This work was supported by grants 53110000117 (to G.W.) and 043222019 (to X.W.) from Tsinghua University.

Please note that all the mouse experiments described here were approved and performed in accordance with the Institutional Animal Care and Use Committee (IACUC) at Tsinghua University.
1. Generation of DR3 Overexpression Cell Lines
<ol>
	<li>Construct the plasmid for DR3 expression (pMXs-IRES-DR3-FLAG) by inserting full-length DR3 cDNA with 3x FLAG at the C-terminus into retroviral vector pMXs-IRES-Blasticidin at the restriction sites of BamHI/XhoI.</li>
	<li>Grow Plat-A cells in 60 mm dishes with 4 mL of Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM). The next day, when the cells reach 80% - 90% confluence, transfect the cells with 2 &#956;g of plasmid pMXs-IRES-DR3-FLAG using transfection reagent (see Table of Materials) according to the manufacturer&#8217;s manual20.</li>
	<li>After 72 h, collect the supernatant, filtrate the media using a 0.45 &#956;m sterile filter, and keep the viral suspension in the dark at 4 &#176;C.</li>
	<li>Grow HT29 cells in DMEM in a 60 mm dish and incubate the cells at 37 &#176;C with 5% CO2.</li>
	<li>The next day, when the cells reach 30% - 50% confluence, infect the cells with 2 mL of viral suspension in the presence of 8 &#956;g of 1,5-Dimethyl-1,5-diazaundecamethylene polymethobromide per milliliter of viral suspension. After 4 - 6 h of incubation at 37 &#176;C, aspirate the viral suspension, add 4 mL of fresh DMEM, and return the dish to the incubator.</li>
	<li>Discard the media from the dishes 24 h postinfection and carefully wash cells with 2 mL of prewarmed PBS. Add 1 mL of trypsin-EDTA to the cells and incubate at 37 &#176;C for 3 min.</li>
	<li>After observing under a microscope at 10X magnification that most of the cells have detached, stop the trypsinization with 2 mL of complete DMEM containing 10% fetal bovine serum (FBS). Collect the cell suspension in a 15 mL tube and centrifuge the cells at 200 x g for 5 min at room temperature (RT).</li>
	<li>Remove the supernatant and resuspend the cell pellet in 10 mL of DMEM. Mix well by gently pipetting up and down. Dilute the cells by factors of 30, 100, and 300. Seed the cells in 150 mm dishes with 20 mL of DMEM containing 1 &#181;g/mL blasticidin. Incubate at a 37 &#176;C incubator with 5% CO2 for ~1 - 2 weeks.</li>
	<li>During this period, observe the colonies every day with an inverted microscope at 10X magnification. Mark the well-isolated colonies at the bottom of the dish when the colony diameter is about 1 - 2 mm.</li>
	<li>Aspirate the medium and wash the cells with 3 mL of pre-warmed PBS. Add 2 milliliters of trypsin-EDTA in a 60 mm dish. Use sterile forceps to pick up autoclaved sterile cloning cylinders, and place them in the 60 mm dish. Pick up the cylinders containing trypsin-EDTA and gently place them over the marked colonies. Make sure every cloning cylinder only contains one colony and avoid contamination with nearby colonies.</li>
	<li>Return the dish to the incubator for 3 minutes.</li>
	<li>After 3 min, check the cells under the microscope at 10X magnification to see whether they have detached. When the cells have lifted up, add 70 &#956;L of culture medium to each cylinder to inactivate the trypsin. Gently mix the cell suspension with a 200 &#956;L pipette. Make sure not to move the cylinder during this process.</li>
	<li>Transfer the cell suspension from each colony to two 24-well plates containing 1 mL of DMEM per well. Add 30 &#956;L of cell suspension per well in plate A and 70 &#956;L in the corresponding wells in plate B. Label the plates properly and put them back in the incubator.</li>
</ol>
2. Verification of the DR3 Overexpression Cell Line
<ol>
	<li>When the cells in plate B are at 90% confluence, remove the media and carefully wash the cells with 1 mL of PBS. Completely remove PBS and lyse the cells with 50 &#956;L of 1x SDS-PAGE loading buffer. Transfer the cell lysates from the 24-well plate to 1.5 mL centrifuge tubes and boil the samples for 10 min at 100 &#176;C.</li>
	<li>Run the samples on 10% sodium dodecyl sulfate (SDS) gel at a constant voltage of 80 V for 15 min and, then, at 120 V for 1 h.</li>
	<li>Transfer the protein to a polyvinylidene fluoride (PVDF) membrane by wet transfer at a constant current of 400 mA for 2 h.</li>
	<li>Test the DR3 expression in different clones with anti-FLAG antibody, using the wild-type HT29 cells as a negative control (Figure 1). Dilute the primary antibody by a factor of 5,000 in 5% nonfat milk that is dissolved in TBST (Tris-buffered saline containing 0.1% Tween-20). Incubate the membrane with FLAG antibody at 4 &#176;C overnight.</li>
	<li>Wash the membrane with TBST by shaking it on a decoloring shaker at 200 rpm at RT. Refresh the TBST every 5 min for a total washing time of 15 min.</li>
	<li>Incubate the membrane with anti-mouse secondary antibody diluted by a factor of 10,000 in 5% nonfat milk at 4 &#176;C for 5-6 h.</li>
	<li>Wash the membrane again as described in step 2.5.</li>
	<li>Detect the FLAG expression using western enhanced chemiluminescence (ECL) substrate and image the membrane with a gel documentation system (see Table of Materials). Identify the clones expressing DR3 as the positive clones.</li>
	<li>Transfer the clones in plate A that correspond to positive clones in plate B to a 6-well plate and continue to amplify the cells until growing them in 10 cm dishes. Make frozen stocks of the cells growing from positive clones and store them at -80 &#176;C for future use.</li>
</ol>
3. Assessment of the Apoptotic Response of Cells to Antimitotic Agents
NOTE: Diazonamide is a new class of marine natural product that has remarkable activity in inhibiting cancer cell growth, which mirrors other tubulin-destabilizing agents21. However, its mode of action remains unclear. To test the cellular response of cells to antimitotic agents, paclitaxel and diazonamide were used to treat wild-type HT29 cells and HT29-DR3 cells. The drugs were dissolved in dimethyl sulfoxide (DMSO) to make 10 mM stocks and, then, further diluted to different concentrations: 30 nM, 100 nM, 300 nM, 1,000 nM, 3,000 nM, and 10,000 nM in DMSO.
<ol>
	<li>Collect HT29 and HT29-DR3 cells by trypsinization. Measure the cell density using an automatic cell counter. Then, seed the cells in 12-well plates in DMEM at a density of 3 x 104 cells per well. The next day, add 10 nM diazonamide and use 1% DMSO as a negative control. After 48 h of treatment, image the cells under a microscope at 10X magnification (Figure 2).</li>
	<li>Seed HT29 and HT29-DR3 cells in 96-well plates in DMEM at a density of 3 x 103 cells per well and allow them to grow overnight at 37 &#176;C. Then, add dose-escalating diazonamide concentrations of 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM, and 100 nM to each well, using 1% DMSO as a negative control.</li>
	<li>After 48 h of treatment, measure the cell viability using a luminescence-based cell viability assay kit according to the manufacturer&#8217;s manual.
	<ol>
		<li>Take out the 96-well plate from the incubator and let it stand at RT for approximately 30 min. Then, add 50 &#181;L of assay reagents to each well, shake the plate at RT for 2 min to lyse the cells, and incubate the plate at RT for another 10 min. Determine the luminescence of each well using a microplate reader (Figure 3). 
		NOTE: The cell viability represents the relative luminescence intensity of each well to that of the control well treated with 1% DMSO.</li>
	</ol>
	</li>
	<li>Use six-weeks-old female BALB/c nude mice for an in vivo study14. 
	NOTE: The mice were maintained under specific pathogen-free conditions.
	<ol>
		<li>Trypsinize HT29 and HT29-DR3 cell and, then, stop the trypsinization using DMEM prewarmed at 37 &#176;C. Collect the cell suspension in a 50 mL tube and centrifuge the cells at 1,000 rpm for 10 min at RT. Discard the supernatant and resuspend the cell pellet in DMEM medium without FBS. Centrifuge the cells again and resuspend the cell pellet in PBS.</li>
		<li>Count the cells using an automatic cell counter and dilute the cells to a concentration of 2.5 x 107 cells/mL using PBS. Generate tumors through a subcutaneous injection of 200 &#956;L of the HT29 or HT29-DR3 cell suspension in the right flank of each mouse (5 x 106 cells/mouse)22.</li>
		<li>Measure the tumor volume (TV) using calipers every 2 d after the transplantation. Calculate the TV using the following formula: TV = 1/2L x W x W (here, W: tumor width, L: tumor length). When the average tumor volume reaches about 100 mm3, randomize the mice into either the control or the treated group (n = 6 per group) and initiate the treatment.</li>
		<li>Prepare the paclitaxel solution as follows. First, dissolve paclitaxel completely in ethanol at 5% of the total volume and, then, add 5% of the final volume of polyoxyl 35 hydrogenated castor oil. Finally, make up the remaining 90% of the volume with D5W (5% dextrose in water, pH 7.4).</li>
		<li>Inject the paclitaxel intravenously at a dose of 20 mg/kg 3x per week for two weeks. Measure the tumor volume and body weight 3x a week14. Terminate the experiment and euthanize the animals if the body weight decreases by 20% or the tumor volume reaches about 2,000 mm3.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to declare.

In this manuscript, we describe a method to generate HT29-DR3 stable cell lines. HT29-DR3 cells provide a model for studying the molecular mechanisms by which DR3 contributes to apoptosis induced by antimitotic agents. This approach is versatile and repeatable. To ensure the success of the procedure, four key steps of the protocol need to be considered. First, in order to produce a high enough virus titer, it is recommended to perform DNA transfection when the Plat-A cells are at 80% - 90% confluence. Second, the viral s...

Heterologous gene expression is often employed to study the function of genes of interest. Two methods, transient and stable transfection, are prevalently used in cells and molecular biology to insert a segment of DNA or RNA into a host cell1,2. Transiently-transfected DNA or RNA cannot be passed on to daughter cells, so the genetic alteration can only be retained for a short period of time. On the other hand, in stably transfected cells, exogenous genes are integrated into the host cell genome, sustaining its expression in the cell line. Thus, stable transfection is a method that is usually reserved for research into long-term genetic regulation. The stable cell line can also be transplanted as xenografts into mouse models for in vivo study3. Stable transfection can be divided into two categories: viral and nonviral. Compared to nonviral transfection, viral infection can transfer genes into a wider variety of human cells with a higher efficiency4,5,6,7. Furthermore, a stable cell line from a single clone offers the advantage of homogeneity and clonal purity.
Uncontrolled cell growth and division are the most distinguishing features of cancer. In clinical settings, antimitotic agents are the primary treatments for many types of tumors8. However, some significant limitations of antimitotic agents cannot be ignored. First, these chemotherapies can also kill normal cells along with the undesired cancerous cells and, thus, can result in severe side effects8. Second, antitubulin drugs are not effective against all types of tumors, despite tubulin&#39;s ubiquitous expression in a wide variety of different tissues as a cytoskeletal protein9,10. It is unclear why antitubulin agents showed promising efficacy against ovarian, lung, and hematological cancers in clinical treatment, but not in kidney, colon, or pancreatic cancers11.&#160;Finally, even patients with the same type of tumor can respond&#160;to the antimitotics differently in an unpredictable manner. There may be a key effector molecule that affects the sensitivity of different patients to antimitotic agents, resulting in the diverse outcomes seen in clinical treatment12. Thus, the potential differential sensitivities of patients to antimitotic treatment should be taken into consideration in order to optimize therapeutic interventions13.
A high-throughput whole-genome siRNA library screen demonstrated that DR3 knockdown could render sensitive cells resistant to antimitotic drugs, implicating a role for DR3 in chemotherapy-induced apoptosis14. As a member of the tumor necrosis factor receptor (TNFR) superfamily, DR3 has been reported to mediate cell apoptosis in various systems15,16,17,18. Thus, we set out to establish stable cell lines overexpressing DR3 to study the molecular mechanisms by which antimitotic drugs induce apoptosis. An appropriate cell model for studying DR3 overexpression can be provided by the human colon cancer cell line HT29, which was found to be DR3-deficient. Additionally, in the clinic, colon cancers are insensitive to antimitotic drugs19.
In this article, we describe a method which allows the generation of an HT29-DR3 stable cell line through retroviral infection. We further show how to validate the DR3 expression in these cells using western blotting and how to assess the sensitivity to antimitotic agents by using a cell viability assay and morphological observation. The cells are amplified from single clones and, thus, have the advantage of a homogeneous genetic background. In addition, the FLAG tag on DR3 allowed for the visualization of cellular DR3 by microscopy and analysis of gene expression and protein interaction by biochemical approaches. Furthermore, the cells can be xenografted in mice to further analyze tumor progression in response to antimitotics in vivo14.
The HT29-DR3 cell system presented here offered an effective tool for studying the molecular mechanisms of how antimitotics kill cancer cells14. Since overexpression and knockdown cell lines are common tools for studying gene function, this protocol can be adapted easily to other genes of interest or other cell lines and, thus, turned into an extensively used approach.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>C11965500BT</td>
			<td>Supplemented with 10% FBS and 1% Penicilin/Streptomycin</td>
		</tr>
		<tr>
			<td>Luminescent Cell Viability Assay</td>
			<td>Promega</td>
			<td>G7571</td>
			<td></td>
		</tr>
		<tr>
			<td>Paclitaxel</td>
			<td>Selleck</td>
			<td>S1150</td>
			<td></td>
		</tr>
		<tr>
			<td>pMXs-IRES-Blasicidin</td>
			<td>Cell Biolabs</td>
			<td>RTV-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>C14190500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>1,5-Dimethyl-1,5-diazaundecamethylene polymethobromide</td>
			<td>Santa Cruz</td>
			<td>sc-134220</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Transfection Reagent</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse secondary antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Flag antibody</td>
			<td>Sigma</td>
			<td>F-3165</td>
			<td></td>
		</tr>
		<tr>
			<td>HT29 cell line</td>
			<td>ATCC</td>
			<td>HTB-38</td>
			<td></td>
		</tr>
		<tr>
			<td>plat-A cell line</td>
			<td>Cell Biolabs</td>
			<td>RV-102</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Immobilon-P</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloning Cylinder</td>
			<td>Sigma</td>
			<td>C1059</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Imaging Multi-Mode Reader</td>
			<td>Biotek</td>
			<td>BTCYT3MV</td>
			<td></td>
		</tr>
		<tr>
			<td>CKX53 Inverted Microscope</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well cell culture plates</td>
			<td>Nest</td>
			<td>712001</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture plates</td>
			<td>Nest</td>
			<td>705001</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Nest</td>
			<td>601052</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m steril filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>Eppendorf</td>
			<td>5810000327</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well White Polystyrene Microplate&#160;</td>
			<td>Corning</td>
			<td>3903</td>
			<td></td>
		</tr>
		<tr>
			<td>Western ECL Substrate</td>
			<td>BIO-RAD</td>
			<td>1705060</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000</td>
			<td>GE Healthcare</td>
			<td>ImageQuant LAS 4000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Decoloring Shaker&#160;</td>
			<td>HINOTECH</td>
			<td>TS-2000A</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>InvivoGen</td>
			<td>ant-bl-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>BIO-RAD</td>
			<td>TC 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher</td>
			<td>31985070</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing cell lines overexpressing dr3 to assess the apoptotic response to anti-mitotic therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

A characterization of HT29 cells stably expressing DR3 is shown in Figure 1. The clones express varying levels of DR3, while the wild-type cells, which serve as a negative control, do not show exogenous gene expression. Here we only show five clones, among which clone 1 and 5 express the highest level of DR3. We chose clone 1 and 5 for the following experiments.
The morphology of HT29 and HT29-DR3 c...

Watch this Scientific Journal Video about Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics at JoVE.com

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

establishing-cell-lines-overexpressing-dr3-to-assess-apoptotic

Research

JoVE Journal

Cancer Research

7.3K Views.  Tsinghua University. Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

Video: Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

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.

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

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

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 rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

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

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.

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. ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...