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>

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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。

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			<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>
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		<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>
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		<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>
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			<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>
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			<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>
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			<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>
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		<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, ...

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

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

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

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

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

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

科研

癌症研究

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

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

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

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

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

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

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

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

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

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

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

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

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

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

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 ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

研究使用细胞外基体水凝胶的正常组织辐射效应

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

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

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange 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

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

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

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 of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

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 ...