V.R.-B. receives funding from the U.S. Department of Health &#38; Human Services, NIH, National Cancer Institute grant number 1 K22 CA207458-01 and J.D.-D. receives funding from U.S. Department of Health &#38; Human Services, NIH, National Cancer Institute grant number 1 R01 CA207311-01.

1. Determination of the Docetaxel Concentration Causing 50% Decrease in Colony Formation Ability (IC50)
NOTE: In this protocol, Docetaxel IC50 concentration has been evaluated using colony formation ability. Alternative methods used to evaluate cell viability (i.e., MTS assays) can be used based on investigators&#39; preferences.
<ol>
	<li>Grow DU145 or 22Rv1 cells in a 75 cm2 flask and prepare enough stock of 10 mM Docetaxel diluted in DMSO to be used in future experiments.</li>
	<li>To plate cells for determination of IC50 of Docetaxel, remove media from the flask, carefully wash cells with 5 mL PBS, and incubate with 2 mL 0.05% Trypsin-EDTA for 3-5 min at 37 &#176;C to detach cells from the flask surface.</li>
	<li>Rinse trypsinized cells from the flask using 5 mL of fresh media and collect the cell suspension in a 15 mL tube. Pellet cells by centrifugation (300 x g, 3 min, room temperature (RT)).</li>
	<li>Remove the supernatant, resuspend the cell pellet in 5 mL of fresh media and count the cells. Dilute the cell suspension to 2-2.5 x 105 cells/mL for DU145 and 4-4.5 x 105 cells/mL for 22Rv1, mix well by pipetting up and down and seed 2 mL of the suspension in each well of two 6-well plates.</li>
	<li>After 24 h, when the cells are at 70-80 % confluence, add dose-escalating Docetaxel concentrations of 1 nM, 2.5 nM, 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, and 1000 nM to each well. Treat the control well with DMSO at the highest volume used for Docetaxel (Figure 2A).</li>
	<li>After 72 h, aspirate drug containing media and add 2 mL of fresh media. Within approximately 4-5 days, the plates will be ready for staining.</li>
	<li>To stain the colonies, wash them gently with 2-3 mL PBS and incubate them with 2-3 mL crystal violet solution (0.1 % w/v in 10 % formalin) for 20 min inside the tissue culture hood or a fume hood.</li>
	<li>Remove staining solution and wash plates with 2-3 mL of H2O, remove H2O and air-dry plates.</li>
	<li>Analyze the results by visualizing the wells to determine which one has the Docetaxel concentration that decreases cell viability by 50% (IC50). Manually count colonies with the help of a marker pen (to avoid counting the same colonies twice) and represent the percentage of cell viability in a graph (Figure 2A). Finally, take digital images of the plates for figure representation (as shown in Figure 3A).</li>
</ol>
2. Generation of Docetaxel-resistant Prostate Cancer Cells
NOTE: Perform cell treatments using the same Docetaxel solution stock used for determination of IC50 drug concentration (prepare enough stock in advance for experimental use). Always keep a small flask of cells from the previous step, just in case something does not work well. Parental cells should be grown in parallel in a small flask throughout the whole procedure and exposed to vehicle (DMSO) in corresponding volumes mimicking the amounts used in the Docetaxel treated flasks.
<ol>
	<li>Plate DU145 or 22Rv1 cells in 150 cm2 flasks containing 20 mL of media (3.5 x 106 cells per flask for DU145 and 6.5*106 cells per flask for 22Rv1). Use of 10 to 20 flasks of cells is recommended to have enough cells at the end of the protocol.</li>
	<li>After 24 h, when cells are at about 70-80% confluence (Figure 2B, before Docetaxel), add Docetaxel at the IC50 concentration determined in step 1 of the protocol (5 nM for the cells described in this protocol).</li>
	<li>After 72 h, aspirate drug containing media and add fresh, Docetaxel-free media (Figure 2B, 72 h after Docetaxel). Change the media every 3-4 days. Within approximately 1-2 weeks, resistant clones will appear evident under the microscope (Figure 2B, 7 and 14 days after Docetaxel).</li>
	<li>Aspirate media, carefully wash cells with 15 mL PBS and incubate with 4 mL 0.05% Trypsin-EDTA for 3-5 min at 37 &#176;C to detach cells from the flask surface.</li>
	<li>Resuspend trypsinized cells from the flask using 8 mL of fresh media. Pool cells from all treated flasks. Pellet cells by centrifugation (300 x g, 3 min, RT).</li>
	<li>Remove the supernatant, resuspend the cell pellet in 20 mL of fresh media and plate the cells in 150 cm2 flasks (3.5 x 106 cells per flask for DU145 and 6.5*106 cells per flask for 22Rv1).</li>
	<li>After 24 h, when cells are at about 70-80% confluence, add 5 nM Docetaxel (initial IC50 concentration) again.</li>
	<li>Repeat steps 2.3 to 2.6 on day 3.</li>
	<li>After 24 h, when cells are at about 70-80% confluence, treat cells with 2X IC50 Docetaxel concentration (10 nM for the cells described in this protocol).</li>
	<li>Repeat steps 2.3 to 2.8, following a dose-escalation of Docetaxel concentrations (25 nM, 50 nM, 100 nM and 250 nM), to generate a stable population of cells growing in your flasks under the final highest concentration. For higher concentrations, the dose-escalation protocol can be implemented for up to 6 months until the 1,000 nM concentration is reached (Figure 1A).</li>
</ol>
3. Functional Characterization of Acquired Docetaxel Resistance by Colony Formation Assay
NOTE: In this protocol, chemoresistance has been evaluated using colony formation assays. Alternative methods used to evaluate cell viability (i.e., MTS assays) can be used based on investigators&#39; preferences.
<ol>
	<li>Plate 2,000 cells (DU145 or 22Rv1, both parental and Docetaxel-resistant) per well in 6-well plates, using 2 mL of media per well.</li>
	<li>After 24 h, add increasing concentrations of Docetaxel (parental cells: 0.25, 0.5, 1, 2.5 and 5 nM; DR cells: 50, 125, 250, 500 and 1,000 nM). For both DU145 and 22Rv1 cell lines. Add DMSO only to one well as a control at the same volume used for the highest Docetaxel dose.</li>
	<li>After 72 h, aspirate drug containing media and add fresh Docetaxel-free media.</li>
	<li>Incubate plates for 1-2 weeks until colonies are visible under the microscope.</li>
	<li>To stain the colonies, wash them gently with 2-3 mL PBS and incubate them with 2-3 mL crystal violet solution (0.1% w/v in 10% formalin) for 20 min inside the tissue culture hood or a fume hood.</li>
	<li>Remove staining solution and wash plates with 2-3 mL of H2O, remove H2O and air-dry plates.</li>
	<li>Analyze result by visualizing the wells and manually counting colonies with the help of a marker pen (to avoid counting the same colonies twice) and represent the percentage of cell viability in a graph. Take digital images of the plates for figure representation (Figure 3A).</li>
</ol>
4. Phenotypic Characterization of Docetaxel Resistance Acquired Phenotype via qRT-PCR Analysis
NOTE: Docetaxel-resistant (DR) cells exhibit a different expression profile compared to their parental cell lines28,29. Therefore, the success of the selection can be verified by qRT-PCR using primers for specific markers (for primer sequences, refer to the materials section; for details, refer to the results section). This should be done after each round of selection starting after the third round. Alternatively, evaluation of Docetaxel-resistant phenotype by Western blotting is also possible.
<ol>
	<li>Extract RNA from both parental and Docetaxel-resistant cells using an RNA extraction kit and following the manufacturer&#39;s instructions (see the Table of Materials and reagents for details). Using a minimum of 1-2 x 106 cells is recommended.</li>
	<li>Quantify RNA at 260 nm absorbance, using a spectrophotometer. For best purity, the 260 nm/280 nm absorbance ratios should be close to 2.0.</li>
	<li>Perform reverse transcription to obtain complementary DNA (cDNA) using the reverse transcription kit and following the manufacturer&#39;s instructions (see the Table of Materials and reagents for details), using 1 &#181;g of RNA in a 20 &#181;L reaction mix (if a higher amount of cDNA is needed, scale up the reaction mix accordingly).</li>
	<li>Prepare the qPCR reaction in triplicate for each gene of interest as follows: 
	- 2 &#181;L of 10 &#181;M forward primer 
	- 2 &#181;L of 10 &#181;M reverse primer 
	- 10 &#181;L SYBR Green PCR master mix 
	- 5 &#181;L H2O 
	- 1 &#181;L of the newly prepared cDNA from step 4.3. 
	NOTE: See Table of Materials for the sequences of primers used in this protocol.</li>
	<li>Set the PCR program as follows: 
	- 95 &#176;C for 15 min 
	- 94 &#176;C for 30 s&#160;&#160;&#160;&#160;&#160;| 
	- 56 &#176;C for 40 s&#160;&#160;&#160;&#160;&#160;|&#160;40 times 
	- 72 &#176;C for 30 s&#160;&#160;&#160;&#160;&#160;|</li>
	<li>To analyze qPCR results and determine changes in gene expression between parental and DR samples, the cycle threshold (Ct) values for each gene of interest are determined in triplicate and the average normalized to the loading control (Actin triplicate average) for DR (Ct DR) and parental cells (Ct parental). Values for the parental cells are normalized to 1. &#916;Ct (Ct DR - Ct parental) is determined to obtain final mRNA fold changes and represented in a graph. An increase of &#62;2 or a decrease of &#60; 0.5 are considered significant and a good validation of the establishment of the Docetaxel acquired resistant phenotype (Figure 3B).</li>
</ol>

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The authors declare that they have no competing financial interests.

In this manuscript, we describe a method to generate Docetaxel-resistant prostate cancer cell model systems that allows for the study of mechanisms contributing to acquisition of the chemoresistance phenotype. While this approach is highly reliable and reproducible, some potential limitations should be considered. Since only a small fraction of the bulk population of cells will exhibit chemoresistance28, it is recommended to start with a large population of cells (we usually plate 20 flasks of 150 cm2, which account for approximately 1.2 x 108 cells). Key steps of the protocol should be considered to ensure success of the procedure. First, it is very important to use the same Docetaxel freshly prepared stock for long term usage (10 mM stock aliquots can be used for years when stored properly in at -80 &#176;C). Slight changes in the Docetaxel aliquot can impact the results of IC50, generation of the cell line timing and the colony formation results. Second, it is crucial to start the protocol determining the IC50 in each individual cell line, since variations can occur when using a new batch of cells. Third, it is essential to monitor that the drug treatment is effective by checking cells under the microscope frequently so any errors can be detected quickly and corrected. Finally, we recommend to always keep a stock of intermediate steps in case of contamination or unexpected problems that could affect the viability of the cells in the middle of the protocol. Importantly, our protocol aims to generate models of drug resistance by exposing prostate cancer cells to high doses of Docetaxel for 72 h followed by long recovery periods, rather than lower constant concentrations of the drug in an attempt to mimic the best clinical scenario reported for prostate cancer treatment with this taxane agent15,16.
Additionally, it should be noted that drug-resistant cells exhibit a high plasticity, which may lead to a progressive loss of acquired resistance properties over time. Therefore, extending the use of these cells for more than 2-3 months in culture is not recommended. If a permanent supply is needed it is advisable to continuously generate new batches of resistant cells. However, if batches of cells have been cultured for an extended time, colony formation assays and qPCR can be used to reevaluate their resistance. Another alternative is to boost waning resistance by treating the cells with a high dose of Docetaxel for 72 h (500-1,000 nM). After a recovery period of one to two weeks, the phenotype can be re-tested by qPCR and colony formation assays. It is also possible, although not recommended, to store aliquots of treated cells in liquid nitrogen (in FBS, 10% DMSO). Please note that after a freeze-thaw cycle, the chemoresistance phenotype should always be re-evaluated with the aforementioned methods.
Despite these caveats, we28,29 and others30,31,32,33 were able to successfully employ these model systems to identify key novel cellular and molecular mechanisms leading to elevated tumor-initiating capacity, cell survival, and aggressiveness in Docetaxel-resistant cells. Using these cancer cell model systems, we discovered that cells exhibiting an undifferentiated phenotype, characterized by the absence of epithelial and prostate-related differentiation markers and overexpression of targetable developmental (Notch and Hedgehog) signaling pathways, survive chemotherapeutic exposure28. In a second study, using these models together with publicly available patient gene data sets24,25, we uncovered that the pioneer transcription factor GATA2 is highly overexpressed in metastatic castration-resistant chemotherapy-resistant prostate cancer cells. GATA2 increases the survival of cells by directly activating an IGF2-dependent downstream kinase signaling network29. Notably, these studies helped to identify novel key roles of GATA2 in advanced metastatic prostate cancer aggressiveness34.
The development of drug resistance is a problem that is not limited to Docetaxel and prostate cancer, as it is prevalent among many other types of cancers treated with a wide array of chemotherapeutic drugs. Indeed, similar restrictions on the availability of experimental models apply, and it is conceivable that our protocol can be adapted to study other cancer types and their respective chemoresistance mechanisms.
The generation of Docetaxel-resistant cells as described in this protocol can be used as a functional tool to identify novel relevant molecular and cellular mechanisms of chemoresistance. This opens the door to finding new targets, upon which the development of new treatments for highly malignant prostate cancer might articulate, pushing once more the boundaries of what we know about this disease and how we treat its patients.

An increased rate of proliferation caused by the loss of cell cycle control is a hallmark of cancer1. This functional property renders cancer cells uniquely dependent on the fidelity of the cell cycle&#39;s key processes during S- and M-phases, which has&#160;led to the development of various cell cycle perturbing drugs that induce DNA damage or mitotic defects. Although numerous anti-mitotic agents, like inhibitors of mitotic kinases or motor proteins, are currently being developed and tested in clinical trials, legacy microtubule targeting agents (MTAs) continue to be the only clinically approved approach for targeting mitosis in cancer2,3,4. MTAs either destabilize (Vinblastine, Vincristine, Vinorelbine) or stabilize (taxane-derived agents Paclitaxel, Docetaxel or Cabazitaxel) microtubules and thereby prevent the formation of a functioning mitotic spindle5,6. These agents trigger the spindle assembly checkpoint7,8, which results in a prolonged mitotic arrest and eventual cell death in cultured cells9,10 and mitotic defects in tumors11,12 and are therefore useful for treating a large variety of cancers, among them prostate cancer13.
Prostate cancer is the most frequently diagnosed cancer and a leading cause of cancer related deaths in men14. Antimitotic agents such as Docetaxel improve prostate cancer patient&#39;s survival and are a mainstay in the treatment of this disease15,16,17. Unfortunately, despite initial tumor shrinkage, tumor cells often develop drug resistance during treatment. Several cellular mechanisms have been implicated in causing the development of chemoresistance, including deregulation of apoptotic and inflammatory pathways, or activation/overexpression of drug efflux pumps like the ATP-binding cassette transporter P-glycoprotein (P-gp/ABCB1), the latter of which results in the export of chemotherapeutic agents out of the cells18,19. Resistance to taxanes has also been linked with other causes, such as changes in the expression of tubulin isotypes or tubulin mutations, which prevent interaction between the drug and its target 20,21. Furthermore, resistance has been related to genome instability accumulation and tumor heterogeneity22,23. However, the mechanisms contributing to taxane resistance are not completely understood, which is evident&#160;by the absence of therapeutic strategies that prevent its appearance. Therefore, there is an urgent need to generate experimental models that assist in the dissection of these mechanisms and to identify novel therapeutic approaches that prevent the development of resistance to these drugs.
In this article we describe a method&#160;which allows the generation of Docetaxel-resistant (DR) prostate cancer cells. We will further describe how to validate the resistance status of these cells using colony formation assays and quantitative RT-PCR. Cells produced by this method have the advantage of recapitulating many of the molecular features found in publicly available human tumor sample databases with the added benefit of providing the versatility to perform a wide variety of experimental assays over longer periods of time than permitted by tumor samples. These cancer models of therapy resistance can be expanded for many weeks in tissue culture, and among other approaches, can be genetically manipulated, visualized by microscopy methods and analyzed for gene expression. Furthermore, they can be xenotrasplanted in mice to further analyze effects in tumor formation and growth in vivo. Currently, the main alternative method to study drug resistance in the context of prostate cancer is the direct analysis of tumor samples. While these samples present a very potent system to gather information about gene expression and mutational status of the given tumors, access to them can be very limited and they are difficult to maintain for further manipulations and experimental analysis, which can easily be done with the cell lines generated with this protocol24,25. Importantly, in the future, alternative approaches such as the generation of human derived organoids directly from patients would be a very powerful tool and alternative to the present method26,27. Since they will recapitulate in a 3D context what a tumor was in its original environment, organoids will be the perfect model between cell lines and human tumors. Yet, the experimental cell models described in this protocol are still more affordable to maintain and suitable for faster analysis. The results obtained by studying these cell lines can be extrapolated to and corroborated in human samples and 3D cultures. Therefore, this protocol may be of interest for researchers seeking cell models to study chemoresistance mechanisms in any cell line, since our protocol can be adapted to the study of other drugs and cancer types. The methods described here are easy to apply in any laboratory, affordable and allow preliminary studies of the molecular and cellular mechanisms of drug resistance.

<table><tbody><tr><td>DU145 cells</td><td>ATCC</td><td>HTB-81</td><td></td></tr><tr><td>22Rv1 cells</td><td>ATCC</td><td>CRL-2505</td><td></td></tr><tr><td>Docetaxel</td><td>Selleck Chemicals</td><td>S1148</td><td>in DMSO (10mM)</td></tr><tr><td>Tissue culture flask 150cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Falcon</td><td>355001</td><td></td></tr><tr><td>6-well tissue culture plates</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Gibco</td><td>11875093</td><td>Supplemented with 10% FBS and 1% Penicilin/Streptomycin</td></tr><tr><td>Foundation B Fetal Bovine Serum</td><td>Gemini</td><td>900208</td><td></td></tr><tr><td>1X Phosphate-Buffered Saline (PBS)</td><td>Corning</td><td>21-040-CM</td><td></td></tr><tr><td>0.05%Trypsin-EDTA</td><td>Gibco</td><td>25300-062</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>SuperScript III first strand synthesis system for RT-PCR</td><td>Invitrogen</td><td>18080051</td><td></td></tr><tr><td>Power SYBR Green PCR Master Mix</td><td>Invitrogen</td><td>4367659</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma-Aldrich</td><td>C0775</td><td></td></tr><tr><td>10% Formalin Solution</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Primers for qRT-PCR:</td><td>Life technologies</td><td></td><td></td></tr><tr><td>CK19 F: CTGCGGGACAAGATTCTTGGT</td><td></td><td></td><td></td></tr><tr><td>CK19 R: CCAGACGGGCATTGTCGAT</td><td></td><td></td><td></td></tr><tr><td>CDH1 F: GGAAGTCAGTTCAGACTCCAGCC</td><td></td><td></td><td></td></tr><tr><td>CDH1 R: AGGCCTTTTGACTGTAATCACACC</td><td></td><td></td><td></td></tr><tr><td>ABCB1 F: TGTTCAAACTTCTGCTCCTGA</td><td></td><td></td><td></td></tr><tr><td>ABCB1 R: CCCATCATTGCAATAGCAGG</td><td></td><td></td><td></td></tr><tr><td>GATA2 F: CATCAAGCCCAAGCGAAGA</td><td></td><td></td><td></td></tr><tr><td>GATA2 R: TTTGACAGCTCCTCGAAGCA</td><td></td><td></td><td></td></tr><tr><td>ACTIN F: CATGTACGTTGCTATCCAGGC</td><td></td><td></td><td></td></tr><tr><td>ACTIN R: CTCCTTAATGTCACGCACGAT</td><td></td><td></td><td></td></tr></tbody></table>

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.

This protocol will lead to the generation of Docetaxel-resistant (DR) prostate cancer cells. The timeline for completion may range from 4 to 7 months as summarized in the schematic representation of the protocol steps in Figure 1A and Figure 1B. The time investment could differ depending on the cell line of choice and the range of drug concentrations used as described in the protocol. Importantly, the determination of the Docetaxel IC50 concentration for each cell line allows the design of the drug escalating concentration range to use in the protocol for the cells of choice (Figure 2A). Once the protocol is started, initial Docetaxel effects on DU145 and 22Rv1 cells can be observed at different time points under the microscope to monitor if the protocol is working properly. Suggested time points to monitor the Docetaxel treatment process are: before Docetaxel exposure (baseline), after Docetaxel exposure for 72 h, 7 days and 14 days. Note that only a minority of tumor cells survive the Docetaxel exposure and generate clones, which can be observed under the microscope (Figure 2B).
Representative colony formation assays and quantification graphs of parental and the newly generated Docetaxel-resistant (DR) cells are shown in Figure 3A. Note that the generated Docetaxel-resistant cells can survive drug concentrations up to 1,000-fold higher than the parental cells.
Finally, the Docetaxel-resistant phenotype can be validated by qRT-PCR (Figure 3B). Note that Docetaxel-resistant cells, compared to the parental cells, display characteristic up-regulation of ABCB1 and GATA2 and down-regulation of CK19 and CDH1. These genes were selected for validation because we have already experimentally shown in our previously published work GATA2 upregulation and downregulation of CK19 together with CDH1 in both DR cell models28,29. The ABCB1 gene was also selected as a staple gene that has been shown by others to be consistently upregulated in drug resistant cells18,19. However, it is important to note that other genes may be chosen according to literature and depending on the cell models and the drugs chosen for generating resistant cells by the researcher.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56327/56327fig1.jpg" /> 
Figure 1: Timeline for the generation of Docetaxel-resistant prostate cancer cell models. (A) Representative diagram of the protocol timeline for generation of Docetaxel-resistant cell models. Illustration depicts the Docetaxel treatment, validation steps, and time needed for each part. (B) Schematic representation of the validation steps of the protocol including functional colony formation assays of parental and Docetaxel-resistant cells treated with the indicated Docetaxel concentrations for 72 h (in red), representative graph of colony percentage and qRT-PCR for phenotypic validation. <a href="https://cloudflare.jove.com/files/ftp_upload/56327/56327fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56327/56327fig2.jpg" /> 
Figure 2: Generation of Docetaxel-resistant prostate cancer cell model systems. (A) Diagram depicting the determination of the Docetaxel IC50 in the parental prostate cancer cell lines DU145 and 22Rv1 (Step 1 of the protocol). Expected percentages of cell viability and the Docetaxel dose-escalating concentrations needed are included. Representative DU145 and 22Rv1 cell viability graphs indicating 5 nM as the IC50 after 72 h of Docetaxel exposure are included. The experiments are triplicates and data represents the mean &#177; SD. (B) Representative bright field microscopy images of DU145 and 22Rv1 cells at indicated times during the generation of Docetaxel resistance (Step 2 of the protocol). Red arrows indicate a Docetaxel resistant clone after the protocol is completed. Scale bars = 50 nm. <a href="https://cloudflare.jove.com/files/ftp_upload/56327/56327fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56327/56327fig3.jpg" /> 
Figure 3: Functional and phenotypic characterization of the Docetaxel-resistant cell models. (A) Representative colony formation assays of parental and Docetaxel-resistant cells treated with the indicated Docetaxel concentrations for 72 h. Percentage of colonies for every treatment concentration is represented in the included graph. The experiments are triplicates and data represents the mean &#177; SD. Scale bars = 5 mm. (B) DR/parental relative mRNA fold increase quantified by qRT-PCR of ABCB1, GATA2, CK19 and CDH1 are shown. All qPCR raw data is normalized to Actin (see protocol for details). The experiments are triplicates and data represent the mean &#177; SD. *p &#60;0.05. <a href="https://cloudflare.jove.com/files/ftp_upload/56327/56327fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel at JoVE.com

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

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 ...

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13.3K Views.  Icahn School of Medicine at Mount Sinai. Weerstand tegen kankertherapieën draagt bij aan de progressie van de ziekte en dood. Het bepalen van de mechanistische onderbouwing van resistentie is cruciaal voor de verbetering van de therapeutische respons. Dit manuscript details het protocol voor het genereren van taxane-resistente cel modellen van prostaatkanker (PC) om te helpen met het ontleden van de trajecten betrokken bij progressie naar Docetaxel resistentie bij patiënten van de PC.

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

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Genetics

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Medicine

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Crystal Violet Staining: A Technique to Visualize Drug-Resistant Cancer Cells Using Colony Formation Assays

Source: Mohr, L. et al., <a href="https://www.jove.com/v/56327">Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel.</a> J. Vis. Exp. (2017)
In this video, we demonstrate a cell viability assay using a crystal violet staining procedure for chemotherapeutic drug-resistant cancer cells.

Source: Mohr, L. et al., Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel. J. Vis. Exp. (2017)
In this video, ...

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Generating Chemoresistant Prostate Cancer Cells: A Procedure for Obtaining Drug-resistant Cancer Cells In Vitro

Source: Mohr, L. et al. <a href="https://www.jove.com/video/56327" target="_blank">Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel.</a> J. Vis. Exp. (2017)
This video describes a protocol for generating taxane-resistant cell models of prostate cancer. These models can help study the pathways involved in progression to Docetaxel resistance in prostate cancer patients.

Source: Mohr, L. et al. Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel. J. Vis. Exp. (2017)
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Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

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