Name: analyzing the communication between monocytes and primary breast cancer cells in an extracellular matrix extract (ecme)-based three-dimensional system
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这项工作得到了委员会 FONSEC SSA/社会保障/ISSSTE 项目 No. 233061 至拉维奇-恩特斯-Pananá和基金 de 支持、医院研究 de 婴儿墨西哥 g ó mez (项目编号费德里克二世) 的支持。埃斯皮诺萨-桑切斯是一名博士生, 来自方案 de Doctorado Ciencias Biomédicas, 国立国立 de 墨西哥 (大学), 并获得了奖学金231663从委员会。也感谢墨西哥社会保障研究所 (社会保障) 提供的财政支持。

从研究 Virología y Cáncer, 医院婴儿 de 墨西哥费德里克二世 g ó mez 的组织银行获得的样本来自。这项研究是由医院的科学、伦理和生物安全审查委员会批准的, 婴儿 de 墨西哥费德里克二世 g ó mez: 研究、Ética、研究和 Bioseguridad。所有患者都有前瞻性的登记, 并被告知研究的性质: 那些愿意参加标本收集之前签署书面知情同意, 并按照道德准则和最佳临床实践的治疗该机构。在研究期间, 参与者的身份?...

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上皮细胞生长在3D 空间构象, 它们与 ECM 蛋白的相互作用是组织稳态的关键。许多癌症研究都是以单分子膜 (2D) 的细胞为基础的, 尽管它们对于了解肿瘤形成和进展的许多方面都是至关重要的, 但单分子膜并没有重述 ECM 强加于细胞的特性,例如: 限制增殖, 黏附依赖细胞生存, 顶端侧极性, ECM 重塑, 细胞分化,等重要的是, 不仅肿瘤细胞和 ECM 之间的相互作用对癌症的进展是必不可少的, 而且在肿?...

肿瘤生物学比以前想象的要复杂得多。越来越多的证据表明, 肿瘤细胞不仅仅是大量失控的增殖细胞;相反, 不同的肿瘤细胞似乎执行不同的功能, 显示高组织和层次结构内的肿块1。肿瘤细胞与 non-transformed 细胞也有密切的联系: 巨噬细胞、成纤维细胞、淋巴细胞、脂肪细胞、内皮干细胞等都被浸润在构成 ECM 的支架蛋白和多糖中。许多直接和间接相互作用建立在被变换的和 non-transformed 细胞之间和与 ECM, 施加对疾病结果的一个强有力的影响2,3。在特定的情况下, 这是特别重要的解剖与 tam 的沟通, 考虑到 tam 已被发现发挥关键作用的肿瘤的演变, 增加转移和疾病复发的风险4 ,5。
为了分析 intra-tumoral 的相互作用及其可能的结果, 新的 3D体外方法是基于 ECM 提取物的使用而开发的, 它提供了更复杂的微环境, 更接近于肿瘤生物学的现实, 相比之下,细胞生长附着在塑料上的传统单层细胞培养。彼得森和比6提供了在层粘连蛋白基底膜上培养的恶性和恶性乳腺上皮细胞的第一个模型, 并首次描述了区分恶性人的3D 器官结构乳腺上皮细胞从他们的恶性对应。十年后, 由 Debnath、Muthuswamy 和布鲁日7、8、9所开发的模型提供了一个有价值的工具来阐明在腺腺的恶性转化过程中受损的生物通路, 如由于大的腺形成由于不受控制的增殖, 域紧密连接蛋白作为损伤细胞极化的证据, 并失去腺腔由于细胞抵抗亡, 一种程序性细胞死亡发生在当与周围的 ECM 分离时, 依赖于锚固的细胞。Sameni、Jedeszko 和斯隆的模型主要关注细胞的蛋白水解活动, 这与侵袭性密切相关, 肿瘤恶性肿瘤的另一个重要特征10,11,12。这些模型依赖于不同的荧光猝灭蛋白基质 (dq-明胶, dq-胶原蛋白 I 和 dq-胶原 IV) 混合的蛋白质基质, 其中荧光信号是胶原蛋白水解降解的指示。3D 模型也用于研究 non-transformed 和肿瘤细胞的干细胞特性, 其中细胞聚集物, 也被称为椭球, 可以被培养悬浮或在 ECM 样蛋白中的细胞分化机制, 不对称细胞分裂, 细胞黏附, 并且细胞运动13,14。入侵检测允许测试肿瘤的内在侵略性和在侵入过程中作为化的分子的鉴定15。总体而言, 3D 模型代表了一种经济实惠的多样化的体外细胞培养, 更密切地反映正常和致癌组织形态发生。
我们设计了一个3D 细胞共培养系统的基础上上述的模型7,10,11, 使用了人类商业零售商的已知侵略性的潜力 (腔和三重阴性类型) 细胞线和主要细胞说明的患者。我们首先开发了一个模型, 无论是非 (MCF-7) 或侵略性 (MDA-MB-231) 的细胞, 共与 U937 单核分子在一个细胞外基质提取物 (ECME) 的基础3D 系统, 允许直接细胞间的相互作用。这些 co-cultures 被用来确定这两个细胞谱系之间的交流如何影响了一系列与癌症侵袭性行为相关的基因的转录。cyclooxygenase-2 (COX-2) 成绩单的显著增加, 与其产品之一, 前列腺素 E2 (PGE2) 的产量增加相吻合, 这一发现凸显了炎症在癌症进展中的作用。还观察到, 当侵袭性 MDA-MB-231 细胞在 DQ-IV 型胶原蛋白中 U937 单核共时, 与更大的胶原蛋白水解相关。值得注意的是, 我们的 co-cultures 不支持假定细胞细胞相互作用机制是胶原蛋白降解所必需的。它更认为, 两个细胞谱系之间的沟通是由分泌分子介导的。此外, 从这些共培养试验中收获的清含有的可溶性因素, 紊乱的腺腺形成的 non-transformed MCF-10A 细胞13。研究发现, 侵袭性的和初级的趋细胞分泌出高水平的单核分子的 MCP-1, GM-脑脊液, 和 RANTES。因此, 我们概述了一个3D 的文化, 其中细胞分离细胞培养插入, 以防止细胞间的相互作用。这些文化被用来解决的间接沟通之间的细胞和单核。对于这些化验, 非和侵略性商业的细胞系和主要的细胞, 和三种不同类型的人单核: 商业 U937 和 THP-1 细胞, 和主要单核 (PMs) 分离的外周血的健康献血者 (所有单核细胞用于激活状态) 被使用。IL-1β和 IL-8 的炎性细胞因子浓度增加, 在共培养中得到了丰富的观察。同样, 发现 MMP-1、MMP-2 和 MMP-10 也增加了在细胞-单核 co-cultures, 从而加强以前的研究结果14。在这份手稿中, 提出了一个点对点的工作流程在3D 文化中的主要的细胞分离和测试, 并具有代表性的结果。这项工作是一个很好的例子的巨大潜力,体外3D 细胞系统提供审问的具体方面的肿瘤生物学。

<table border="0" cellpadding="0" cellspacing="0" style="width:1108px;" width="1108">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">U937</td>
			<td style="width:260px;">American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">CRL-1593.2</td>
			<td style="width:463px;">Monocytic cell line/histiocytic lymphoma&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">THP-1</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">TIB-202</td>
			<td>Monocytic cell line/acute monocytic leukemia&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MCF-10A</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td>CRL-10317</td>
			<td>Non-transformed breast cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MCF-7</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-22</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">T47D</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-133</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">HS578T</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-126</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MDA-MB-231</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-26</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">RPMI 1640 medium</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DMEM High Glucose&#160;</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">11964-092</td>
			<td>4.5 g/L glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DMEM/F12</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">11039-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Antibiotic/Antimycotic</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">15240-062</td>
			<td>100 U/mL penicillin, 100 &#181;g/mL streptomycin, and 0.25 &#181;g/mL Fungizone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Fetal Bovine Serum</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Horse serum&#160;</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td>16050114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">0.05% Trypsin-EDTA 1X</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">25300-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS 1X (Phosphate Buffered Saline</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF)</td>
			<td>PeproTech</td>
			<td>AF-100-15 (1.00mg)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td>SIGMA-ALDRICH</td>
			<td>I1882-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrocortisone</td>
			<td>SIGMA-ALDRICH</td>
			<td>H088-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholera toxin Vibrio cholerae</td>
			<td>SIGMA-ALDRICH</td>
			<td>C8052-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Matrigel&#160;</td>
			<td>Corning Inc</td>
			<td>356237</td>
			<td style="width:463px;">Engelbreth-Holm-Swarm (EHS) mouse sarcoma, extracellular matrix extract, Store at -20&#176;C until use at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">GM-CSF</td>
			<td>PeproTech</td>
			<td>300-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MCP-1</td>
			<td>PeproTech</td>
			<td>300-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">RANTES</td>
			<td>PeproTech</td>
			<td>300-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IL-8</td>
			<td>PeproTech</td>
			<td>200-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IL-1&#946;</td>
			<td>PeproTech</td>
			<td>200-01B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Crystal-violet</td>
			<td>Hycel M&#233;xico</td>
			<td>541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Transwell permeable supports (inserts)</td>
			<td>Corning Inc</td>
			<td style="width:143px;">3422</td>
			<td>6.5 mm diameter, 8 &#181;m pore size/24-well plates&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Transwell permeable supports (inserts)</td>
			<td>Thermofisher Scientific</td>
			<td style="width:143px;">140620</td>
			<td>6.5 mm diameter, 0.4 &#181;m pore size/24-well plates&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Monocyte Isolation Kit II Human&#160;</td>
			<td style="width:260px;">Miltenyi Biotec</td>
			<td style="width:143px;">130-091-153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">LS Columns</td>
			<td style="width:260px;">Miltenyi Biotec</td>
			<td style="width:143px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Human Cytokine/Chemokine Magnetic Bead Panel Kit 96 Well Plate Assay</td>
			<td style="width:260px;">EMD Millipore</td>
			<td style="width:143px;">HCYTOMAG-60K</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Magpix with xPonent software (laser based fluorescent analytical test instrumentation)</td>
			<td style="width:260px;">Luminex Corporation</td>
			<td style="width:143px;">40-072</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Lab-Tek chamber slide with cover (8 well)</td>
			<td style="width:260px;">Nalge Nunc International</td>
			<td style="width:143px;">177402</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Glass bottom microwell 35mm petri&#160; dishes</td>
			<td style="width:260px;">MatTek Corp.</td>
			<td style="width:143px;">P35G-1.5-14-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing the communication between monocytes and primary breast cancer cells in an extracellular matrix extract (ecme)-based three-dimensional system

嵌入细胞外基质 (ECM), 正常和肿瘤上皮细胞密切沟通与造血和造血细胞, 从而大大影响正常的组织稳态和疾病的结果。在乳腺癌中, 肿瘤相关巨噬细胞 (tam) 在疾病进展、转移和复发中起关键作用;因此, 了解单核细胞 chemoattraction 对肿瘤微环境的机制及其与肿瘤的相互作用对控制该病具有重要意义。在这里, 我们提供了一个三维 (3D) 的人乳腺癌 (细胞) 共培养系统的详细描述和人单核。在与单核细胞共培养时, 在活化、正常 T 细胞表达和分泌 (RANTES)、单核蛋白趋化 protein-1 (MCP-1) 和粒细胞-巨噬群刺激因子 (g-csf) 的基础上, 有较高的基底水平,炎细胞因子白细胞介素 (IL)-1 β (IL-1β) 和 IL-8 是丰富的结合基质金属蛋白酶 ()-1, MMP-2, 和 MMP-10。这种肿瘤基质微环境促进了 MCF-10A 3D 腺样结构、单核细胞 chemoattraction 和侵袭性的亡的抗性。这里提出的协议提供了一个负担得起的替代研究内通信, 是一个巨大的潜力的例子,在体外3D 细胞系统提供来审问肿瘤生物学的特定特征与肿瘤有关侵略.

3D 文化中的细胞形态学分析:
在5天的时间内, 研究了低密度和高密度3D 培养的初生细胞生长的形态学变化。在前48小时, 细胞坚持 ECME 和保持低密度。在这个时间点, 它可以清楚地看到, 细胞呈现出一个细长的纺锤样的形状, 一些有两个或更多的长细胞质预测和不显示明显的细胞细胞接触。经过5天的培养, 细胞增殖, ?...

Watch this Scientific Journal Video about Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System at JoVE.c

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

基于细胞外基质提取物 (ECME) 的三维系统中单核细胞与原代乳癌细胞的通讯分析

在这里, 我们描述了一个三维的培养方法来分析原发性乳腺癌细胞的形态学, 以及研究他们的直接/间接相互作用的单核和结果, 如胶原降解, 免疫细胞招募, 细胞侵袭和促进癌症相关的炎症。

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Research

JoVE Journal

Cancer Research

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

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

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

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

舌细胞外基质的制备与舌鳞癌体外重建

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

利用成像质谱技术捕获组织与细胞之间的小分子通信

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

三维流状入侵检测中的全人肿瘤基基质

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

小鼠乳腺皮下HC11和EpH4细胞的分化

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

使用微生理系统模拟人体乳房组织中的乳腺癌

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...