Name: analyzing the communication between monocytes and primary breast cancer cells in an extracellular matrix extract (ecme)-based three-dimensional system
Uploaded: 2018-01-08T13:00:00.000+00:00
Duration: 10 min 55 s
Description: 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

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, 参与者的身份是匿名的。患者包括被诊断为浸润性导管癌, 组织学等级2和临床阶段 II, 没有早先新辅助疗法在组织切除之前。患者均为女性, 平均年龄在56.8 岁 (范围42到 75),14。
1. 3D 细胞培养
<ol><li>种子 0.1 x 106 MCF-10A 单元格或主要的在25厘米2培养烧瓶中的 DMEM/F12 培养基补充5% 马血清, 100 U/毫升青霉素, 100 µg/毫升链霉素, 100 ng/毫升霍乱毒素, 0.5 µg/毫升可的松, 10 µg/毫升胰岛素, 20 ng/毫升表皮生长因子 (EGF)。种子 0.1 x 106商业 MCF-7 细胞, 75 厘米2培养皿中的 MDA-MB-231 细胞, DMEM/F12 培养基补充 10% FBS, 100 U/毫升青霉素, 100 µg/毫升链霉素。在37° c 的湿润 5% CO2环境中孵育。</li>
	<li>48小时后, 用10毫升无菌1x 磷酸缓冲盐水 (PBS) 冲洗细胞单层。处理在37° c 的情况下, 用0.05% 的胰蛋白酶和0.48 毫米乙酸酸 (EDTA) 的3毫升溶液进行细胞单层化。添加200µL 的相应血清, 以停止 trypsinization 和7毫升的培养基没有补充剂。通过移重细胞, 取出分10µL, 在 Neubauer 室中计数细胞。</li>
	<li>在一个8井室滑动系统的每一个井, 传播40µL 基地纯 ECME, 其次是孵化30分钟在37° c。</li>
	<li>在每个井, 安置800细胞悬浮在400µL DMEM/F12 (没有酚红色) 补充如在步骤 1.1, 但与以下变动: 为主要的细胞和 MCF-10A 细胞, 增加 4 ng/毫升 EGF 和 2% ECME;对于 MCF-7 和 MDA-MB-231, 只添加 2% ECME。</li>
	<li>在湿润的 5% CO2环境中孵育37° c 的区域性。每48小时替换一次区域性媒体。</li>
	<li>用光学显微镜记录每24小时15天细胞的形态学变化。为了分析主要的细胞形态学, MCF-10A 和 MDA-MB-231 细胞分别是有序腺泡形成和侵袭性癌样结构的好的参考细胞系。</li>
	<li>在100X 和200X 的放大率下获得明亮场显微镜下的细胞图像, 并将细胞形态学与参考 MCF-10A 和 MDA-MB-231 细胞系进行比较 (图 1)。</li>
</ol>2. 从肿瘤组织中获取原癌细胞
注意:获得肿瘤上皮细胞的组织从切除原发肿瘤的患者没有以前新辅助疗法;避免坏死区域, 并至少使用 0.5 cm3的肿瘤组织。
<ol><li>用无菌 1x PBS 冲洗肿瘤组织, 并用手术刀将其机械地分解成1-2 毫米的碎片。</li>
	<li>在室温 (RT) 2 毫升的无菌20毫升玻璃小瓶中, 用以下溶液消化组织: 1 毫克/毫升胶原酶 I 和 100 u/毫升透明质酸 DMEM/F12 培养基中含有 100 u/毫升青霉素和100µg/毫升链霉素, 不断搅拌。</li>
	<li>通过经消毒的薄纱片过滤产生的悬浮液, 消除大块的未消化组织。然后通过灭菌的100µm 孔膜过滤细胞悬浮液。</li>
	<li>将 430 x g 的细胞颗粒在 RT 5 分钟, 用无菌 1x PBS 冲洗两次。DMEM/F12 培养基中的培养初级细胞, 辅以5% 匹马血清、100种 U/毫升青霉素、100µg/毫升链霉素、100 ng/毫升霍乱毒素、0.5 µg/毫升氢化可的松、10µg/毫升胰岛素和 EGF 的 20 ng/毫升。每48小时将培养基更换为37° c, 在湿润的 5% CO2环境中保持培养。</li>
	<li>确保分离的细胞是上皮细胞与一个标准的免疫组织化学的协议14。测试三上皮标志物: 蛋白 (PanCK)、mucin-1 (Muc-1) 和上皮细胞黏附分子 (EpCAM) 的面板。</li>
</ol>3. 从外周血中分离 PM 细胞
<ol><li>提取大约40毫升的外周血从一个健康的志愿者, 稀释的血液在1:3 的比例无菌内毒素-免费 1x PBS, 并服从它的密度梯度分离。</li>
	<li>在锥形管中, 放置2毫升的聚和钠影培养基, 密度为1.077 克/毫升。小心, 慢慢地覆盖8毫升的稀释血液。离心机为30分钟, 765 x g 在 RT. 使用离心机最慢的加速减速速度。 注意:离心后, 四层将形成自下而上: 红血球颗粒, 密度梯度介质层, 单核细胞层 (它看起来像一个优良的白色环), 和等离子体层。</li>
	<li>用不育的巴斯德吸管小心翼翼地从渐变中检索单个核细胞层, 然后用 1x PBS 洗涤三次, 每次在 RT 时依次进行较慢的离心 (430、275和 191 x g)。</li>
	<li>使用负选择协议避免激活单元格。此类协议的一个示例如下:<ol>
<li>一次用洗涤缓冲液 (0.5% 牛血清白蛋白, 2 毫米 EDTA 在 1x PBS) 清洗单个核细胞。计算 Neubauer 腔室中的单元格, 并将它们调整为 1 x 107 cells/30 µL 的密度。</li>
		<li>对于每 1 x 107单元格, 添加10µL FcR 阻断试剂和10µL 单核生物素抗体鸡尾酒, 其中包含人 CD3、CD7、CD16、CD19、CD56、CD123 和血型 a (包括在商业套件中)。</li>
		<li>混合细胞, 孵育15分钟, 在4° c。添加一个额外的30µL 洗涤缓冲液加20µL 的 anti-biotin 微 (包括在套件);混合细胞, 孵育20分钟, 在4° c。</li>
		<li>用缓冲溶液冲洗细胞一次, 在 RT 时离心 430 x g, 在1.5 毫升的磁分离溶液中重。</li>
		<li>在 pre-rinsed 磁分离柱上加载单元悬浮, 并添加7毫升缓冲溶液。在一个15毫升的锥形管中收集单核细胞浓缩的分数, 计数的单元格, 如果没有立即培养, 冻结在密度为 2 x 106 cells/1 毫升的 DMEM/F12 培养基补充 50% FBS 和10% 亚砜在−80° c。 注意:避免在超过2月的冷冻后使用单核细胞, 因为它们的生存能力可能受到损害。</li>
	</ol>
</li>
	<li>在 DMEM/F12 培养基中, 以 6% FBS、100 U/毫升青霉素和100µg/毫升链霉素为补充, 在37° c 的湿润 5% CO2环境中维持 PMs 的培养。</li>
	<li>从至少两个不同的捐赠者那里独立地执行每组实验, 因为来自不同捐献者的单核细胞会导致单核细胞活化。</li>
</ol>4. 建立3D 共同文化
<ol><li>
3D co-cultures 与间接交互 注:使用0.4 µm 孔径膜的聚碳酸酯细胞培养板, 在24井底培养基中进行这些检测。在这个试验中, 清和细胞都可以在实验结束时被检索到。<ol>
<li>培养 2 x 106 U937 和 THP-1 单核细胞10毫升的 RPMI 1640 培养基补充 10% FBS, 1% 抗生素/抗, 并培养新的 PMs 补充 DMEM/F12 培养基 (如前所示, 在步骤 3.5), 在37° c 在湿化 5% CO2环境.</li>
		<li>板 4 x 105单核细胞在1毫升/其相应的培养基 (补充与 2% ECME 和 2% FBS U937 和 THP-1 单核细胞, 或 2% ECME 和 6% FBS 的 PMs)。</li>
		<li>在每个井中放置一个插入物, 并在相应的培养基中添加0.9 毫升的 4 x 105的细胞悬浮液 (辅以 2% ECME 和 2% FBS, 用于商业细胞系或5% 匹马血清用于初级的细胞介素)。将总介质的一半替换为每48小时。</li>
		<li>在37° c 的湿润 5% CO2环境中孵育5天, 并恢复清。如果执行后续分析, 则按 trypsinization 检索单元格。</li>
		<li>将单个单元格区域性的控件包含在各自的媒体中。</li>
	</ol>
</li>
	<li>
3D co-cultures 与直接互动
	<ol>
<li>在细胞培养前对不同荧光染料的细胞和单核蛋白进行标记, 以便在培养后对每个细胞谱系进行独立分类, 以分析 mRNA 和蛋白质的表达。使用可自由通过活细胞细胞膜的香豆素和罗丹明的商用衍生物。标签的一般协议如下:<ol>
<li>准备一单层的权益细胞 (2 x 106细胞在 25 cm2培养烧瓶中), 用足够的5ml 工作溶液取代标准介质 (1:2, 000 的荧光染料在标准基质中没有任何补充)。在37° c 的湿润 5% CO2环境中孵育30分钟。抽出染料溶液, 用足够的 1x PBS 轻轻冲洗。吸入 PBS 并添加标准介质。</li>
			<li>处理在37° c 的情况下, 用0.05% 的胰蛋白酶和0.48 毫米 EDTA 的2毫升溶液进行细胞单层化。添加200µL FBS, 以停止 trypsinization 和7毫升的通讯员媒体没有补充。重细胞移。取一分10µL, 在 Neubauer 室中计数细胞。在细胞计数后继续与共培养协议。</li>
			<li>在 RT 中, 在 430 x g 处对5分钟的细胞进行颗粒 (这首先是因为在悬浮液中单核生长)。用3毫升的荧光染料的5ml 工作溶液 (1:2, 000 的荧光染料在不加补充的标准基质介质中) 丢弃上清和轻度重细胞。在37° c 的湿润 5% CO2环境中孵育30分钟。</li>
			<li>再次颗粒细胞, 丢弃染料工作溶液, 轻轻重5-7 毫升的 1x PBS。再一次, 丢弃 PBS, 在5-7 毫升的标准培养基中重细胞。以分10µL 细胞悬液计数细胞在 Neubauer 室。在细胞计数后继续与共培养协议。</li>
		</ol>
</li>
		<li>设置共培养如下:: 在4井室滑动系统的每个井中, 在井底铺上足够的 ECME, 形成均匀的层。板20µL 的一个单一的细胞悬浮包含 5 x 105标记的每井细胞。</li>
		<li>在37° c 的孵育15-20 分钟后, 在5µL 试验培养基中加入 2.5 x 1080的悬浮液, 并附上 60% ECME。</li>
		<li>允许 ECME 在37° c 固化15-20 分钟。</li>
		<li>加入1毫升的1:1 混合的细胞和单核培养培养基。</li>
		<li>在37° c 的湿润 5% co2环境中孵育 co-cultures, 用于24小时、48小时或5天来跟踪不同时间点的变化。</li>
		<li>降解 ECM 蛋白, 从培养基中回收细胞。吸气并丢弃培养基, 加入0.5 毫升的 1x PBS, 0.1% 胰蛋白酶和 0.25% EDTA, 在37° c 下孵育3小时。孵育后, 加入0.5 毫升的 1x PBS 与 10% FBS 中和胰蛋白酶, 并重细胞移大力获得一个单细胞悬浮。</li>
		<li>颗粒细胞在 765 x g, 5 分钟在 RT, 丢弃上清, 并重5毫升的 1x PBS 与 10% FBS 细胞。重复此步骤一次。</li>
		<li>用适当的仪器将细胞悬浮到荧光活化的细胞分类 (外地)。确保最后的种群至少有95% 的纯度)。</li>
		<li>分拣后, 用无菌 1x pbs、颗粒 (RT 5 分钟 430 x g) 冲洗细胞, 在无菌 1x pbs 中重。细胞可以根据特定的协议进行 RNA 分离或蛋白质分析。</li>
		<li>重复每个化验三次, 包括每个细胞谱系的3D 文化作为控制。</li>
	</ol>
</li>
	<li>
3D co-cultures 对胶原降解的直接交互作用
	<ol>
<li>建立3D 细胞 co-cultures, 如步骤4.2 所述, 附加步骤添加32.5 µg/毫升的 IV 型胶原标记荧光素异硫氰酸 ECME。ECME 中 IV. 型胶原的最终浓度为0.5%。在培养皿中种植35毫米的片。</li>
		<li>在培养皿的底部散布一层40µL 的 ECME。允许 ECME 在37° c 固化。</li>
		<li>在介质的10µL 中添加 2.5 x 105的单元格, 并允许单元格安定下来。</li>
		<li>在40µL 测定培养基中加入 1.25 x 105 U937 单核细胞悬浮液 (补充60% 的 DQ-胶原蛋白 IV 标记-ECME)。</li>
		<li>允许 ECME 在37° c 固化15-20 分钟。</li>
		<li>加入2毫升的1:1 混合的细胞和单核培养培养基。</li>
		<li>在37° c 的湿润 5% co2环境中孵育 co-cultures 5 天。每48小时替换一次区域性媒体。</li>
		<li>分析了共焦扫描显微镜中的荧光发射, 并测量了每50µm2的集成光学密度 (IOD)。将 IODs 与单个区域性和区域性进行比较, 时间为零 (图 2)。</li>
	</ol>
</li>
</ol>5. 与间接相互作用的3D 共文化中的清分析
<ol><li>经过5天的共培养, 恢复清从上部和下部车厢的 3D co-cultures, 并从单独的3D 文化控制。用移把每一上清液拌匀。分, 并储存在−20°C 上清, 直到使用 (最多一个月)。 注意:如果存储时间超过一个月, 请将清保留在−80°C。</li>
	<li>分析清与复用化验平台, 酶联免疫吸附试验 (ELISA), 或 bead-based 免疫按照制造商的推荐程序。 注意:清也可以用印迹或通过特定的孔隙过滤器进行浓缩, 以获得大小丰富的蛋白质组分来进行酶分析16,17。另外, 如下所述, 使用清作为条件介质进行其他实验。条件媒体通常是从5天的区域性 (图 3) 中获得的。</li>
</ol>6. 清原细胞对腺形成和腺结构影响的表征
<ol><li>在一个8井室滑动系统的每个井传播一个40µL 基地 ECME 并且让它凝固为30分钟在37° c。</li>
	<li>种子 800 MCF-10A 细胞/井在400µL 补充 DMEM/F12 培养基, 如步骤1.2 所述。</li>
	<li>增加400µL 的条件培养基或补充 DMEM/F12 培养基与 20 ng/毫升的人类重组 IL-1β。</li>
	<li>将房间滑入一个玻璃培养皿中, 其中含有35毫米的 petri 皿, 里面装满2毫升的 PBS, 以创造一个湿度环境。</li>
	<li>每48小时更换一次介质/上清液。</li>
	<li>用光学显微镜记录每24小时14天的腺腺形成。</li>
	<li>经过14天的培养, 染色腺与100µL 4 ', 6-diamidino-2-吲 (DAPI) 在 1x PBS 浓度 100 nM 观察细胞核。在恒定搅拌的 RT 中孵育25分钟。冲洗三次与 1x PBS 5 分钟, 每次在不断搅拌在 RT. 安装的准备与安装的介质专门用于荧光染色。用透明的抛光剂密封, 在4° c 下保持免受光线的保护。</li>
	<li>用共焦显微镜对腺进行分析, 在不同深度的腺 (图 4a、B、C) 中拍摄横向堆栈的图像。</li>
	<li>在腺中使用适当的染色协议来可视化不同的细胞蛋白。例如, e-钙黏蛋白被染色, 以评估细胞粘附, 细胞极性, 和/或流明形成18 (图 4D)。</li>
</ol>7. 迁移化验
注意:使用8µm 孔径膜的聚碳酸酯细胞培养材料, 在24层底培养板上进行 U937、THP-1 和新鲜 PMs 的迁移测定。这是先前证明的趋化因子 GM-CSF, MCP-1, 和 RANTES 被发现在高浓度的个体3D 文化的积极的细胞系。有人建议, 这些细胞因子是关键的吸引单核瘤的部位的原发肿瘤;这是测试的迁移化验使用细胞因子作为化。
<ol><li>文化 U937, THP-1, 或新的 PMs, 如步骤4.1.1 所述。</li>
	<li>每次用200µL RPMI 1640, 无血清即可冲洗膜。</li>
	<li>用50µL 的冷 ECME, 将插入物放在24井底培养板上, 并允许 ECME 在37° c 时聚合1小时。</li>
	<li>添加180µL RPMI 无 FBS 在板的上部车厢。添加在较低的会议厅没有起泡800µL 的 RPMI 补充 100 ng/毫升的 GM CSF, MCP-1, 或 RANTES 作为趋化因子。使用不含细胞因子的培养基作为阴性对照。孵育30分钟, 在37° c 建立一个趋化因子梯度。</li>
	<li>在无菌管中放置每种单核细胞的 1.5 x 105 , 用1毫升的 1x PBS 冲洗两次, 在 RT 上为430分钟离心 5 x g. 重单核细胞在20µL RPMI 没有 FBS, 安置他们在插入物, 并且非常仔细地质细胞悬浮 (上部房间的最后的容量是200µL)。</li>
	<li>允许在湿润的 5% CO2环境中的单元迁移在37° c 时进行24小时的移动。 注意:由于单核细胞是换药的, 因此迁徙的单元通常会在下层的介质中。</li>
	<li>使用 Neubauer 腔或流式细胞仪移除插入物、检索介质以及在2、4、6和 24 h 处计数单元格;或者, 特别是如果在介质中找不到单元格, 则使用数码相机获取插入物下部的图像。3随机字段中的平均单元格计数 (在100X 放大倍数) 用于分析 (图 5)。</li>
</ol>8. 入侵化验
注意:利用8µm 孔径膜的聚碳酸酯细胞培养材料, 对24层底培养皿中的细胞进行入侵检测。在我们最初的研究中, IL-8 是一个细胞因子丰富的清的细胞/单核 3D co-cultures。这种细胞因子是否参与了对细胞系侵入的检测。
<ol><li>在 75 cm2烧瓶中展开 "零售商" 单元格。MCF-7、T47D、HS578T 和 MDA-MB-231, 如步骤1.2 所述 (但不含 ECME) 和步骤2.4 中所述的主要的 "细胞"。</li>
	<li>一次洗涤每一次聚碳酸酯8µm-孔膜细胞培养插入与200µL 培养基没有血清 (使用的媒介取决于被测试的细胞线) 水合膜。</li>
	<li>用50µL 的冷 ECME (1:4 稀释 ECME: 无 FBS 的冷介质) 填充插入物, 在24井底培养板上放置刀片, 并允许 ECME 在37° c 处1小时内聚合。</li>
	<li>一旦 ECME 有聚合, 添加180µL 各自的细胞介质没有 FBS。添加800µL 的媒体不 FBS 通过插入的狭缝。孵育30分钟, 在37° c, 建立一个 IL-8 梯度。 注意:避免在媒体中创建气泡。使用的媒体是没有 FBS, 但补充 100 ng/毫升的 IL-8 作为一个趋化, 或纯媒体没有 FBS, 作为负控制。</li>
	<li>获取每个单元格行的总计 6 x 105单元格, 如下所示:<ol>
<li>丢弃清, 冲洗一次与10毫升的 1x PBS, 并添加2毫升0.05% 胰蛋白酶/0.48 毫米 EDTA 2 分钟在37° c 分离的细胞。添加4毫升补充培养基, 以停止胰蛋白酶反应和重大力移。</li>
	</ol>
</li>
	<li>采取10µL 分细胞悬浮计数细胞在一个 Neubauer 室。取一卷包含 6 x 105单元的单元格挂起。离心机在 430 x g 为5分钟, 在 RT, 丢弃上清, 并冲洗1毫升的 1x PBS 细胞。再重复一次冲洗。</li>
	<li>重的细胞在20µL 各自媒介没有 FBS 和地方在插入物包含180µL 各自媒介没有 FBS。仔细质移的细胞悬浮。</li>
	<li>在湿润的 5% CO2环境中, 允许细胞侵入在37° c 的48小时内进行。</li>
	<li>48小时后, 取出插入物, 丢弃上清液, 用200µL 1x PBS 仔细冲洗一次。用棉签除去 PBS 的过量。 注意:侵入细胞通常附着在插入物的另一侧, 在这种情况下细胞是附着的。</li>
	<li>通过将插入物放置在一个24井底培养板的井中, 在 RT 中放入1毫升的4% 甲醛. 之后, 用 1x PBS 冲洗一次刀片。</li>
	<li>将插入物放在一个24井底培养板的井中, 在 RT 中放置1毫升的 1x PBS, 0.2% 结晶紫, 将其染色. 小心, 用棉签将所有的 ECME 从膜上取下, 其中含有不迁移的细胞, 用蒸馏水仔细冲洗, 以避免清除固定的侵入细胞。</li>
	<li>通过观察倒置显微镜下插入物的下侧来计数侵入细胞。使用3随机场 (100X 放大倍数) 的平均单元数。在细胞侵入成簇且难以单独计数的情况下, 用数码相机从3随机场 (100X 放大倍数) 中获取图像。用软件对图像进行分析, 并使用 IOD 来量化细胞入侵 (图 6)。</li>
</ol>

<ol>
	<li>Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+understanding+tumor+hierarchy+and+heterogeneity.">Cancer stem cells: understanding tumor hierarchy and heterogeneity.</a> Medicine (Baltimore). 95 (1), 2-7 (2016).</li><li>Wang, 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=Role+of+tumor+microenvironment+in+tumorigenesis.">Role of tumor microenvironment in tumorigenesis.</a> J Cancer. 8 (5), 761-773 (2017).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nat Med. 19 (11), 1423-1437 (2013).</li><li>Sica, 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=Macrophage+polarization+in+tumour+progression.">Macrophage polarization in tumour progression.</a> Semin Cancer Biol. 18 (5), 349-355 (2008).</li><li>Mantovani, A., Marchesi, F., Malesci, A., Laghi, L., Allavena, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-associated+macrophages+as+treatment+targets+in+oncology.">Tumour-associated macrophages as treatment targets in oncology.</a> Nat Rev Clin Oncol. , (2017).</li><li>Petersen, O. W., Ronnov-Jessen, L., Howlett, A. R., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+with+basement+membrane+serves+to+rapidly+distinguish+growth+and+differentiation+pattern+of+normal+and+malignant+human+breast+epithelial+cells.">Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.</a> Proc Natl Acad Sci U S A. 89 (19), 9064-9068 (1992).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+oncogenesis+of+MCF-10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures.">Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> Methods. 30 (3), 256-268 (2003).</li><li>Debnath, 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=The+role+of+apoptosis+in+creating+and+maintaining+luminal+space+within+normal+and+oncogene-expressing+mammary+acini.">The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini.</a> Cell. 111 (1), 29-40 (2002).</li><li>Muthuswamy, S. K., Li, D., Lelievre, S., Bissell, M. J., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ErbB2,+but+not+ErbB1,+reinitiates+proliferation+and+induces+luminal+repopulation+in+epithelial+acini.">ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini.</a> Nat Cell Biol. 3 (9), 785-792 (2001).</li><li>Sameni, M., Dosescu, J., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+proteolysis:+stromal+and+inflammatory+cells+increase+tumor+proteolysis.">Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis.</a> Mol Imaging. 2 (3), 159-175 (2003).</li><li>Sameni, 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=MAME+models+for+4D+live-cell+imaging+of+tumor:+microenvironment+interactions+that+impact+malignant+progression.">MAME models for 4D live-cell imaging of tumor: microenvironment interactions that impact malignant progression.</a> J Vis Exp. (60), (2012).</li><li>Jedeszko, C., Sameni, M., Olive, M. B., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+protease+activity+in+living+cells:+from+two+dimensions+to+four+dimensions.">Visualizing protease activity in living cells: from two dimensions to four dimensions.</a> Curr Protoc Cell Biol. 4, 20 (2008).</li><li>Chimal-Ramirez, G. 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=MMP1,+MMP9,+and+COX2+expressions+in+promonocytes+are+induced+by+breast+cancer+cells+and+correlate+with+collagen+degradation,+transformation-like+morphological+changes+in+MCF-10A+acini,+and+tumor+aggressiveness.">MMP1, MMP9, and COX2 expressions in promonocytes are induced by breast cancer cells and correlate with collagen degradation, transformation-like morphological changes in MCF-10A acini, and tumor aggressiveness.</a> Biomed Res Int. 2013, 279505 (2013).</li><li>Espinoza-Sanchez, N. A., Chimal-Ramirez, G. K., Mantilla, A., Fuentes-Panana, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1beta,+IL-8,+and+Matrix+Metalloproteinases-1,+-2,+and+-10+Are+Enriched+upon+Monocyte-Breast+Cancer+Cell+Cocultivation+in+a+Matrigel-Based+Three-Dimensional+System.">IL-1beta, IL-8, and Matrix Metalloproteinases-1, -2, and -10 Are Enriched upon Monocyte-Breast Cancer Cell Cocultivation in a Matrigel-Based Three-Dimensional System.</a> Front Immunol. 8, 205 (2017).</li><li>Li, Y., Wang, L., Pappan, L., Galliher-Beckley, A., Shi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1beta+promotes+stemness+and+invasiveness+of+colon+cancer+cells+through+Zeb1+activation.">IL-1beta promotes stemness and invasiveness of colon cancer cells through Zeb1 activation.</a> Mol Cancer. 11, 87 (2012).</li><li>Gallagher, S., Winston, S. E., Fuller, S. A., Hurrell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblotting+and+immunodetection.">Immunoblotting and immunodetection.</a> Curr Protoc Mol Biol. 10, 18 (2008).</li><li>Troeberg, L., Nagase, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymography+of+metalloproteinases.">Zymography of metalloproteinases.</a> Curr Protoc Protein Sci. 21, 15 (2004).</li><li>Arevalo-Romero, H., Meza, I., Vallejo-Flores, G., Fuentes-Panana, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+CagA+and+IL-1beta+Promote+the+Epithelial-to-Mesenchymal+Transition+in+a+Nontransformed+Epithelial+Cell+Model.">Helicobacter pylori CagA and IL-1beta Promote the Epithelial-to-Mesenchymal Transition in a Nontransformed Epithelial Cell Model.</a> Gastroenterol Res Pract. 2016, 4969163 (2016).</li><li>Reed, J. R., Leon, R. P., Hall, M. K., Schwertfeger, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1beta+and+fibroblast+growth+factor+receptor+1+cooperate+to+induce+cyclooxygenase-2+during+early+mammary+tumourigenesis.">Interleukin-1beta and fibroblast growth factor receptor 1 cooperate to induce cyclooxygenase-2 during early mammary tumourigenesis.</a> Breast Cancer Res. 11 (2), 21 (2009).</li><li>Ma, L., 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=Epidermal+growth+factor+(EGF)+and+interleukin+(IL)-1beta+synergistically+promote+ERK1/2-mediated+invasive+breast+ductal+cancer+cell+migration+and+invasion.">Epidermal growth factor (EGF) and interleukin (IL)-1beta synergistically promote ERK1/2-mediated invasive breast ductal cancer cell migration and invasion.</a> Mol Cancer. 11, 79 (2012).</li><li>Grande, S. M., Bannish, G., Fuentes-Panana, E. M., Katz, E., Monroe, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonic+B-cell+and+viral+ITAM+signaling:+context+is+everything.">Tonic B-cell and viral ITAM signaling: context is everything.</a> Immunol Rev. 218, 214-234 (2007).</li><li>Benton, G., Arnaoutova, I., George, J., Kleinman, H. K., Koblinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+from+discovery+and+ECM+mimicry+to+assays+and+models+for+cancer+research.">Matrigel: from discovery and ECM mimicry to assays and models for cancer research.</a> Adv Drug Deliv Rev. 79-80, 3-18 (2014).</li><li>Worthington, P., Pochan, D. J., Langhans, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+Hydrogels+-+Versatile+Matrices+for+3D+Cell+Culture+in+Cancer+Medicine.">Peptide Hydrogels - Versatile Matrices for 3D Cell Culture in Cancer Medicine.</a> Front Oncol. 5, 92 (2015).</li><li>Tibbitt, M. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+extracellular+matrix+mimics+for+3D+cell+culture.">Hydrogels as extracellular matrix mimics for 3D cell culture.</a> Biotechnol Bioeng. 103 (4), 655-663 (2009).</li><li>Schlaermann, 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=A+novel+human+gastric+primary+cell+culture+system+for+modelling+Helicobacter+pylori+infection+in+vitro.">A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro.</a> Gut. 65 (2), 202-213 (2016).</li><li>Pompaiah, M., Bartfeld, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+Organoids:+An+Emerging+Model+System+to+Study+Helicobacter+pylori+Pathogenesis.">Gastric Organoids: An Emerging Model System to Study Helicobacter pylori Pathogenesis.</a> Curr Top Microbiol Immunol. 400, 149-168 (2017).</li><li>Takebe, 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=Vascularized+and+functional+human+liver+from+an+iPSC-derived+organ+bud+transplant.">Vascularized and functional human liver from an iPSC-derived organ bud transplant.</a> Nature. 499 (7459), 481-484 (2013).</li><li>Takasato, 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=Directing+human+embryonic+stem+cell+differentiation+towards+a+renal+lineage+generates+a+self-organizing+kidney.">Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney.</a> Nat Cell Biol. 16 (1), 118-126 (2014).</li></ol>

作者声明他们没有任何利益冲突。

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

analyzing-communication-between-monocytes-primary-breast-cancer-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

10.9K 次观看.  Hospital Infantil de México Federico Gómez. 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。

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

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

球体的检测，以测量TGF -β诱导的入侵

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

科研

医学

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

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 fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

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 ...

癌症研究

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

制备及分析<em&gt;体外</em&gt;三维乳腺癌代理项

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

生物工程

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

<meta charset="utf8"></head><body>检查 3D 胶原基质设置中的巨噬细胞-肿瘤球状体相互作用

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

<meta charset="utf8"></head><body>巨噬细胞-肿瘤球状体共侵袭测定

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

巨噬细胞-肿瘤球状体共侵袭测定

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

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

摘要

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此视频中的章节

球体的检测，以测量TGF -β诱导的入侵

使用微生理系统模拟人体乳房组织中的乳腺癌

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

乳腺癌细胞侵袭定量使用三维（3D）模型

制备及分析

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

巨噬细胞-肿瘤球状体共侵袭测定

巨噬细胞-肿瘤球状体共侵袭测定

巨噬细胞-肿瘤球状体共侵袭测定

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭