Name: 3-d cell culture system for studying invasion and evaluating therapeutics in bladder cancer
Uploaded: 2018-09-13T14:45:00
Description: Watch this Scientific Journal Video about 3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer at JoVE.com

The authors would like to thank the laboratory of Dr. Howard Crawford (University of Michigan) for technical support and providing materials and equipment for this study, and Alan Kelleher for technical support. 
This work was funded by grants from the University of Michigan Rogel Cancer Center Core Grant CA046592-26S3, NIH K08 CA201335-01A1 (PLP), BCAN YIA (PLP), NIH R01 CA17483601A1 (DMS).

1. Growing Cancer Spheroids
<ol>
	<li>Growing from cell lines
	<ol>
		<li>Culture human bladder cancer cells under conventional adherent cell culture conditions and maintain in a 37 &#176;C incubator supplied with 5% CO2. Maintain cells at &#60;90% confluency. 
		NOTE: Culture media used is Dulbecco&#39;s modified minimum essential media (DMEM) containing 4.5 g/L D-glucose, L-Glutamine, 110 mg/L sodium pyruvate, and supplied with 10% fetal bovine serum (FBS) throughout this protocol.</l...

<ol>
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Here we describe a 3-D tumor spheroid model that allows real-time observation of bladder cancer invasion which is critical for cancer progression and metastasis. This system is amenable to the incorporation of various stromal and cellular components to allow investigators to better recapitulate the tissue microenvironment where bladder cancer invasion takes place. Bladder cancer spheroids can be generated from various sources such as cell lines (including genetically modified cell lines useful for the examination of sign...

Invasion is a critical step in cancer progression, which is required for metastasis, and is associated with lower survival and poor prognosis in patients. In human bladder cancer, the most common malignancy of the urinary tract which causes about 165,000 deaths per year worldwide, cancer stage, treatment and prognosis are directly related to the presence or absence of invasion1. Around 75% of the cases of bladder cancer are non-muscle invasive and are managed with local resection. In contrast, muscle-invasive bladder cancers (about 25% of all cases) are aggressive tumors with high metastatic rates and are treated with aggressive multimodality therapy2,3. Therefore, understanding the molecular pathways that trigger invasion is essential to better characterize the risk of invasive progression and to develop therapeutic interventions which can prevent invasive progression.
Tumor invasive progression occurs in a complex three-dimensional (3-D) environment and involves tumor cell interaction with other tumor cells, stroma, basement membrane, and other types of cells including immune cells, fibroblasts, muscle cells and vascular endothelial cells. Permeable support (e.g., Transwell) assay systems are commonly employed to quantitate cancer cell invasion4, but these systems are limited because they do not allow microscopic monitoring of the invasion process in real-time and the retrieval of samples for further staining and molecular analysis is challenging. Development of a 3-D bladder tumor spheroid system to study invasion is desirable because it allows the incorporation of defined microenvironmental components with the convenience of in vitro systems.
In this protocol, we describe a system to interrogate the invasive processes of human bladder cancer cells using a 3-D spheroid invasion assay incorporating collagen-based gel matrices and confocal microscopy to allow investigators to monitor cell motility and invasion in real-time (Figure 1A). This system is versatile and can be modified to interrogate various stromal/tumor settings. It can incorporate most bladder cancer cell lines or primary bladder tumors and additional stromal cells such as cancer associated fibroblasts and immune cells5,6,7. This protocol describes a matrix composed of type-1 collagen, but can be modified to incorporate other molecules such as fibronectin, laminin, or other collagen proteins. Invasive processes can be followed for 72 h or longer depending on the capability of the microscope and system used. Fixation and immunofluorescence staining of the tumor embedded in the 3-D matrix before, during, and after invasion allows the interrogation of proteins upregulated in invasive cells, thus providing crucial information that usually absent or difficult to gather using other 3-D culture models. This system can also be utilized to screen compounds which block invasion, and to delineate signaling pathways affected by such compounds.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Human bladder cancer cell lines UM-UC9, UM-UC13, UM-UC14, UM-UC18, 253J</td>
			<td style="width:203px;"></td>
			<td style="width:169px;"></td>
			<td style="width:612px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">DMEM cell culture medium</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">11995065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic-Antimycotic (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">15240062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:169px;">A3803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS), pH 7.4&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar Ultral-low attachment 6-well cluster&#160;</td>
			<td>Corning</td>
			<td align="right" style="width:169px;">3471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conventional inverted microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;">491206-0001-000</td>
			<td>General use for cell culture and checking spheroids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen type 1 from rat tail, high concentration&#160;</td>
			<td>Corning</td>
			<td align="right" style="width:169px;">354249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab-Tek II Chambered Coverglass</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">155382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;">LSM800</td>
			<td>A confocal miscoscope with climate chamber, multi-location imaging, and Z-stack scanning function&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat micromtome</td>
			<td>Leica Biosystems</td>
			<td style="width:169px;">CM3050 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zen 2 Image processing software&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde solution</td>
			<td>Electron Microscopy Sciences</td>
			<td align="right" style="width:169px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImmEdge Hydrophobic Barrier PAP Pen</td>
			<td>Vector Laboratories&#160;</td>
			<td style="width:169px;">H4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">O.C.T compound&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">23730571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 solution&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">62249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-ATDC (Trim29) antibody</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:169px;">HPA020053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-Cytokeratin 14 antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:169px;">ab7800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-Vimentin antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:169px;">ab24525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Diamond&#160;</td>
			<td style="width:203px;"></td>
			<td style="width:169px;"></td>
			<td>Mounting medium</td>
		</tr>
	</tbody>
</table>

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

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. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

Successful creation of invasive bladder cancer tumor spheroid requires the formation of appropriately sized tumor spheroids from cell lines or primary tumors. Figure 2A shows appropriately sized spheroids developed from four human bladder cancer cell lines (UM-UC9, UM-UC13, UM-UC14, 253J, and UM-UC18). Figure 2B shows a tumor spheroid from a BBN-generated mouse bladder tumor embedded in collagen ...

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

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

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. ...

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