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
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	<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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		<col />
		<col />
	</colgroup>
	<tbody>
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			<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>
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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. ...

Invasive progression is a crucial determinant of clinical outcome in patients with bladder cancer. Here we will describe a simple in vitro method, which utilizes tumor spheroids generated using cell lines or primary tumors to study invasion. The main advantage of this technique is that we are able to monitor the process of cancer invasion in a real-time fashion, and also use immunofluorescent staining to interrogate the samples at the end of the experiment.This technique can be tailored to incorporate other cell types, such as fibro blast or immune cells. It can also be used to test compounds which might be useful to block invasion. Critical factors for success include optimization of tumor spheroid formation and embedding in collagen use of appropriate microscopy with correct working distance and appropriate immunostaining procedures.To begin generating spheroids from cell lines, culture human bladder cancer cells under conventional adherence cell culture conditions. One day prior to the experiment, trypsinize the cells and distribute 1, 000, 000 cells in three milliliters of cell culture media to each well of a six well plate. Next, incubate the cells in low attachment conditions at 37 degrees Celsius for at least 16 hours.To generate spheroids from primary tumors, collect and wash 0.5 millimeter cubed tumor pieces in five milliliters of ice-cold PBS. Centrifuge the tumor pieces at 200 times G for five minutes at four degrees Celsius. After this, collect the tumor pieces via centrifugation at 200 times G for 10 minutes at four degrees Celsius.Then remove the supernatant and add five milliliters of DMEM supplemented with 10%FBS. Transfer the tumor spheroid media mixture to a six-well ultra low attachment plate. Then incubate the plate at 37 degrees Celsius for at least 16 hours.First, dilute type one collagen in DMEM supplemented with 10%FBS to make a two milligrams per milliliter mixture. Then quickly coat the wells of a chamber slide with the mixture. Keep the chamber slide stationary at room temperature for 15 minutes.After this, gently pipette 500 microliters of spheroid media from the ultra-low attachment plate into a 1.5 milliliter micro centrifuge tube. Then allow the spheroids to settle at the bottom of the tube for two minutes. Carefully remove the supernatant from the tube.Then quickly add 500 milliliters of fresh collagen DMEM mixture to the spheroids. Pipette up and down to gently mix the spheroids and collagen. Next add 250 microliters of the spheroid collagen mixture to the wells of the collagen coated chamber slide.Allow the collagen matrix containing the spheroid to solidify completely. After the collagen matrix solidifies, add one milliliter of DMEM supplemented with FBS to each well of the chamber slides. Then incubate the chamber slide at 37 degrees Celsius with 5%CO2.Prepare the confocal microscope according to the manufacturer's instructions and ensure that the climate chamber reaches 37 degrees Celsius and is supplied with 5%CO2. Then, transfer the chamber slide to the microscope slide adapter. Using a low power objective, locate the spheroids of interest then start imaging with high power objective and perform time lapse imaging for 24 to 72 hours.First, use small forceps to lift the block of collagen gel and spheroids from the chamber slide. Then place the collagen gel block in a plastic histology mold. Rinse the collagen gel block with 1x PBS briefly.And fix it with 4%PFA and PBS for 30 minutes at room temperature. After this, wash the collagen gel block with 1.5 milliliters of 1x PBS on a shaker for 15 minutes. Apply a thin layer of OTC compound to the bottom of a new plastic histology mold.Then place the fixed and washed collagen gel block on top of the OTC compound before carefully filling the rest of the mold with OTC compound. Keep the mold at four degrees Celsius for one hour. After this, flash freeze the mold on a 100 millimeter petri dish floating on liquid nitrogen and store the samples at minus 80 degrees Celsius.Transfer the frozen sample block to the minus 20 degrees Celsius chamber of a cryostat. Then perform conventional frozen sectioning with a seven micrometer section interval. After this, air dry the slides for one hour at room temperature.Then permeabilize the samples for 15 minutes with PBS containing 0.5%Triton X-100. Next, wash the samples with PBS three times for 10 minutes each. Then encircle the samples with a hydrophobic barrier pen.Treat the samples with blocking solution for one hour at room temperature. Then apply 40 microliters of primary antibody containing solution to each sample. Incubate the slides for one hour at 37 degrees Celsius.After this, wash the sample three times with PBS for 15 minutes per wash. Next apply 40 microliters of secondary antibody containing solution to each sample. Then wash the samples three times with PBS for 15 minutes per wash.Stain the sample with Hoechst 33342 solution at room temperature for 10 minutes. Then wash the samples with PBS for five minutes. Mount the samples with mounting medium and cover them with cover slips.Finally, leave the slides in the dark at room temperature for 24 hours before performing confocal microscopy. In this protocol, invasive bladder cancer tumor spheroids were formed from cell lines and primary tumors. Representative time lapse video demonstrates bladder cancer tumor spheroid invasion into the collagen matrix.Representative time lapse video demonstrates invasive behavior of GFP labeled bladder cancer tumor spheroids alone or co-cultured with RFP labeled fibroblasts. To demonstrate the feasibility of using immunofluorescent staining of protein markers, the spheroids were fixed, sectioned and stained at 24 or 72 hours. The higher magnification view of the UM-UC-14 spheroids, shows filamentous staining for ataxia telangiectasia group D associated protein.The highly invasive cells disseminated from UM-UC-18 spheroids after 24 hours of invasion, express a high level of Vimentin a marker of Epithelial-to-Mesenchymal transition. This assay is useful for limited screens of pharmacologic inhibitors of bladder cancer invasion. Cytochalasin D is used here to represent pharmacologic inhibition of invasion.Here we describe a model that allows real-time observation of bladder cancer invasion. This system is amenable to incorporation of various stromal and cellular components, which allows investigators to better recapitulate the tissue microenvironment where bladder cancer invasion takes place. This system not only provides a useful tool for studying the invasion in 3D environment, but also is suitable for limited screening of pharmalogic compounds that have potential to block cancer cell invasion.

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A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living ...

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

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

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

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

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

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 ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...