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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