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
<ol>
	<li>Thaw basement membrane matrix (e.g., Matrigel) at 4 &#176;C overnight before starting the experiment. Keep it on ice during use.</li>
	<li>Place culture plates at 37 &#176;C for 24 h prior to experiments. This will help the basement membrane matrix dome (hereafter referred to as matrix dome) to polymerize. Plating organoids is described in step 2.1.2.</li>
	<li>Prepare the organoid medium according to the established protocol5.</li>
	<li>Prepare the organoid medium without the addition of epidermal growth factor (EGF; see Table of Materials for components). EGF suppresses the AR transcriptional output and confers anti-androgen resistance5.</li>
	<li>Prepare Dulbecco&#39;s Modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). 
	NOTE: This media is used to inhibit the enzymatic digestion with trypsin replacement in sections 2-4.</li>
	<li>Prepare drug solutions according to the manufacturer&#39;s protocol. For enzalutamide/mdv3100 (hereafter referred to as second generation anti-androgen), prepare a stock solution of 100 &#181;M in dimethyl sulfoxide (DMSO). Stock can be stored at -20 &#176;C for up to 6 months and does not have to be made fresh.</li>
</ol>
2. Isolation, enzymatic digestion, and establishment of organoids
<ol>
	<li>Isolate prostate organoids from mouse or human tissue according to the previously established protocols5,7. A brief description is provided below.
	<ol>
		<li>Mince and enzymatically digest prostate tissue to produce a single cell suspension. In this experiment, 1 mL of 5 mg/mL collagenase type II in ADMEM/F12 was used for the digestion of 50 mg of prostate tissue.</li>
		<li>Collect cells by centrifugation at 300 x g for 5 min, count the cells, resuspend them in the basement membrane matrix, and plate at the appropriate density5,7 in the matrix domes on pre-warmed organoid culture plates (plating method is shown in Figure 1A).</li>
		<li>Allow the domes to solidify and add media onto the tops of the domes so that they are completely covered.</li>
	</ol>
	</li>
	<li>Grow organoids to the desired quantity for downstream applications. Cell number can be determined by standard counting methods. See the application-specific section of the protocol for additional details on density.</li>
	<li>Using a P1000 pipette, draw up the medium and pipette up and down to disrupt. When basement membrane matrix is fully disrupted, transfer the suspension to a 15 mL conical tube. Do not place more than 10 domes per single 15 mL conical tube. Centrifuge at 300 x g for 5 min.</li>
	<li>Draw off the supernatant and wash the cell pellet with 5 mL of PBS. Centrifuge at 300 x g for 5 min.</li>
	<li>Draw off the supernatant and resuspend the pellet in 4 mL of trypsin replacement. Digest for 5-10 min with shaking at 37 &#176;C. Add an equal volume of organoid medium + 10% FBS to inhibit the trypsin replacement. Centrifuge at 300 x g for 5 min.</li>
	<li>Draw off the supernatant and resuspend in 1 mL of PBS.</li>
	<li>Filter the suspension with a 40 &#181;m filter to ensure a single cell suspension. Quantify the cell number using a hemocytometer or equivalent counting device. 
	NOTE: If obtaining viable single cells is difficult, use flow sorting to obtain a single cell solution.</li>
</ol>
3. Assessing organoid formation capacity
NOTE: To determine the percentage of cells that can generate an organoid, a seeding assay can be performed as a proxy for the stem/progenitor potential. The organoid formation capacity is also important for defining a cell seeding number for the viability assays.
<ol>
	<li>Dilute the cell suspension obtained in step 2.7 to 100 cells per 10 &#181;L of the suspension using organoid medium containing 10 &#181;M Rho kinase inhibitor Y-27632.</li>
	<li>Transfer 1,100 cells (110 &#181;L of suspension) to a new conical tube.</li>
	<li>Add 285 &#181;L of basement membrane matrix and resuspend the cells. This will result in a ~70% matrix concentration. 
	NOTE: Dilution of the basement membrane matrix during seeding greatly reduces the variation in dome size, caused by the viscosity of the protein matrix.</li>
	<li>Seed cells in 35 &#181;L of matrix domes in a pre-warmed 24 well plate, resulting in 200 cells/well. Plate 3-5 replicates per sample (also see plating method in Figure 1A and step 2.1.2).</li>
	<li>To ensure that the cells remain within the matrix dome, flip the plate and place it in a cell incubator to solidify the basement membrane matrix.</li>
	<li>After 10 min, remove the plate from the incubator and add medium containing the Rho kinase inhibitor.</li>
	<li>Refresh the medium every 2 days. After 7 days, quantify the number of organoids. Keep the Rho kinase inhibitor in media throughout the experiment.</li>
	<li>Count the number of organoids established per dome and calculate the percent of organoids formed out of the total number of cells plated (200 cells). 
	NOTE: Organoid establishment ratios vary from 3%-60% depending on the cell type and genotype.</li>
</ol>
4. Determining pharmacological responses of organoids
<ol>
	<li>Continuing from step 2.7, seed 1,000-10,000 cells in a matrix dome. Use the organoid formation efficiency and growth speed as a proxy for determining the final cell number. 
	NOTE: Recommended cell numbers are provided in Table 1. Use three to five replicates per condition per analysis. Use a final concentration of 70% basement membrane matrix to reduce pipetting errors induced by the viscosity.</li>
	<li>Seed 35 &#181;L of matrix domes in a 24 well plate and let the domes solidify as done in section 3 (also see plating method in Figure 1A and step 2.1.2). Add medium containing the Rho kinase inhibitor and drug of choice. This method can be applied to all drugs, but in this protocol, a second-generation anti-androgen is used at 10 &#181;M for an example. To determine half maximal inhibitory concentration (IC50), perform a log10 incremental, and as a control, use the vehicle in which the drug was dissolved.</li>
	<li>Refresh the medium every two or three days and analyze the organoids on day 7 to determine the pharmacological response of the drug. The timepoints may vary among the choice of experiment and drug. 
	NOTE: Organoids do not have to be trypsinized to perform these assays.</li>
	<li>To keep organoids intact, using a P1000 pipette, draw up the medium and pipette up and down to disrupt the basement membrane matrix.</li>
	<li>When the basement membrane matrix is fully disrupted, transfer the suspension to a 15 mL conical tube. Do not transfer more than 10 matrix domes per 15 mL conical tube.</li>
	<li>Centrifuge at 300 x g for 5 min. Draw off the supernatant and wash with 5 mL of PBS.</li>
	<li>Resuspend organoids in 1 mL of PBS and disrupt the organoids using trituration and a glass Pasteur pipette.</li>
	<li>Quantify the number of organoid fragments. Seed 5 replicates containing 100 organoid fragments as described in step 2.1.2.</li>
	<li>Perform the cell viability assay as described below in section 7.</li>
</ol>
5. RNA isolation from organoids
NOTE: Commercially available column-based methods yield good quantity and quality of RNA. To ensure good quantity RNA, use a minimum of one dome per sample; however, using three domes is recommended, which can be seeded in a single well of a 12 well plate.
<ol>
	<li>Add &#223;-mercaptoethanol (1%) to the glutathione lysis buffer in the RNA isolation kit.</li>
	<li>Draw off the medium from the basement membrane domes containing organoids and add 750 &#181;L of this buffer. Pipette up and down using a P1000 pipette. Check that all the basement membrane matrix has been dissolved.</li>
	<li>Add 750 &#181;L of 70% ethanol and mix by pipetting. Subsequently transfer 700 &#181;L of the mixture to the column, centrifuge at 12,000 x g for 1 min, and repeat with the remainder of the lysate.</li>
	<li>Perform washes and on-column DNase treatment according to manufacturer&#39;s instructions. Elute RNA in 30-50 &#181;L of RNAse-free water.</li>
	<li>Measure the concentrations using a fluorometer at OD = 260 nm and 280 nm and store at -80 &#176;C or continue with downstream applications.</li>
</ol>
6. Protein isolation from organoids
NOTE: For protein isolation, prepare standard RIPA buffer containing phosphatase and protease inhibitors (Table of Materials). Using at least three domes is recommended, which can be seeded in a single 12 well.
<ol>
	<li>Using a P1000 pipette, draw up the medium from the cell with the basement membrane domes containing organoids and pipette up and down to disrupt the basement membrane matrix.</li>
	<li>When fully disrupted, transfer the suspension to a 15 mL conical tube. Centrifuge at 300 x g for 5 min.</li>
	<li>Draw off the supernatant and wash with 5 mL of ice-cold PBS. Centrifuge at 300 x g for 5 min.</li>
	<li>Draw off the supernatant and resuspend the pellet in 4 mL of trypsin replacement. Digest for 5-10 min while shaking at 37 &#176;C.</li>
	<li>Add an equal volume of organoid medium + 10% FCS to inhibit the trypsin replacement. Centrifuge at 300 x g for 5 min. 
	NOTE: Post-centrifugation, no basement membrane matrix should be visible in the pellet.</li>
	<li>Draw off the supernatant and wash with 5 mL of ice-cold PBS. Draw off the supernatant and resuspend the cell pellet in 300 &#181;L of lysis buffer using a P1000 pipet, then transfer to a 1.5 mL microcentrifuge tube.</li>
	<li>Incubate on ice for 10 min and subsequently sonicate 2x for 30 s each at cooled water with a temperature of 4 &#176;C. Place the tube back on ice and perform protein quantification using standard methods.</li>
	<li>Denature the protein by adding sodium dodecyl sulfate (SDS) containing loading dye and boil for 5 min at 95 &#176;C. Store lysates at -80 &#176;C or continue with downstream applications.</li>
</ol>
7. Cell viability assay with organoids
NOTE: Cell viability can be assessed using the commercially available cell viability assay kit and a luminometer. Prepare buffers according to the manufacturer&#39;s instructions. Five replicates per condition is recommended: one replicate consisting of one 35 &#181;L basement membrane matrix dome in one well of a 24 well plate.
<ol>
	<li>Draw off the medium of the organoid culture, being careful to leave the matrix domes intact.</li>
	<li>Add 65 &#181;L of PBS and pipette up and down to disrupt the matrix dome.</li>
	<li>Add 100 &#181;L of the cell viability assay kit buffer and resuspend by pipetting.</li>
	<li>Incubate at room temperature (RT) for 10 min with shaking.</li>
	<li>Transfer 100 &#181;L of mixture to a non-translucent plate suitable for the luminometer and perform reading according to the manufacturer&#39;s instructions for the cell viability assay kit.</li>
</ol>
8. Preparation of organoids for xenografting
NOTE: Organoids are also amenable for subcutaneous grafting in both immune compromised animals, as well as, isogenic mice. To ensure injected organoids are distinguishable in vivo, label organoids with a constitutively expressing fluorophore5. It is recommended to perform a pilot experiment for grafting using 5 x 105 cells to 2 x 106 cells per injection, with increments of 5 x 106 cells, as grafting efficiency varies between organoid lines.
<ol>
	<li>Using a P1000 pipette, draw up the medium and pipette up and down to disrupt the basement membrane matrix. When fully disrupted, transfer the suspension to a 15 mL conical tube. Centrifuge at 300 x g for 5 min. 
	NOTE: Do not transfer more than 10 matrix domes per 15 mL conical tube.</li>
	<li>Draw off the supernatant and wash with 5 mL of PBS. Centrifuge at 300 x g for 5 min.</li>
	<li>Draw off the supernatant and resuspend the pellet in 4 mL of trypsin replacement. Digest for 5-10 min while shaking at 37 &#176;C.</li>
	<li>Add equal volume of organoid medium + 10% FBS to inhibit trypsin replacement. Centrifuge at 300 x g for 5 min.</li>
	<li>Draw off the supernatant and resuspend in 1 mL of PBS. Filter the suspension with a 40 &#181;m filter to ensure a single cell suspension. Quantify cells using standard methods.</li>
	<li>Spin down and resuspend the cells in PBS + Rho inhibitor to a concentration of 2 x 106 cells per 100 &#181;L (see Table 2 for cell concentrations and absolute cell number needed for varying concentrations). Use an equal volume of basement membrane matrix to generate a 1:1 suspension. Place the suspension on ice.</li>
	<li>Inject cells according to standard protocols and monitor xenograft growth using standard methods8.</li>
</ol>

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C.L.S serves on the board of directors of Novartis; is a cofounder of ORIC Pharmaceuticals and coinventor of enzalutamide and apalutamide; is a science advisor to Agios, Beigene, Blueprint, Column Group, Foghorn, Housey Pharma, Nextech, KSQ, Petra, and PMV; and is a cofounder of Seragon, purchased by Genentech/Roche in 2014. W.R.K. is a coinventor and patent holder of organoid technology.

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 ...

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JoVE Journal

Cancer Research

10.9K Views.  Memorial Sloan Kettering Cancer Center. 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.

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

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 ...

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 ...

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Optimizing HCC Organoid Formation for Target Identification and Drug Development

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents necessitate further research into its pathogenesis and treatment. Organoids, a novel model that closely resembles native tumor tissue and can be cultured in vitro, have garnered significant interest in recent years, with numerous reports on the development of organoid models for liver cancer. In this study, we have successfully optimized the procedure and established a culture protocol that enables the formation of larger-sized HCC organoids with stable passaging and culture conditions. We have comprehensively outlined each step of the procedure, covering the entire process of HCC tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation, and provided detailed precautions in this paper. These organoids exhibit genetic similarity to the original HCC tissues and can be utilized for diverse applications, including the identification of potential therapeutic targets for tumors and subsequent drug development.

Author Spotlight: Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids 

We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate effectiveness.

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...