养来自临床样品的CSC的可重复性有限，因为多次传代后球状体的破坏。最近，已经开发了从患者身上培养CSC的实验方法，用于多种应用4，5，6，7。特别是，通过利用CSCs在无血清培养基的超低附着板中形成球状体来生长的特性，诱导培养的CSC表达在具有多谱系分化潜力的正常肿瘤细胞中未表达的干细胞表面标志物8，9。
最近的数据表明，在腹膜上可视化为播散的休眠卵巢（O）CSC的持续存在与其作为复发性肿瘤的再生有关10。因此，了解OCSC的分子和生物学特征可以有效地靶向和根除这些细胞，从而产生潜在的肿瘤缓解。特别是，关于CSC在血管生成中的作用的细胞和分子机制特征知之甚少11。因此，在本协议中，我们在体外环境中使用患者来源的OCSCs，使用共培养模型研究内皮细胞的血管生成特性，该模型可能模拟CSC和内皮细胞的肿瘤微环境在临床环境中转移部位。最终，由于新生血管形成是支持肿瘤生长和转移所必需的关键过程，因此更好地了解其机制将允许在转移部位为OCSC开发一种新的靶向疗法。
在这里，我们提出了一个协议，以时间过程的方式可视化围绕CSC的新生血管形成步骤。该方案的优点包括允许使用3D共培养系统NICO-1进行完全可重复的研究，从而允许观察OCSC衍生的肿瘤起始能力在内皮细胞血管生成过程中对患者的影响。

<table><tbody><tr><td>0.025% Trypsin&amp;nbsp;</td><td>Thermo</td><td>R001100</td><td></td></tr><tr><td>10 mL Pipet</td><td>Thermo</td><td>170356N</td><td></td></tr><tr><td>1250 &amp;micro;L Pipet tip</td><td>QSP</td><td>T112XLRS-Q</td><td></td></tr><tr><td>15 mL tube</td><td>Nunc</td><td>339650</td><td></td></tr><tr><td>200 &amp;micro;L Pipet tip</td><td>QSP</td><td>T110RS-NEW</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Thermo (Gibco)</td><td>21985023</td><td></td></tr><tr><td>5 mL Pipet</td><td>Thermo</td><td>170366N</td><td></td></tr><tr><td>50 mL tube</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>AccuMAX</td><td>Innovative Cell Technologies</td><td>AM105</td><td></td></tr><tr><td>BioCoatTM Collagen I 60mm Dish</td><td>Corning</td><td>356401</td><td></td></tr><tr><td>Centrifuge</td><td>KUBOTA</td><td>2800</td><td></td></tr><tr><td>Costar 6 Well Clear Flat Bottom Ultra Low Attachment Multiple Well Plates</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>Endothelial Cell Growth Medium 2&amp;nbsp;</td><td>PromoCell</td><td>C-22011&amp;nbsp;</td><td></td></tr><tr><td>Ethanol</td><td>WAKO</td><td>057-00456</td><td></td></tr><tr><td>FGF-Basic</td><td>Thermo (Gibco)</td><td>PHG0021</td><td></td></tr><tr><td>Histodenz</td><td>SIGMA</td><td>D2158</td><td></td></tr><tr><td>HUEhT-1 cell</td><td>JCRB Cell Bank</td><td>JCRB1458</td><td></td></tr><tr><td>ICCP Filter 0.6 &amp;micro;m</td><td>Ginrei Lab.</td><td>2525-06</td><td></td></tr><tr><td>Insulin, human</td><td>SIGMA (Roche)</td><td>11376497001</td><td></td></tr><tr><td>Luminometer</td><td>PerkinElmer</td><td>ARVO MX-flad</td><td></td></tr><tr><td>Matrigel Matrix</td><td>Corning</td><td>356234</td><td></td></tr><tr><td>Microscope</td><td>Yokogawa</td><td>CQ-1</td><td></td></tr><tr><td>NICO-1</td><td>Ginrei Lab.</td><td>2501-02</td><td></td></tr><tr><td>OptiPlate-96</td><td>PerkinElmer</td><td>6005290</td><td></td></tr><tr><td>P1000 Pipet</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>P200 Pipet</td><td>Gilson</td><td>F123601</td><td></td></tr><tr><td>PBS</td><td>Thermo (Gibco)</td><td>14190-144</td><td></td></tr><tr><td>StemPro hESC SFM</td><td>Thermo (Gibco)</td><td>A1000701</td><td></td></tr><tr><td>Transfer Pipet</td><td>FALCON</td><td>357575</td><td></td></tr><tr><td>Y-27632</td><td>WAKO</td><td>253-00513</td><td></td></tr></tbody></table>

evaluating the angiogenetic properties of ovarian cancer stem-like cells using the three-dimensional co-culture system, nico-1

癌症干细胞（CSC）位于支持性生态位中，构成由相邻基质细胞，血管和细胞外基质组成的微环境。CSC参与内皮发育的能力构成了一个重要特征，直接有助于对肿瘤发生和肿瘤转移机制的一般理解。这项工作的目的是建立一种可重复的方法，以研究卵巢癌干细胞（OCSC）的肿瘤起始能力。本文使用体外共培养模型NICO-1研究了内皮细胞和OCSC之间的新生血管形成机制以及内皮细胞的形态变化。该协议允许以时程方式可视化OCSC周围的新生血管形成步骤。该技术可以提供有关OCSC在肿瘤转移中的血管生成特性的见解。

我们收集了晚期卵巢癌患者在手术或穿刺过程中获得的腹水，以便对球状体进行长期稳定的培养。在这里，我们介绍了卵巢CSC的长期球状体培养案例，称为CSC1和CSC2。两种细胞系具有相同的诊断和组织学特征。OCSC与内皮细胞相互作用的机制作用仍然是诱导OCSC周围内皮细胞新生血管所必需的。因此，我们旨在阐明转移部位CSC血管生态位发育的过程。我们使用体外共培养模型NICO-1检查了内皮细胞（HUEhT-1）和OCSC之间的相互作用。 图1 显示了CSC1和CSC2诱导的管形成活性的比较。随着时间的推移，在与CSC2共培养中形成的血管管的数量显着增加（图2A）。对于阳性对照，我们提出了 图2B ，它显示了在没有CSC2共培养的情况下处理VEGF（10ng / mL）后HuEhT-1的血管生成特性。我们现在正在澄清结果背后的详细机制。 图3 显示了使用 NICO-1 的 OCSC 与内皮细胞共培养模型的代表性图像。将HUEhT-1细胞与CSC2共培养20小时，并捕获延时视频图像。 图3A 显示了共培养20小时前后CSC2的表型。 图3B 显示了同时共培养的HUEhT-1细胞。值得注意的是，HUEhT-1细胞在与CSC2共培养过程中形成血管管 （图3C:视频剪辑）
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61751/61751fig1v2.jpg"> 图 1:OCSC 血管生态位模型。 OCSC诱导内皮细胞管形成（血管化）的机制作用仍然未知。我们使用体外共培养模式NICO-1检查了内皮细胞（HUEhT-1）和OCSCs之间的相互作用。该系统的右侧隔室由一个插入物组成，该插入物将OCSC与细胞培养基一起固定。左隔室由含有内皮细胞和HUVEC的孔组成，其培养基与右孔相同。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61751/61751fig2v2.jpg"> 图2:OCSC诱导的新生血管活动比较。 随着时间的推移，与CSC2共培养时形成的血管管的数量显着增加。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61751/61751fig3v2.jpg"> 图 3:使用 NICO-1 与 CSC2 共培养的 HUEhT-1 细胞的血管形成。 HUEhT-1细胞在与CSC2共培养过程中形成血管管。

Watch this Scientific Journal Video about Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1 at JoVE.com

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

卵巢癌干细胞 （OCSC） 负责癌症的发生、复发、治疗耐药性和转移。OCSC血管生态位被认为可促进OCSC的自我更新，导致化学耐药性。该协议为体外建立可重复的OCSC血管生态位模型提供了基础。

使用三维共培养系统NICO-1评估卵巢癌干细胞样细胞的血管遗传特性

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

evaluating-angiogenetic-properties-ovarian-cancer-stem-like-cells

Nanyang Technological University

Research

JoVE Journal

Biology

2.5K 次观看.  Teikyo University School of Medicine. 卵巢癌干细胞 （OCSC） 负责癌症的发生、复发、治疗耐药性和转移。OCSC血管生态位被认为可促进OCSC的自我更新，导致化学耐药性。该协议为体外建立可重复的OCSC血管生态位模型提供了基础。

视频: Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

科研

生物工程

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

医学

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

癌症研究

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

发育生物学

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

评价的人卵巢癌细胞干细胞特性使用多和单细胞为基础的化验球

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

模拟卵巢癌多细胞球体行为动态三维微型腹膜

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

在卵巢癌肿瘤起始细胞体外富集

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...