作者感谢霍华德-克劳福德博士 (密歇根大学) 实验室的技术支持, 为这项研究提供材料和设备, 艾伦凯莱赫技术支持。
这项工作由密歇根大学 Rogel 癌症中心核心赠款 CA046592-26S3、nih K08 CA201335-01A1 (进工党)、BCAN YIA (进工党)、nih R01 CA17483601A1 (DMS) 资助。

1. 生长中的癌症球体
<ol><li>
从细胞线生长
	<ol>
<li>培养人膀胱癌细胞在常规黏附细胞培养情况下和维护在37°c 孵化器供应与 5% CO2。保持细胞在 < 90% 融合。 注: 所使用的培养基是 Dulbecco 的改良最小必需培养基 (DMEM), 含有4.5 克/升 d-葡萄糖, l-谷氨酰胺, 110 毫克/升丙酮钠, 并在本议定书中提供10% 胎牛血清 (血清)。</li>
		<li>在实验开始前的一天, 胰蛋白酶处理 (通过使用0.25% 胰蛋白酶-EDTA) 细胞, 量化细胞浓度, 并在3毫升培养基中分布 1 x 106细胞, 每一个井中都有一高的超低附着板。</li>
		<li>在低附着条件下孵化细胞在37°c ≥16 h。这应该允许足够的时间让细胞聚集成球体的大多数膀胱癌细胞株。 注: 球体的形成可以通过倒亮场显微镜观察。球体的最佳大小和数量可能取决于个别实验, 但我们发现, 直径从50µm 到150µm 的球体通常适用于使用20X 物镜的时间推移成像。</li>
		<li>要将其他细胞类型 (如成纤维细胞或免疫细胞) 纳入球体, 在超低附着板中混合所需的细胞和癌细胞, 并在37摄氏度内孵育≥16 h 以允许形成混合细胞类型球体。</li>
	</ol>
</li>
	<li>
从原发肿瘤生长 注: 肿瘤球体也可从原发膀胱肿瘤来源, 如由致癌性膀胱肿瘤模型开发的肿瘤8。例如, 用 n-丁基 n (4-丁基) 中亚硝胺 (BBN) 喂养的小鼠产生的膀胱肿瘤, 可以用无菌手术器械收割和碎成小块 (约0.5 毫米3)。本系统还可以研究消化性膀胱癌的主要标本。<ol>
<li>收集和洗涤0.5 毫米3肿瘤片在5毫升冰冷磷酸盐缓冲生理盐水 (PBS)。离心在 200 x g 5 分钟在4摄氏度。重复这个洗涤步骤一次。</li>
		<li>以 200 x g为10分钟, 以4摄氏度的离心法收集肿瘤片。取出上清液, 加入5毫升的 DMEM, 含10% 的血清。将肿瘤球体/介质混合物转移到6井超低附着板上。在嵌入胶原基质之前, 在37摄氏度的≥16中孵化 (见步骤 2.3)。</li>
	</ol>
</li>
</ol>2. 准备3维文化室
<ol><li>
稀释 type-1 胶原蛋白 (来源于鼠尾) 在 DMEM 含有10% 的血清, 使2毫克/毫升混合物根据制造商的指示。
	<ol>
<li>简而言之, 根据制造商的指示, 以达到生理 pH 值, 将胶原蛋白与适量的 1 N 氢氧化钠溶液和 DMEM 混合在一起。</li>
		<li>在混合了胶原和 DMEM 后, 快速地涂上腔板的水井 (对于大多数应用来说, 在凝固前用胶原-DMEM 混合物 (200 µL/井), 带1.5 盖玻璃的会议厅幻灯片是最佳的)。允许涂布室滑动在室温下固定15分钟。 注: 根据实验的目的, 通过添加其他细胞外基质成分, 如纤维粘连蛋白或层粘连蛋白, 可以对胶原基基质的组成进行修改。</li>
	</ol>
</li>
	<li>轻轻吸管500µL 的媒体包含球体 (或肿瘤片断, 如果主要肿瘤样本使用) 从6井超低附着板成空1.5 毫升离心管。等待2分钟让球体安定到管子的底部。</li>
	<li>小心地从试管中取出上清液。准备另一管 type-1 胶原蛋白 (2 毫克/毫升) 混合 DMEM 含有10% 的血清, 并迅速添加500µL 的这种混合物球体。用慢吹打轻轻地混合球体和胶原蛋白。</li>
	<li>添加250µL 的球体/胶原蛋白混合物的一个良好的胶原预涂膜室幻灯片。允许含有球体的胶原基质完全凝固 (约30分钟37摄氏度)。</li>
	<li>胶原基质固化后, 添加1毫升的 DMEM, 其中每一个井含10% 个血清 (图 1B)。孵化的房间幻灯片在37摄氏度孵化器提供 5% CO2 , 直到准备成像。</li>
</ol>3. 活细胞时间推移成像
<ol><li>根据制造商的指示打开共焦显微镜, 确保气候室达到37摄氏度, 并提供 5% CO2。 注: 我们的显微镜和腔室通常需要1小时才能达到系统平衡。</li>
	<li>小心地将会议厅滑动到附着在显微镜上的滑动适配器。</li>
	<li>使用低功耗目标 (如5X) 定位感兴趣的球体, 并以更高的功率目标开始成像 (在当前协议中, 20X 目标用于大多数活细胞成像, 以提高图像质量)。 注: 对于原发性膀胱肿瘤标本, 可能需要5X 和10X 的目标, 以覆盖较大的成像区域。对于我们的时间推移成像, 成像间隔设置为30分钟, Z 堆栈用于图像的整个球体。通常, z 切片之间的距离设置为20X 目标的4µm, 而 z 轴的总距离约为200µm. 图像可以使用 DIC 和荧光如果细胞/组织海港荧光蛋白标记。</li>
	<li>执行 24–72 h 的时间推移成像。 注意: 持续时间由细胞类型和实验需要决定。</li>
</ol>4. 用于冰冻组织切片的肿瘤球体样品块的制备
<ol><li>用小镊子小心地将胶原凝胶块和球体从腔内滑出。将胶原凝胶块置于塑料组织学模型中。</li>
	<li>简单地用 1x pbs 冲洗胶原凝胶, 然后在室温下用4% 多聚甲醛 (粉煤灰) 在 pbs 中固定30分钟。 注意: 粉煤灰是潜在的致癌物, 应该小心处理。</li>
	<li>用1.5 毫升的 1x pbs 在振动筛上洗15分钟的胶原凝胶. 更换 PBS 并重复上一个洗涤步骤3x。</li>
	<li>应用一个薄层 (约3毫米) 的最佳切削温度 (OCT) 化合物覆盖底部的一个新的塑料组织学模具。将固定的和水洗的胶原凝胶放在 oct 化合物的顶部, 然后用 oct 化合物填充霉菌, 并避免在 oct 中形成任何气泡, 仔细地嵌入整个凝胶。</li>
	<li>将模具保持在4摄氏度, 1 小时。</li>
	<li>将含有样品的霉菌和 OCT 化合物放在漂浮在液氮上的100毫米培养皿上。允许样品完全闪光冷冻。</li>
	<li>将冷冻样品块存放在-80 摄氏度, 供将来使用。</li>
</ol>5. 冰冻切片肿瘤球体免疫荧光成像
<ol><li>将冰冻样品块从-80 °c 冷藏库转移到恒温器的-20 °c 室。</li>
	<li>
通过将节间隔设置为7µm 来执行常规的冻结切片。让切片样品附着在没有皱纹或被困空气的玻璃滑梯上。
	<ol>
<li>将幻灯片存放在-80 摄氏度前, 再进行染色。 注意: 不同的间隔时间可以用来适应实验需要。</li>
	</ol>
</li>
	<li>空气干燥的幻灯片在室温下1小时。</li>
	<li>Permeabilize 样品与 PBS 包含0.5% 海卫 X-100 15 分钟。</li>
	<li>洗涤样品3x 与 PBS 为10分钟每洗涤。</li>
	<li>用疏水性屏障笔将样品用疏水屏障包围在滑梯上。</li>
	<li>用阻断溶液 (1x PBS 含5% 牛血清白蛋白) 治疗样品, 室温1小时。</li>
	<li>应用40µL 的原发性抗体溶液 (1x PBS, 含 5% BSA, 1:100 稀释原抗体), 每张幻灯片上的样品。</li>
	<li>以37°c 为1小时, 或在室温下, 在一夜之间孵化出初级抗体的幻灯片 (潜伏期和条件因原抗体而异)。</li>
	<li>用 PBS 清洗样品 3x (每次清洗) 15 分钟。</li>
	<li>应用40µL 的二次抗体含液 (1x PBS 含有5% 的 BSA 1:300 次抗体稀释) 的每一个样本在幻灯片上。</li>
	<li>用 PBS 清洗样品 3x (每次清洗) 15 分钟。</li>
	<li>将样品与赫斯特33342溶液 (1 µg/毫升, 在 PBS 稀释) 在室温下染色10分钟。然后用 PBS 冲洗样品5分钟。</li>
	<li>装上安装介质的样品并盖上适当大小的盖板。在室温下将幻灯片保持在24小时的黑暗中, 然后进行共焦显微术。</li>
</ol>

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作者声明没有竞争的财政利益。

在这里, 我们描述了一个3维的肿瘤球体模型, 允许实时观察膀胱癌侵袭, 这是至关重要的癌症进展和转移。该系统可以将各种基质和细胞成分结合在一起, 使研究者能够更好地复述发生膀胱癌侵袭的组织微环境。膀胱癌球体可以从各种来源, 如细胞系 (包括基因修饰细胞线, 用于检查影响侵袭过程的信号通路) 和小鼠原发性肿瘤 (这里描述)。它也有可能适应人类原发性肿瘤的分析。根据所使用的成像?...

侵袭是癌症进展的一个关键步骤, 这是转移的需要, 并与较低的生存和预后较差的患者。在人类膀胱癌中, 泌尿道最常见的恶性肿瘤每年导致大约16.5万人死亡, 癌症分期、治疗和预后直接关系到入侵1的存在或缺失。大约75% 的膀胱癌病例为非肌肉侵入, 并以局部切除术进行管理。相反, 肌肉侵袭性膀胱癌 (约25% 的所有病例) 是积极的肿瘤, 转移率高, 并以积极的综合治疗2,3治疗。因此, 了解触发入侵的分子通路, 对于更好地表征侵入性进展的危险和发展能够预防侵入性进展的治疗干预是至关重要的。
肿瘤侵袭性进展发生在复杂的三维 (3) 环境中, 与其他肿瘤细胞、基质、基底膜和其他类型的细胞 (包括免疫细胞、成纤维细胞、肌肉细胞和血管) 相互作用。内皮细胞。渗透性支持 (例如Transwell) 检测系统通常用于定量癌细胞入侵4, 但这些系统是有限的, 因为它们不允许对入侵过程进行实时的显微监测和对样品进行进一步染色和分子分析的检索具有挑战性。研制一种3维膀胱肿瘤球体系统来研究入侵是可取的, 因为它允许在体外系统的方便的情况下纳入已定义的微环境成分。
在本议定书中, 我们描述了一个系统, 以检查人体膀胱癌细胞的侵袭过程, 使用3维球形入侵检测, 包括胶原基凝胶基质和共焦显微镜, 以允许调查人员监测细胞活力和实时入侵 (图 1A)。该系统是多才多艺的, 可以修改, 以询问各种基质/肿瘤的设置。它可以纳入大多数膀胱癌细胞系或原发性膀胱肿瘤和其他基质细胞, 如癌症相关的成纤维细胞和免疫单元5,6,7。该协议描述了由 type-1 胶原组成的基质, 但可以被修改以加入其他分子, 如纤维粘连蛋白, 层粘连蛋白, 或其他胶原蛋白。侵入过程可以遵循72小时或更长的时间取决于显微镜和系统使用的能力。3维基质中嵌入的肿瘤的固定和免疫荧光染色允许对侵入细胞中的蛋白质上调进行审问, 从而提供通常不存在或难以收集的关键信息。使用其他3维文化模型。该系统还可用于屏蔽入侵的化合物, 并描绘受此类化合物影响的信号通路。

<table><tbody><tr><td>Human bladder cancer cell lines UM-UC9, UM-UC13, UM-UC14, UM-UC18, 253J</td><td></td><td></td><td></td></tr><tr><td>DMEM cell culture medium</td><td>Thermo Fisher Scientific</td><td>11995065</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Thermo Fisher Scientific</td><td>15240062</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Thermo Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A3803</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS), pH 7.4&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Costar Ultral-low attachment 6-well cluster&amp;nbsp;</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>Conventional inverted microscope&amp;nbsp;</td><td>Carl Zeiss</td><td>491206-0001-000</td><td>General use for cell culture and checking spheroids</td></tr><tr><td>Collagen type 1 from rat tail, high concentration&amp;nbsp;</td><td>Corning</td><td>354249</td><td></td></tr><tr><td>Nunc Lab-Tek II Chambered Coverglass</td><td>Thermo Fisher Scientific</td><td>155382</td><td></td></tr><tr><td>Confocal microscope&amp;nbsp;</td><td>Carl Zeiss</td><td>LSM800</td><td>A confocal miscoscope with climate chamber, multi-location imaging, and Z-stack scanning function&amp;nbsp;</td></tr><tr><td>Cryostat micromtome</td><td>Leica Biosystems</td><td>CM3050 S</td><td></td></tr><tr><td>Zen 2 Image processing software&amp;nbsp;</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Paraformaldehyde solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>ImmEdge Hydrophobic Barrier PAP Pen</td><td>Vector Laboratories&amp;nbsp;</td><td>H4000</td><td></td></tr><tr><td>O.C.T compound&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>23730571</td><td></td></tr><tr><td>Hoechst 33342 solution&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>62249</td><td></td></tr><tr><td>Anti-ATDC (Trim29) antibody</td><td>Sigma-Aldrich</td><td>HPA020053</td><td></td></tr><tr><td>Anti-Cytokeratin 14 antibody</td><td>Abcam</td><td>ab7800</td><td></td></tr><tr><td>Anti-Vimentin antibody</td><td>Abcam</td><td>ab24525</td><td></td></tr><tr><td>ProLong Diamond&amp;nbsp;</td><td></td><td></td><td>Mounting medium</td></tr></tbody></table>

3-d cell culture system for studying invasion and evaluating therapeutics in bladder cancer

膀胱癌是一个重要的健康问题。据估计, 今年美国将有超过1.6万人死于膀胱癌。虽然75% 的膀胱癌是无创性的, 而且不太可能转移, 但有25% 的进展是侵入型生长模式。多达半数的侵袭性癌症患者将会产生致命的转移性复发。因此, 了解膀胱癌侵袭性进展的机制对于预测患者预后和预防致死性转移是至关重要的。在本文中, 我们提出了一个三维的癌症入侵模型, 允许合并肿瘤细胞和基质成分, 模仿在体内的条件发生在膀胱肿瘤微环境。该模型提供了利用时间推移成像实时观察侵入过程的机会, 并利用共焦荧光成像和筛查化合物, 以阻断入侵的可能性, 对所涉及的分子通路进行审问。虽然本议定书的重点是膀胱癌, 但也有可能采用类似的方法来检查其他肿瘤类型的侵袭和运动。

成功地创建浸润性膀胱癌肿瘤球体需要形成适当大小的肿瘤球体从细胞系或原发肿瘤。图 2A显示适当大小的球体开发从四人膀胱癌细胞线 (UM-UC9, UM-UC13, UM-UC14, 253J 和 UM-UC18)。图 2B显示一个肿瘤球体的 BBN 产生的小鼠膀胱肿瘤嵌入胶原基质。这些球体被嵌入如上文所述, 有代表性的图像被捕获使用一个显?...

Watch this Scientific Journal Video about 3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer at JoVE.com

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

3维细胞培养系统研究膀胱癌侵袭和评价治疗方法

控制膀胱癌的过程代表了生物标志物和治疗发展的机会。在这里, 我们提出了一个膀胱癌入侵模型, 其中包括3维的肿瘤球体文化, 时间推移成像和共焦显微术。该技术可用于定义侵入过程的特征和筛选治疗药物。

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

3-d-cell-culture-system-for-studying-invasion-evaluating-therapeutics

Research

JoVE Journal

Cancer Research

8.8K 次观看.  University of Michigan Medical Center. 控制膀胱癌的过程代表了生物标志物和治疗发展的机会。在这里, 我们提出了一个膀胱癌入侵模型, 其中包括3维的肿瘤球体文化, 时间推移成像和共焦显微术。该技术可用于定义侵入过程的特征和筛选治疗药物。

视频: 3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

三维细胞培养模式的测量肿瘤细胞浸润间质流体流动的影响

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

科研

生物工程

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

小鼠膀胱肿瘤器官的培养、操作和正交移植

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

癌症研究

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

监测三D培养中的癌细胞入侵和T细胞毒性

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Culture of Bladder Cancer Organoids as Precision Medicine Tools

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as two-dimensional (2D) cultures on tissue culture plastic. There is a critical need for more representative models of tumor complexity that can accurately predict therapeutic response and sensitivity. The development of three-dimensional (3D) ex vivo culture of patient-derived organoids (PDOs), derived from fresh tumor tissues, aims to address these shortcomings. Organoid cultures can be used as tumor surrogates in parallel to routine clinical management to inform therapeutic decisions by identifying potential effective interventions and indicating therapies that may be futile. Here, this procedure aims to describe strategies and a detailed step-by-step protocol to establish bladder cancer PDOs from fresh, viable clinical tissue. Our well-established, optimized protocols are practical to set up 3D cultures for experiments using limited and diverse starting material directly from patients or patient-derived xenograft (PDX) tumor material. This procedure can also be employed by most laboratories equipped with standard tissue culture equipment. The organoids generated using this protocol can be used as ex vivo surrogates to understand both the molecular mechanisms underpinning urological cancer pathology and to evaluate treatments to inform clinical management.

Patient-derived organoids (PDOs) are a powerful tool in translational cancer research, reflecting both the genetic and phenotypic heterogeneity of the disease and response to personalized anti-cancer therapies. Here, a consolidated protocol to generate human primary bladder cancer PDOs in preparation for the evaluation of phenotypic analyses and drug responses is detailed.

膀胱癌类器官作为精准医疗工具的文化

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as ...

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 ...

医学

癌症生物学中的 3D 模型:异球体中的侵袭动力学

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

作者聚焦:破译头颈癌进展过程中癌症和免疫细胞之间的串扰

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

分析 3D 培养物体外细胞侵袭的替代策略

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

癌细胞球体检测，以评估入侵的3D设置

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

3维细胞培养系统研究膀胱癌侵袭和评价治疗方法

摘要

探索更多视频

此视频中的章节

三维细胞培养模式的测量肿瘤细胞浸润间质流体流动的影响

小鼠膀胱肿瘤器官的培养、操作和正交移植

监测三D培养中的癌细胞入侵和T细胞毒性

膀胱癌类器官作为精准医疗工具的文化

乳腺癌细胞侵袭定量使用三维（3D）模型

分析 3D 培养物体外细胞侵袭的替代策略

分析 3D 培养物体外细胞侵袭的替代策略

分析 3D 培养物体外细胞侵袭的替代策略

三维（3D）肿瘤球体侵袭试验

癌细胞球体检测，以评估入侵的3D设置