Name: prostate organoid cultures as tools to translate genotypes and mutational profiles to pharmacological responses
Uploaded: 2019-10-24T15:00:00
Description: Watch this Scientific Journal Video about Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses at JoVE.com

K.P. is supported by NIH 1F32CA236126-01. C.L.S. is supported by HHMI; CA193837; CA092629; CA224079; CA155169; CA008748; and Starr Cancer Consortium. W.R.K. is supported by Dutch Cancer Foundation/KWF Buit 2015-7545 and Prostate Cancer Foundation PCF 17YOUN10.

All work described in this protocol has been performed with previously established murine organoids and patient-derived organoids. All animal work was performed in compliance with the guidelines of Research Animal Resource Center of Memorial Sloan Kettering Cancer Center (IACUC: 06-07-012). All patient-derived tissues were collected in compliance with rules and regulations of Memorial Sloan Kettering Cancer Center (IRB: 12001).
1. Medium and buffer preparation
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	<li>Thaw basement membran...

<ol>
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Understanding the molecular mechanisms underlying anti-androgen resistance and discovering potential therapeutic vulnerabilities requires testing of pharmacological responses in model systems mimicking prostate cancer. Described here is a detailed protocol for the reliable analysis of pharmacological responses in patient-derived and genetically engineered prostate organoids and preparation of these organoid samples for downstream applications.
There are two critical steps in this protocol. The...

Drug resistance is one of the major clinical problems in cancer treatment. Metastatic prostate cancer (PCa) treatment is primarily directed at the androgen-signaling axis. Next-generation anti-androgen therapies (e.g., enzalutamide and abiraterone) have showed great clinical success, but virtually all PCa eventually progresses towards an androgen-independent state, or castration resistant prostate cancer (CRPC).
Recent genomic and transcriptomic profiling of CRPC revealed there are three general mechanisms of resistance in prostate cancer: 1) activating mutations resulting in the restoration of androgen receptor (AR) signaling1; 2) activation of bypass signaling, as exemplified in a pre-clinical model for next-generation anti-androgen therapy resistance in which activation of the glucocorticoid receptor (GR) can compensate for loss of AR signaling2; and 3) the recently identified process of lineage plasticity, in which tumor cells acquire resistance by switching lineages from a cell type dependent on the drug target to another cell type that is not dependent on this (which, in PCa, is represented as AR-negative and/or neuroendocrine disease [NEPC])3,4. However, the molecular mechanisms that cause drug resistance are not understood. Moreover, acquired anti-androgen resistance may lead to therapeutic vulnerabilities that can be exploited. Therefore, it is essential to evaluate drug responses in model systems that mimic patient phenotypes and genotypes.
Prostate organoids are organotypic cultures grown in a 3D protein matrix with a defined medium. Importantly, prostate organoids can be established from benign and cancerous tissue of murine or human origin, and they retain phenotypic and genotypic features found in vivo5,6. Importantly, both anti-androgen sensitive PCa and CRPC cells are represented in the current compendium of organoids. Moreover, prostate organoids are easily genetically manipulated using CRISPR/Cas9 and shRNA5. Thus, prostate organoids are a suitable model system for testing drug responses and elucidating resistance mechanisms. Here, a detailed protocol is described to perform drug testing and analyze pharmacological responses using prostate organoids.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>Organoid medium component: Final concentration 200 nM</td>
		</tr>
		<tr>
			<td>ADMEM/F12</td>
			<td>Gibco/Life technologies</td>
			<td>12634028</td>
			<td>Organoid medium component</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco/Life technologies</td>
			<td>17504-044</td>
			<td>Organoid medium component</td>
		</tr>
		<tr>
			<td>Cell culture plates</td>
			<td>Fisher</td>
			<td>657185</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Titer Glo</td>
			<td>Promega</td>
			<td>G7571</td>
			<td></td>
		</tr>
		<tr>
			<td>DHT</td>
			<td>Sigma-Aldrich</td>
			<td>D-073</td>
			<td>Organoid medium component: Final Concentration 1 nM</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>315-09</td>
			<td>Organoid medium component: Final concentration 50 ng/ml for mouse, 5 ng/nl for Human</td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td>Human specific organoid medium component: Final concentration 10 ng/ml</td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Human specific organoid medium component: Final concentration 5 ng/ml</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco/Life technologies</td>
			<td>35050079</td>
			<td>Organoid medium component</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>MADE IN-HOUSE</td>
			<td>N/A</td>
			<td>Organoid medium component: Final concentration 10 mM</td>
		</tr>
		<tr>
			<td>Matrigel (Growthfactor reduced &#38; Phenol Red free)</td>
			<td>Corning</td>
			<td>CB-40230C</td>
			<td>Organoid medium component</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td>Organoid medium component: Final concentration 1.25 mM</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td>Human specific organoid medium component: Final concentration 10 mM</td>
		</tr>
		<tr>
			<td>NOGGIN</td>
			<td>Peprotech or stable transfected 293t cells with Noggin construct (Karthaus et al. 2014)</td>
			<td>120-10C</td>
			<td>Organoid medium component: Final Concentration 10% conditioned medium or 100 ng/ml</td>
		</tr>
		<tr>
			<td>Penicillin/Streptavidin</td>
			<td>Gemini Bio-Products</td>
			<td>400-109</td>
			<td>Organoid medium component</td>
		</tr>
		<tr>
			<td>Phospatase inhibitors</td>
			<td>Merck Millipore</td>
			<td>524629</td>
			<td></td>
		</tr>
		<tr>
			<td>Prostaglandin E2</td>
			<td>Tocris</td>
			<td>3632464</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitors</td>
			<td>Merck Millipore</td>
			<td>539131</td>
			<td></td>
		</tr>
		<tr>
			<td>R-SPONDIN</td>
			<td>Peprotech or stable transfected 293t cells with R-Spondin1 construct (Karthaus et al. 2014)</td>
			<td>120-38</td>
			<td>Organoid medium component: Final Concentration 10% conditioned medium or 500 ng/ml</td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>Merck</td>
			<td>20-188</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA-easy minikit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Aldrich</td>
			<td><a href="https://www.sigmaaldrich.com/catalog/search?term=152121-30-7&amp;interface=CAS%20No.&amp;lang=en&amp;region=US&amp;focus=product">152121-30-7</a></td>
			<td>Human specific organoid medium component: Final concentration 10 &#956;M</td>
		</tr>
		<tr>
			<td>TryplE</td>
			<td>ThermoFisher</td>
			<td>12605036</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleckchem</td>
			<td>S1049</td>
			<td>Organoid medium component: Final Concentration 10 &#956;M</td>
		</tr>
	</tbody>
</table>

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.

Seeding efficiency 
Organoid formation capacity is determined by phenotype and genotype. Wild-type (WT) prostate basal cells showed superior organoid formation capacity (30%-40%) compared to luminal cells (3%) (Figure 1A). After organoid establishment, the formation capacity increased drastically. Typically, 25%-30% of cells derived from a WT organoid can form a new organoid (Figure 1<strong...

Watch this Scientific Journal Video about Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses at JoVE.com

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

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 ...

This protocol provides standard and reproducible methods to assess pharmacological responses in prostate organoid cultures. Prostate organoid cultures provide an in vitro system that preserves many aspects of in vivo biology and pharmacological response by allowing cells to adopt a three-dimensional structure in a basement membrane matrix. We are particularly excited about using these methods to assess how genotype dictates pharmacological response in the prostate and to model drug resistance.These methods can be applied to organoid culture systems that use the dome plating method. However, the components in the media such as growth factors may differ depending on tissue type. Visual demonstration will allow for a more specific and detailed discussion of the protocol steps and how they differ from the many other published organoid culture systems available to researchers.Organoid culture is typically more time consuming than two-dimensional cell culture. Be sure to allocate extra time for these methods. Start by isolating prostate organoids from mouse or human tissue.Mince and enzymatically digest the tissue to produce a single cell suspension, then collect the cells by centrifugation at 300 times g for five minutes. Count the cells and resuspend them in the basement membrane matrix. Then plate them at the appropriate density in the matrix domes on pre-warmed organoid culture plates.Domes should be two millimeters apart from one another and from the side of the well. Once solidified, gently add media from the side of the well to avoid disrupting the matrix. After the domes have solidified, add media on top of the dome so that they are completely covered.Grow organoids to the desired quantity for downstream applications determining cell number with standard counting methods. When the organoids are ready to use, draw up the medium with a Pten00 pipette and pipette it up and down to disrupt the basement membrane matrix. Transfer the suspension to a 15 milliliter conical tube and centrifuge at 300 times g for five minutes.Aspirate the supernatant and wash the cell pellet with five milliliters of PBS. Repeat the centrifugation, then resuspend the pellet in four milliliters of trypsin replacement and allow it to digest for five to 10 minutes while shaking at 37 degrees Celsius. Add an equal volume of organoid medium with 10%FBS to inhibit the trypsin replacement and centrifuge the tube at 300 times g for five minutes.Aspirate the supernatant and resuspend the cells in one milliliter of PBS. Then strain the cells with a 40 micrometer filter to ensure single cell suspension and count them using a hemocytometer. Dilute the cell suspension to 100 cells per 10 microliters using organoid medium with 10 micromolar rho kinase inhibitor Y27632.Transfer 1, 100 cells to a new conical tube and add 285 microliters of basement membrane matrix which will result in a 70%matrix concentration. Next, seed the cells in 35 microliters of matrix domes in a pre-warmed 24 well plate making sure to plate three to five replicates per sample. Flip the plate and place it in a cell incubator to solidify the basement matrix.After 10 minutes, remove the plate from the incubator and add medium containing the rho kinase inhibitor. Refresh the medium every two days and after seven days, count the number of organoids established per dome and calculate the percent of organoids formed out of the total number of cells. After isolating the organoids as previously described, seed 1, 000 to 10, 000 cells in the matrix dome using organoid formation efficiency and growth speed as a proxy for determining the final cell number.Then seed 35 microliters of matrix domes in a 24 well plate and let the dome solidify. Add medium containing the rho kinase inhibitor and the drug of choice. Perform a log 10 incremental to determine half maximal inhibitory concentration using the vehicle in which the drug was dissolved as a control.Refresh the medium every two to three days and analyze the organoids on day seven to determine pharmacological response to the drug by performing a cell viability assay as described in the manuscript. To plate organoid fragments without trypsinization, use a Pten00 pipette to aspirate the medium and pipette up and down to disrupt the basement membrane matrix. When the matrix is fully disrupted, transfer the suspension to a 15 milliliter conical tube and centrifuge at 300 times g for five minutes.Aspirate the supernatant and add five milliliters of PBS. After the wash, resuspend the organoids in one milliliter of PBS and disrupt the organoids by trituration with a glass Pasteur pipette. Quantify the number of organoid fragments and seed five replicates with 100 fragments as previously described.Seven days later, perform a cell viability assay according to manuscript directions. Wild type prostate basal cells demonstrated superior organoid formation compared to luminal cells. A minor increase in organoid formation was achieved with CRISPR-Cas9-mediated loss of Pten or P53 and loss of both further increased formation capacity.The effects of anti-androgenic molecules on growth were tested in murine organoids with different genotypes. Loss of P53 did not cause resistance to the anti-androgenic molecules, but loss of Pten increased resistance. Dual loss of P53 and Pten, however, resulted in complete resistance.Androgen receptor inhibition also altered phenotypes. Wild type Pten deleted and P53 deleted organoids demonstrated the decrease in organoid lumen size while organoids with loss of both Pten and P53 were phenotypically unaffected. As expected, when these cells were grafted subcutaneously in the flank, only the double deleted Pten and P53 organoids grew.Two distinct human prostate cancer organoids MSKPCA2 and MSKPCA3 were tested for their response to anti-androgenic molecules. Proliferation of MSKPCA2 organoids was strongly inhibited whereas MSKPCA3 organoids remained unaffected. MSKPCA2 expressed high levels of androgen receptors and FKBP5 as well as hallmark luminal proteins.MSKPCA3 also expressed basal and mesenchymal markers and showed no expression of FKBP5 suggesting that these organoids model a non-luminal androgen-independent phenotype. Organoids needs to be disrupted periodically either by trypsinization or trituration with a glass pipette in order to remain viable. Frequency depends on species origin and genotype.Any type of next generation sequencing or proteomics experiment can be performed following the procedures described. These experiments would investigate the molecular basis of the phenotypes and pharmacological responses. This three-dimensional organoid culture technique allows researchers to study pharmacological responses in a highly controlled genetic context in vitro.This can be a reliable alternative to lengthy in vivo studies.

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Research

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Cancer Research

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

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 ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Culture of Bladder Cancer Organoids as Precision Medicine Tools

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as two-dimensional (2D) cultures on tissue culture plastic. There is a critical need for more representative models of tumor complexity that can accurately predict therapeutic response and sensitivity. The development of three-dimensional (3D) ex vivo culture of patient-derived organoids (PDOs), derived from fresh tumor tissues, aims to address these shortcomings. Organoid cultures can be used as tumor surrogates in parallel to routine clinical management to inform therapeutic decisions by identifying potential effective interventions and indicating therapies that may be futile. Here, this procedure aims to describe strategies and a detailed step-by-step protocol to establish bladder cancer PDOs from fresh, viable clinical tissue. Our well-established, optimized protocols are practical to set up 3D cultures for experiments using limited and diverse starting material directly from patients or patient-derived xenograft (PDX) tumor material. This procedure can also be employed by most laboratories equipped with standard tissue culture equipment. The organoids generated using this protocol can be used as ex vivo surrogates to understand both the molecular mechanisms underpinning urological cancer pathology and to evaluate treatments to inform clinical management.

Patient-derived organoids (PDOs) are a powerful tool in translational cancer research, reflecting both the genetic and phenotypic heterogeneity of the disease and response to personalized anti-cancer therapies. Here, a consolidated protocol to generate human primary bladder cancer PDOs in preparation for the evaluation of phenotypic analyses and drug responses is detailed.

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as ...

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is essential to understand the process and related mechanisms of prostate cancer bone metastasis for developing new therapeutics. For this, an animal model of bone metastasis is an essential tool. Here, we report detailed procedures to generate a bone metastasis mouse model via intra-cardiac injection of prostate cancer cells. A bioluminescence imaging system can determine whether prostate cancer cells have been accurately injected into the heart and monitor cancer cell metastasis since it has great advantages in monitoring metastatic lesion development. This model replicates the natural development of disseminated cancer cells to form micro-metastases in the bone and imitates the pathological process of prostate cancer bone metastasis. It provides an effective tool for further exploration of the molecular mechanisms and the in vivo therapeutic effects of this disease.

Here, we present a protocol for the intra-cardiac injection of human prostate cancer cells to generate a mouse model with bone metastasis lesions.

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...