Carcinoma della prostata in fase avanzata: problemi per i pazienti
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An unexpected error occurred. Issue doi: Determining the mechanistic underpinnings of resistance is crucial for adenocarcinoma della prostata step 2 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 adenocarcinoma della prostata step 2 PC patients.
Mohr, L. 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 PCresistance to MTAs adenocarcinoma della prostata step 2 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.
An increased rate of proliferation caused by the loss of cell cycle control is a hallmark of cancer 1. Although numerous anti-mitotic agents, like inhibitors adenocarcinoma della prostata step 2 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 cancer 234.
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 spindle 56. These agents trigger the spindle assembly checkpoint 78which results in a prolonged mitotic arrest and eventual cell death in cultured cells 910 and mitotic defects in tumors 1112 and are therefore useful for treating a large variety of cancers, among them prostate cancer Prostate cancer is the most frequently diagnosed cancer and a leading cause of cancer related deaths in men Antimitotic agents such as Docetaxel improve prostate cancer patient's survival and are a mainstay in the treatment of this disease 1516 Unfortunately, despite initial tumor shrinkage, tumor cells often develop drug resistance during treatment.
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 Furthermore, resistance has been related adenocarcinoma della prostata step 2 genome instability accumulation and tumor heterogeneity 22 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.
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 adenocarcinoma della prostata step 2 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 protocol 24 Importantly, in the future, alternative approaches such as the generation of human derived organoids adenocarcinoma della prostata step 2 from patients would be a very powerful tool and alternative to the present method adenocarcinoma della prostata step 2 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. Alternative methods used to evaluate cell viability i. 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 adenocarcinoma della prostata step 2 Docetaxel treated flasks. NOTE: In this protocol, chemoresistance has been evaluated using colony formation assays.
NOTE: Docetaxel-resistant DR cells exhibit a different expression profile compared to their parental cell lines 28 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 adenocarcinoma della prostata step 2 round. Alternatively, evaluation of Docetaxel-resistant phenotype by Western blotting is also possible.
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 DU and 22Rv1 cells can be observed at different time points under the microscope to monitor if the protocol is working adenocarcinoma della prostata step 2. Suggested time points to monitor the Docetaxel treatment process are: before Docetaxel exposure baselineafter Docetaxel exposure for 72 h, 7 days and 14 days.
Adenocarcinoma della prostata step 2 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 adenocarcinoma della prostata step 2 are shown in Figure 3A.
Note that the generated Docetaxel-resistant cells can survive drug concentrations up to 1,fold higher than the parental cells. 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 models 28 The ABCB1 gene was also selected as a staple gene that has been shown by others to be consistently upregulated in drug resistant cells 18 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.
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 redrepresentative graph of colony percentage and qRT-PCR for phenotypic validation. Please adenocarcinoma della prostata step 2 here to view a larger version of this figure. Figure 2: Generation of Docetaxel-resistant prostate cancer cell model systems.
Expected percentages of cell viability and the Docetaxel dose-escalating concentrations needed are included. B Representative bright field microscopy images of DU and 22Rv1 cells at indicated times during the generation adenocarcinoma della prostata step 2 Docetaxel resistance Step 2 of the protocol.
Red arrows indicate a Docetaxel resistant clone after the protocol is completed. 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.
All qPCR raw data is normalized to Actin see protocol for details. In this manuscript, we adenocarcinoma della prostata step 2 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 chemoresistance 28it is recommended to start with a large population of cells we usually plate 20 flasks adenocarcinoma della prostata step 2 cm 2which account for approximately 1.
Key steps of the protocol should be considered to ensure success of the procedure. 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 adenocarcinoma della prostata step 2 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 adenocarcinoma della prostata step 2 this taxane agent 15 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 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 adenocarcinoma della prostata step 2, nM. After a recovery period of one to two weeks, the phenotype can be re-tested by qPCR and colony formation assays.
Please note that after a freeze-thaw cycle, the chemoresistance phenotype should always be re-evaluated with the aforementioned methods. Adenocarcinoma della prostata step 2 these caveats, we 2829 and others 30313233 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 exposure In a second study, using these models together with publicly available patient gene data sets 2425we 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 network Notably, these studies helped to identify novel key roles of GATA2 in advanced metastatic prostate cancer aggressiveness 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 adenocarcinoma della prostata step 2 more the boundaries of what we know about this disease and how we treat its patients.
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