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>

<ol>
	<li>Robinson, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+Clinical+Genomics+of+Advanced+Prostate+Cancer.">Integrative Clinical Genomics of Advanced Prostate Cancer.</a> Cell. 162 (2), 454 (2015).</li><li>Arora, V. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoid+Receptor+Confers+Resistance+to+Antiandrogens+by+Bypassing+Androgen+Receptor+Blockade.">Glucocorticoid Receptor Confers Resistance to Antiandrogens by Bypassing Androgen Receptor Blockade.</a> Cell. 155 (6), 1309-1322 (2013).</li><li>Ku, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rb1+and+Trp53+cooperate+to+suppress+prostate+cancer+lineage+plasticity,+metastasis,+and+antiandrogen+resistance.">Rb1 and Trp53 cooperate to suppress prostate cancer lineage plasticity, metastasis, and antiandrogen resistance.</a> Science. 355 (6320), 78-83 (2017).</li><li>Mu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX2+promotes+lineage+plasticity+and+antiandrogen+resistance+in+TP53-+and+RB1-deficient+prostate+cancer.">SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer.</a> Science. 355 (6320), 84-88 (2017).</li><li>Karthaus, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Multipotent+Luminal+Progenitor+Cells+in+Human+Prostate+Organoid+Cultures.">Identification of Multipotent Luminal Progenitor Cells in Human Prostate Organoid Cultures.</a> Cell. 159 (1), 163-175 (2014).</li><li>Gao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+Cultures+Derived+from+Patients+with+Advanced+Prostate+Cancer.">Organoid Cultures Derived from Patients with Advanced Prostate Cancer.</a> Cell. 159 (1), 176-187 (2014).</li><li>Drost, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+culture+systems+for+prostate+epithelial+and+cancer+tissue.">Organoid culture systems for prostate epithelial and cancer tissue.</a> Nature Protocols. 11 (2), 347-358 (2016).</li><li>Bose, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERF+mutations+reveal+a+balance+of+ETS+factors+controlling+prostate+oncogenesis.">ERF mutations reveal a balance of ETS factors controlling prostate oncogenesis.</a> Nature. 546 (7660), 671-675 (2017).</li><li>Platt, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas9+Knockin+Mice+for+Genome+Editing+and+Cancer+Modeling.">CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.</a> Cell. 159 (2), 440-455 (2014).</li><li>Carver, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+feedback+regulation+of+PI3K+and+androgen+receptor+signaling+in+PTEN-deficient+prostate+cancer.">Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer.</a> Cancer Cell. 19 (5), 575-586 (2011).</li><li>Puca, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient+derived+organoids+to+model+rare+prostate+cancer+phenotypes.">Patient derived organoids to model rare prostate cancer phenotypes.</a> Nature Communications. 9 (1), 2404 (2018).</li><li>Gao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+Cultures+Derived+from+Patients+with+Advanced+Prostate+Cancer.">Organoid Cultures Derived from Patients with Advanced Prostate Cancer.</a> Cell. 159 (1), 176-187 (2014).</li><li>Puca, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delta-like+protein+3+expression+and+therapeutic+targeting+in+neuroendocrine+prostate+cancer.">Delta-like protein 3 expression and therapeutic targeting in neuroendocrine prostate cancer.</a> Science Translational Medicine. 11 (484), eaav0891 (2019).</li><li>Dijkstra, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Tumor-Reactive+T+Cells+by+Co-culture+of+Peripheral+Blood+Lymphocytes+and+Tumor+Organoids.">Generation of Tumor-Reactive T Cells by Co-culture of Peripheral Blood Lymphocytes and Tumor Organoids.</a> Cell. 174 (6), 1586-1598 (2018).</li><li>van de Wetering, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+derivation+of+a+living+organoid+biobank+of+colorectal+cancer+patients.">Prospective derivation of a living organoid biobank of colorectal cancer patients.</a> Cell. 161 (4), 933-945 (2015).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lgr5(+ve)+stem+cells+drive+self-renewal+in+the+stomach+and+build+long-lived+gastric+units+in+vitro.">Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.</a> Cell stem cell. 6 (1), 25-36 (2010).</li><li>Bartfeld, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+of+human+gastric+epithelial+stem+cells+and+their+responses+to+bacterial+infection.">In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection.</a> Gastroenterology. 148 (1), 126-136 (2015).</li><li>Huch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+genome-stable+bipotent+stem+cells+from+adult+human+liver.">Long-term culture of genome-stable bipotent stem cells from adult human liver.</a> Cell. 160 (1-2), 299-312 (2015).</li><li>Boj, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Schutgens, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubuloids+derived+from+human+adult+kidney+and+urine+for+personalized+disease+modeling.">Tubuloids derived from human adult kidney and urine for personalized disease modeling.</a> Nature Biotechnology. 37 (3), 303-313 (2019).</li><li>Sachs, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Living+Biobank+of+Breast+Cancer+Organoids+Captures+Disease+Heterogeneity.">A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity.</a> Cell. , 1-25 (2017).</li></ol>

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><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 &amp;amp; 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>&lt;a href=&quot;https://www.sigmaaldrich.com/catalog/search?term=152121-30-7&amp;amp;interface=CAS%20No.&amp;amp;lang=en&amp;amp;region=US&amp;amp;focus=product&quot;&gt;152121-30-7&lt;/a&gt;</td><td>Human specific organoid medium component: Final concentration 10 &amp;mu;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 &amp;mu;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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Cancer Research

11.0K 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

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living biobanks&#34; of patient derived cell lines. For many types of epithelial cells, various three dimensional (3D) culture approaches have been described that support an improved differentiated state. While CRCs retain their lineage commitment to the tissue from which they are isolated, they fail to express many of the differentiation markers associated with the tissue of origin when grown under normal two dimensional (2D) culture conditions. To enhance the application of patient-derived CRCs for prostate cancer research, a 3D culture format has been defined that enables a rapid (2 weeks total) luminal cell differentiation in both normal and tumor-derived prostate epithelial cells. Herein, a filter insert-based format is described for the culturing and differentiation of both normal and malignant prostate CRCs. A detailed description of the procedures required for cell collection and processing for immunohistochemical and immunofluorescent staining are provided. Collectively the 3D culture format described, combined with the primary CRC lines, provides an important medium- to high- throughput model system for biospecimen-based prostate research.

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living ...

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

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

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

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

Genetics

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 ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...