This study was supported by the Leo and Anne Albert Charitable Foundation and JM Foundation. We thank the University of California San Diego Moores Cancer Center members, Dr. Jing Yang and Dr. Kay T. Yeung for allowing us the use of their microtome and Randall French, Department of Surgery for technical expertise.

This study was carried out in strict accordance with the recommendations in the Guide for the University of California San Diego (UCSD) Institutional Review Board (IRB). IRB #090401 Approval was received from the UCSD Institutional Review Board (IRB) to collect surgical specimen from patients for research purposes. An informed consent was obtained from each patient and a surgical bone prostate cancer metastasis specimen was obtained from orthopedic repair of a pathologic fracture in the femur. Animal protocols were performed under the University of California San Diego (UCSD) animal welfare and Institutional Animal Care and Use Committee (IACUC) approved protocol #S10298. Cells from mechanically and enzymatically dissociated patient tumor tissue were intra-femorally injected into 6 to 8 week old male Rag2-/-;&#947;c-/- mice as previously described17. Xenograft tumor volume was determined using an in vivo bioluminescence imaging system and caliper measurements. Upon tumor growth up to 2.0 cm (the maximal allowable size approved by IACUC), the tumor was harvested for 3D organoids establishment.
NOTE: Figure 1 shows the workflow for establishing 3D Organoids and a protocol number for each step of the experimental procedures.
1. Processing of patient derived xenograft (PDX) tumor tissues
NOTE: This is an initial step for organoid establishment for a tumor derived from a xenograft mouse model. This protocol is adapted from a previous publication by Drost et al.5 and we have modified the media conditions to include serum supplementation to organoid media.
<ol>
	<li>Process the tumor specimen as described below.
	<ol>
		<li>Mince tumor samples to 1-3 mm3 sized pieces and digest the samples with 10 mL of cell dissociation solution for 45 min at room temperature.</li>
		<li>To terminate digestion, add 20 mL of DMEM complete media to the samples.</li>
		<li>Filter the suspension through a 70 &#956;m cell strainer. Use a sterile plunger flange to push any leftover tissue on the top of 70 &#956;m cell strainer.</li>
		<li>Centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Wash the cell pellet three times with fresh adDMEM complete media. Match the volume of media for wash and resuspension with the volume of media suggested in Table 3. For example, for a 24 well plate culture condition, the volume for the wash should be 500 &#181;L. 
		NOTE: As shown in Table 3, the protocol is applicable to different culture conditions.</li>
		<li>Determine final cell counts using Trypan blue dye and a hemocytometer.</li>
		<li>After obtaining cell counts, re-suspend cell pellet in 80 &#181;L of 2% FBS in PBS per 2 x 106 tumor cells.</li>
		<li>Add 20 &#181;L of Mouse Cell Depletion Cocktail per 2 x 106 tumor cells. Mix well and incubate for 15 min at 2-8 &#176;C.</li>
		<li>Adjust the volume to 500 &#181;L with 2% FBS in PBS buffer per 2 x 106 tumor cells. 
		NOTE: Up to 1 x 107 tumor cells in 2.5 mL of cell suspension can be processed on one LS column.</li>
		<li>Load the LS columns on the magnetic column separator and place a 15 mL conical tube on a rack underneath to collect the flow-through.</li>
		<li>Rinse each column with 3 mL of 2% FBS in PBS buffer. Discard the conical tube with the wash flow-through and replace with a new, sterile 15 mL conical tube.</li>
		<li>Add the cell suspension (up to 2.5 mL of 1 x 107 tumor cells) onto the column. Collect flow-through that will be the enriched with human tumor cells.</li>
		<li>Wash the column twice with 1 mL of 2% FBS in PBS buffer. 
		NOTE: It is important to perform wash steps as soon as the column is empty. Also, try to avoid forming air bubbles.</li>
	</ol>
	</li>
	<li>Aliquot the appropriate volume of cell suspension to a 1.5 mL tube for the desired culture set up (Table 3).</li>
	<li>Centrifuge the 1.5 mL tube at 300 x g and 4 &#176;C for 5 min.</li>
	<li>Carefully remove and discard the supernatant.</li>
</ol>
2. Processing of patient primary tumor tissues
NOTE: This is an initial step for organoid establishment.
<ol>
	<li>Follow all of step 1 except the mouse cell depletion process, which is not necessary for processing of patient primary tumor tissues.</li>
</ol>
3. Forming an attached round dome on the plate
NOTE: This manuscript describes three ways to make a dome from a mixture of the cell pellet and the basement membrane (e.g., Matrigel) as shown in Figure 1 and Figure 2. In Steps 2-4, the cells and the basement membrane should be kept on ice to prevent solidification of the basement membrane.
<ol>
	<li>Resuspend the cell pellet in the appropriate volume of basement membrane (e.g., 40 &#181;L) for a 24 well plate set up (Table 3).</li>
	<li>Pipette up and down gently to ensure that the cells are re-suspended well in the basement membrane.</li>
	<li>Pipette the appropriate volume (Table 3) of the cell-basement membrane mixture (and optional 10 &#181;L of adDMEM complete media) into the center of the pre-warmed tissue culture plate.</li>
	<li>Invert the plate and immediately place the plate upside down in the CO2 cell culture incubator set at 5% CO2, 37 &#176;C for 15 min. This prevents cells from settling and adhering to the plate bottom while allowing the basement membrane to solidify.</li>
	<li>Pipette the appropriate volume (Table 3) of pre-warmed medium containing 10 &#956;M Y-27632 dihydrochloride into each well.</li>
	<li>Place the plate right side up inside the CO2 cell culture incubator (5% CO2, 37 &#176;C).</li>
	<li>Change the media every 3-4 days. After 5-7 days, use culture medium without 10 &#956;M Y-27632 dihydrochloride to maintain the cultures.</li>
</ol>
4. Forming a floating dome from an attached round dome on the plate
<ol>
	<li>After step 3.7, detach the dome using a cell scraper.</li>
</ol>
5. Forming floating beads
NOTE: This protocol is named as floating beads since the mixture of basement membrane, media, and organoids look like beads.
<ol>
	<li>Cut a 2 inch x 4 inch piece of paraffin film.</li>
	<li>Place the paraffin film on the top of the divots of an empty tip-holding rack from a 1000 &#181;L plastic pipette tip box.</li>
	<li>Gently press down on the paraffin film to trace the divots using a gloved index finger but without breaking through the paraffin film.</li>
	<li>Spray the paraffin film with 70% ethanol and turn on the UV lamp in the cell culture hood to sterilize the prepared paraffin film for at least 30 min.</li>
	<li>Prepare a mixture of cells and 20 &#181;L of basement membrane. Seeding density can be 50,000 - 250,000 cells per dome.</li>
	<li>Pipette the mixture of cells processed from step 1 or 2, and 20 &#181;L of basement membrane into the mold of the divot formed in the prepared paraffin film.</li>
	<li>Resuspend the cell pellet in basement membrane and pipette the cell suspension in the prepared paraffin filmed divots.</li>
	<li>Place the solidified beads and paraffin film into a 6-well plate. One well in a 6-well plate can fit up to 5 beads.</li>
	<li>Pipette 3-5 mL of pre-warmed medium containing 10 &#956;M Y-27632 dihydrochloride into each well while gently brushing beads off of the paraffin film. 
	NOTE: As a minimum volume, 3 mL is recommended. For a maximum number of beads (N=5) per well, 5 mL of medium is recommended.</li>
	<li>Place the plate inside a CO2 incubator (5% CO2, 37 &#176;C).</li>
	<li>Change the organoid media every 3-4 days. After 5-7 days, use culture medium without 10 &#956;M Y-27632 dihydrochloride to maintain the cultures.</li>
</ol>
6. In vivo organoids image stitching using microscope8
NOTE: Certain microscopes are unable to reach the outer perimeter of the cell plate (edge wall); therefore, we suggest using the wells close to the perimeter of the cell plate when image stitching.
<ol>
	<li>Place the cell culture plate in an upward position into the plate holder in the Keyence microscope.</li>
	<li>Place the lens on the center of the target dome.</li>
	<li>Set up the automatic stitching process by selecting number of the frames. For examples, 3 x 3 or 5 x 5 can be chosen to generate 9 images or 25 images total.</li>
	<li>Press the capture button to initiate imaging process.</li>
	<li>Open the image viewer software and load a group of images taken by step 4.</li>
	<li>Click Image Stitching to create a high-resolution stitched image. 
	NOTE: Capturing of serial 9 or 25 images can be performed either by manual or automatic set up to focus the cells.</li>
</ol>
7. Organoid processing for histology: the agarose spin down method
NOTE: This protocol is adapted from a previous publication by Vlachogiannis et al.7. We have added a step involving agarose embedding to successfully embed all populations of organoids.
<ol>
	<li>Remove existing media from the well. Be careful not to aspirate the basement membrane domes.</li>
	<li>Add an equal (equal to the volume of media removed from step 1) volume of cell recovery solution and incubate for 60 min at 4 &#176;C.</li>
	<li>Dislodge the basement membrane dome using a pipette and crush the basement membrane dome using a pipet tip. Collect the dissociated dome and cell recovery solution in a 1.5 mL tube.</li>
	<li>Centrifuge at 300 x g and 4 &#176;C for 5 min.</li>
	<li>Remove the supernatant (cell recovery solution). Save all supernatants in separate tubes until the end when the presence of organoids is confirmed in the final pelleting step.</li>
	<li>Add desired volume (Table 3) of cold PBS and gently pipette up and down to mechanically disturb pellet.</li>
	<li>Centrifuge at 300 x g and 4 &#176;C for 5 min.</li>
	<li>Remove the supernatant (PBS).</li>
	<li>Fix the pellet in a matched volume (e.g., 500 &#181;L for one pellet from the 24 well plate culture condition, Table 3) of 4% PFA for 60 min at room temperature.</li>
	<li>Following fixation, centrifuge at 300 x g and 4 &#176;C for 5 min.</li>
	<li>Remove the supernatant (PFA).</li>
	<li>Wash with matched volume (e.g., 500 &#181;L for one pellet from the 24 well plate culture condition, Table 3) of PBS and centrifuge at 300 x g and 4 &#176;C for 5 min.</li>
	<li>Prepare warm agarose (2% agarose in PBS). 
	NOTE: Here, cell pellets for frozen sections can be directly re-suspended in 200 &#181;L of OCT compound without further steps in Protocol 7.</li>
	<li>Re-suspend the cell pellet in 200 &#181;L of agarose (2% in PBS).
	<ol>
		<li>Immediately after adding agarose, gently detach the cell pellet from the wall of the 1.5 mL tube using the 25 G needle attached to 1 mL syringe. As shown in Figure 3, if the cell pellet is not physically detached from the wall of the 1.5 mL tube, then there is a risk of losing all or part of the cell pellet during the agarose embedding process.</li>
	</ol>
	</li>
	<li>Wait until the 2% agarose in PBS is completely solidified.</li>
	<li>Detach the solidified agarose block from the 1.5 mL tube using a 25 G needle attached to the 1 mL syringe.</li>
	<li>Transfer the detached agarose block containing the cell pellet to a new 1.5 mL tube.</li>
	<li>Fill the tube with 70% EtOH and proceed further using the conventional protocol for tissue dehydration and paraffin embedding.</li>
</ol>
8. Histology and Immunofluorescent cytochemistry (IFC) of organoids
<ol>
	<li>Select the slide(s) for histology or IFC.</li>
	<li>Before initiating the staining process, find out where the cells are located on the slide and draw a circle around the cells on the slide using a marker.</li>
	<li>Draw the perimeter around the edge or boarder of the slide and where circles are located on the slide in a laboratory notebook to record their locations.</li>
	<li>Perform desired staining. 
	NOTE: During this process, marked circles disappear since regular maker is not resistant to the chemicals. Even some histology permanent markers may be erased during staining process.</li>
	<li>After the staining process, place the slide over the drawing in the laboratory notebook to find the locations of cells on the slide.</li>
</ol>

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Sanghee Lee and Christina A.M. Jamieson are the guest editors of JoVE Methods Collection.

3D organoids derived from patient bone metastasis prostate cancer cells are still relatively rare. Here, we describe strategies and further optimized protocol to successfully established serial 3D patient derived organoids (PDOs) of BMPC. In addition, protocols are described to secure the organoids in samples with lower cell density for IFC and IHC analysis. Differential phenotypes in the form of cyst, spheroids and more complex organoids indicate that this protocol provides culture conditions that allows for heterogonou...

Three-dimensional cultured organoids have drawn attention for their potential to recapitulate the in vivo architecture, cellular functionality and genetic signature of their original tissues1,2,3,4,5. Most importantly, 3D organoids established from patient tumor tissues or patient derived xenograft (PDX) models provide invaluable opportunities to understand mechanisms of cellular signaling upon tumorigenesis and to determine the effects of drug treatment on each cell population6,7,8,9,10,11,12,13. Drost et al.5 developed a standard protocol for establishment of human and mouse prostate organoids, which has been widely adopted in the field of urology. In addition, significant effort has been dedicated for further characterization of 3D organoids and to understand the detailed mechanisms of tumorigenesis and metastasis4,12,14,15. In addition to the previously established and widely accepted protocol for 3D organoids cultures, we describe here a step-by-step protocol for the 3D culture of PDO using three different doming methods in optimized culture conditions.
In this manuscript, 3D organoids were established as an ex vivo model of bone metastatic prostate cancer (BMPC). The cells used for these cultures came from the Prostate Cancer San Diego (PCSD) series and were derived directly from patient prostate cancer bone metastatic tumor tissues (PCSD18 and PCSD22) or patient derived xenograft (PDX) tumor models (samples named PCSD1, PCSD13, and PCSD17). Because spontaneous bone metastasis of prostate cancer cells is rare in genetically engineered mouse models16, we used direct intra-femoral (IF) injection of human tumor cells into male Rag2-/-&#947;c-/- mice to establish the PDX models of bone metastatic prostate cancer17.
Once 3D organoids are established from heterogeneous patient tumor cells or patient derived xenografts, it is essential to confirm their identity as prostate tumor cells and to determine their phenotypes in the 3D organoid cultures. Immunofluorescence chemistry (IFC) allows the visualization of protein expression in situ in each cell, often indicating the potential functions for specific cell populations2,4. In general, IFC protocols for a vast majority of samples including tissues and cells are straightforward and fully optimized. However, the cell density and number of organoids can be significantly lower than that of conventional culture. Therefore, the IFC protocol for organoids requires additional steps to ensure proper processing and embedding in paraffin for all organoids in the samples. We describe additional steps for an agarose pre-embedding process and tips to label the location of sectioned organoids on the slide that increases the success rate of IFC on organoids especially when the samples of organoids have lower cell density than desired.

<table><tbody><tr><td>1 mL Pipettman</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>1 mL Syringe</td><td>BD Syringe</td><td>329654</td><td></td></tr><tr><td>1.5 mL tube</td><td>Spectrum Lab Products</td><td>941-11326-ATP083</td><td></td></tr><tr><td>25G Needle</td><td>BD PrecisionGlide Needle</td><td>305122</td><td></td></tr><tr><td>4% Paraformaldehyde (PFA)</td><td>Alfa Aesar</td><td>J61899</td><td></td></tr><tr><td>70% Ethanol (EtOH)</td><td>VWR</td><td>BDH1164-4LP</td><td></td></tr><tr><td>A83-01</td><td>Tocris Bioscience</td><td>2939</td><td></td></tr><tr><td>Accumax</td><td>Innovative Cell Technologies, Inc.</td><td>AM105</td><td></td></tr><tr><td>adDMEM</td><td>Life Technologies</td><td>12634010</td><td></td></tr><tr><td>Agarose</td><td>Lonza</td><td>50000</td><td></td></tr><tr><td>Antibody -for Cytokeratin 5</td><td>Biolegend</td><td>905901</td><td></td></tr><tr><td>Antibody for Cytokeratin 8</td><td>Biolegend</td><td>904801</td><td></td></tr><tr><td>B27</td><td>Life Technologies</td><td>17504044</td><td></td></tr><tr><td>Bioluminescence imaging system, IVIS 200</td><td>Perkin Elmer Inc</td><td>IVIS 200</td><td></td></tr><tr><td>Cell Culture Plate - 24 well</td><td>Costar</td><td>3524</td><td></td></tr><tr><td>Cell Culture Plate - 48 well</td><td>Costar</td><td>3548</td><td></td></tr><tr><td>Cell Culture Plate - 6 well</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>Cell Dissociation Solution, Accumax</td><td>Innovative Cell Technologies, Inc.</td><td>AM105</td><td></td></tr><tr><td>Cell Recovery Solution</td><td>Corning</td><td>354253</td><td></td></tr><tr><td>Cell Scraper</td><td>Sarstedt</td><td>83.180</td><td></td></tr><tr><td>Cell Strainer</td><td>Falcon (Corning)</td><td>352350</td><td></td></tr><tr><td>CO2 incubator</td><td>Fisher Scientific</td><td>3546</td><td></td></tr><tr><td>DAPI</td><td>Vector Vectashield</td><td>H-1200</td><td></td></tr><tr><td>DHT</td><td>Sigma-Aldrich</td><td>D-073-1ML</td><td></td></tr><tr><td>dPBS</td><td>Corning/Cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>EGF</td><td>PeproTech</td><td>AF-100-15</td><td></td></tr><tr><td>FBS</td><td>Gemini Bio-Products</td><td>100-106</td><td></td></tr><tr><td>FGF10</td><td>PeproTech</td><td>100-26</td><td></td></tr><tr><td>FGF2</td><td>PeproTech</td><td>100-18B</td><td></td></tr><tr><td>Forceps</td><td>Denville Scientific</td><td>S728696</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi</td><td>130-0420401</td><td></td></tr><tr><td>Magnetic Column Seperator: QuadroMACS Separator</td><td>Miltenyi</td><td>130-090-976</td><td></td></tr><tr><td>Marker</td><td>VWR</td><td>52877-355</td><td></td></tr><tr><td>Matrigel (Growth Factor Reduced)</td><td>Mediatech Inc. (Corning)</td><td>356231</td><td></td></tr><tr><td>Matrigel (High Concentration)</td><td>BD (Fisher Scientific)</td><td>CB354248</td><td></td></tr><tr><td>Microscope Imaging Software, Keyence</td><td>BZ-X800 (newest software) BZ-X700 (old software)</td><td></td><td></td></tr><tr><td>Microscope, Keyence</td><td>BZ-X700 (model 2016-2017)/BZ-X710 (model 2018-2019)</td><td></td><td></td></tr><tr><td>Mouse Cell Depletion Kit</td><td>Miltenyi</td><td>130-104-694</td><td></td></tr><tr><td>N-Acetylcysteine</td><td>Sigma-Aldrich</td><td>A9165-5G</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N0636-100G</td><td></td></tr><tr><td>Noggin</td><td>PeproTech</td><td>120-10C</td><td></td></tr><tr><td>OCT Compound</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Parafilm</td><td>American National Can</td><td>N/A</td><td></td></tr><tr><td>Pen-Strep</td><td>Mediatech Inc. (Corning)</td><td>30-002-CI-1</td><td></td></tr><tr><td>Pipette tipes for 1 mL (Blue Tips)</td><td>Fisherbrand Redi-Tip</td><td>21-197-85</td><td></td></tr><tr><td>Plunger (from 3 mL syringe)</td><td>BD Syringe</td><td>309657</td><td></td></tr><tr><td>Prostaglandin E2</td><td>Tocris Bioscience</td><td>2296</td><td></td></tr><tr><td>R-Spondin 1</td><td>Trevigen</td><td>3710-001-01</td><td></td></tr><tr><td>SB2021190</td><td>Sigma-Aldrich</td><td>S7076-25MG</td><td></td></tr><tr><td>Small Table Top Centrifuge</td><td>ThermoFisher Scientific</td><td>75002426</td><td></td></tr><tr><td>Water Bath</td><td>Fisher Sci</td><td>2320</td><td></td></tr><tr><td>Y-27632 Dihydrochloride</td><td>Abmole Bioscience</td><td>M1817</td><td></td></tr></tbody></table>

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.

3D organoids were successfully established from a patient derived xenograft (PDX) model of bone metastatic prostate cancer (BMPC) as well as directly from patient bone metastatic prostate cancer tissue (Figure 4). Briefly, our PDX models of BMPC were established by intra-femoral (IF) injection of tumor cells into male Rag2-/- c-/- mice and then PDX tumors were harvested and processed as described in this manuscript. As shown in Figure 4, PD...

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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.

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 ...

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Research

JoVE Journal

Cancer Research

9.3K Views.  University of California, San Diego. 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.

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

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

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.

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 ...

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials (&#8220;Phase II-like mouse clinical trials&#8221;); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 &#181;m cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 &#181;M Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers&#8217; method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Medicine

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 ...

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. ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Handling and Assessment of Human Primary Prostate Organoid Culture

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

Bioengineering

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

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

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