We would like to thank members of the Spector lab for critical discussions throughout the course of this work. We thank Norman Sachs and Hans Clevers (Hubrecht Institute, Netherlands) for initially providing us with their organoid culturing protocol. We acknowledge the CSHL Cancer Center Histology and Microscopy Shared Resources for services and technical expertise (NCI 2P3OCA45508). We thank Dr. Qing Gao for assistance with histological sample preparation. We are grateful for the support of Dr. Karen Kostroff (Northwell Health) for providing patient tumor samples. We also appreciate the efforts of the Northwell Health Biobanking team for sample acquisition, and we thank the patients and their families for donating tissues for research. This research was supported by CSHL/Northwell Health (D.L.S.), NCI 5P01CA013106-Project 3 (D.L.S.), and Leidos Biomedical HHSN26100008 (David Tuveson and D.L.S).

Tumor resections from breast cancer patients, along with the distal and adjacent normal tissue, were obtained from Northwell Health in accordance with Institutional Review Board protocols IRB-03-012 and IRB 20-0150, and with written informed consent from the patients.
NOTE: All procedures mentioned below were performed in a mammalian tissue culture BSL2 room designated for patient samples upon approval of the biosafety committee. All procedures should be performed following safety protocols maintaining aseptic conditions in biosafety cabinets. Each centrifugation step is performed at room temperature (RT) unless stated otherwise. Tissue/organoids and basement membrane matrix stocks are always placed on ice unless stated otherwise. New plates are incubated overnight for pre-warming. Plating domes on pre-warmed plates ensures the best results to obtain rounded domes that don&#39;t flatten out while plating or lifting off later from the plate surface.
1. Medium preparation and recipes
<ol>
	<li>Prepare R-spondin1 conditioned media as described below (alternatively can be bought commercially).
	<ol>
		<li>Prepare two separate growing media composed of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), 10% FBS, 5% penicillin-streptomycin, and with or without 300 &#181;g/mL zeocin supplementation.</li>
		<li>Thaw a vial of HEK293T-HA-Rspondin1-Fc cells (obtained from Calvin Kuo&#39;s lab at Stanford University and also commercially available) in a 75 cm2 tissue culture flask with 15 mL of medium.</li>
		<li>Split the cells into 2 x 175 cm2 culture flasks, each with 35 mL of medium containing zeocin.</li>
		<li>Passage the cells again to obtain as many flasks as needed for generating the desired batch of R-spondin1 conditioned medium. Use growth medium without zeocin.</li>
		<li>When the flasks containing medium without zeocin are confluent, remove and replace the growing medium with Ad-DF+++ (step 1.2; Table 1). Place the cells in a 37 &#176;C incubator with 5% CO2 for 1 week.</li>
		<li>Spin down the medium at 400 x g for 5 min to remove any unattached cells. Filter the medium through a 0.22 &#181;m filter. Make 25 mL aliquots and store them at -20 &#176;C (can be stored for up to 6 months).</li>
		<li>Using HEK293T cells, perform a dual luciferase TOPFLASH41,42 assay using a commercial DNA transfection reagent at a 4.8:1 reagent to DNA ratio with the diluent DMEM (high glucose, pyruvate). Add 100 &#181;L of R-spondin1 conditioned medium (or basal medium for the negative control) to 100 &#181;L of transfected cells. Then, perform the dual luciferase assay as per the manufacturer&#39;s protocol to verify Wnt activity of the R-spondin1 conditioned medium. 
		NOTE: The assay uses a TOP plasmid with Firefly luciferase activity read-out, and the FOP plasmid is used as a negative control.</li>
	</ol>
	</li>
	<li>Prepare basal medium (Ad-DF+++ medium, as previously published by Sachs et al.36).
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td>Reagent</td>
				<td>Stock Concentration</td>
				<td>Final Concentration</td>
			</tr>
			<tr>
				<td>Advanced DMEM/F12</td>
				<td>1x</td>
				<td>1x</td>
			</tr>
			<tr>
				<td>GlutaMax</td>
				<td>100x</td>
				<td>1x</td>
			</tr>
			<tr>
				<td>HEPES</td>
				<td>1 M</td>
				<td>10 mM</td>
			</tr>
			<tr>
				<td>Penicillin-Streptomycin</td>
				<td>10,000 U/mL; 10,000 &#181;g/mL</td>
				<td>100 U/mL; 100 &#181;g/mL</td>
			</tr>
		</tbody>
	</table>
	 
	Table 1: Basal Ad-DF+++ medium composition.</li>
	<li>Prepare complete medium for patient-derived breast organoids as previously published by Sachs et al.36.
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td>Reagent</td>
				<td>Stock Concentration</td>
				<td>Final Concentration</td>
			</tr>
			<tr>
				<td>Ad-DF+++ medium</td>
				<td>1x</td>
				<td>1x</td>
			</tr>
			<tr>
				<td>R-spondin in-house</td>
				<td>100%</td>
				<td>10%</td>
			</tr>
			<tr>
				<td>B-27 supplement</td>
				<td>50x</td>
				<td>1x</td>
			</tr>
			<tr>
				<td>Nicotinamide</td>
				<td>1 M</td>
				<td>5 mM</td>
			</tr>
			<tr>
				<td>NAC</td>
				<td>500 mM</td>
				<td>1.25 mM&#160;</td>
			</tr>
			<tr>
				<td>Primocin</td>
				<td>50 mg/mL</td>
				<td>50 &#181;g/mL</td>
			</tr>
			<tr>
				<td>Noggin</td>
				<td>100 &#181;g/mL</td>
				<td>100 ng/mL</td>
			</tr>
			<tr>
				<td>Human EGF</td>
				<td>5 &#181;g/mL</td>
				<td>5 ng/mL</td>
			</tr>
			<tr>
				<td>Human Heregulin &#946;1/Neuregulin1</td>
				<td>75 &#181;g/mL</td>
				<td>37.5 ng/mL</td>
			</tr>
			<tr>
				<td>Y-27632 Dihydrochloride (Rho-Kinase)</td>
				<td>100 mM</td>
				<td>5 &#181;M</td>
			</tr>
			<tr>
				<td>A83-01</td>
				<td>5 mM</td>
				<td>500 nM</td>
			</tr>
			<tr>
				<td>Human FGF-7</td>
				<td>100 &#181;g/mL</td>
				<td>5 ng/mL</td>
			</tr>
			<tr>
				<td>Human FGF-10</td>
				<td>1 mg/mL</td>
				<td>20 ng/mL</td>
			</tr>
			<tr>
				<td>p38i</td>
				<td>30 mM</td>
				<td>498 nM</td>
			</tr>
		</tbody>
	</table>
	 
	Table 2: Complete medium composition for patient-derived breast organoids.</li>
	<li>Prepare 2 mg/mL collagenase IV solution by weighing the required amount of collagenase IV powder and solubilizing it in Ad-DF+++ basal medium. Filter through a 0.22 &#181;m filter before use.</li>
	<li>Prepare 0.5% bovine serum albumin (BSA) solution from 7.5% stock solution using 1x Dulbecco&#39;s phosphate buffered saline (DPBS) as a diluent. Filter through a 0.22 &#181;m filter before use.</li>
</ol>
2. Establishing breast tumor/normal organoids from resected tissue (Figure 1)
<ol>
	<li>Transport a surgically resected breast tumor/normal tissue specimen to the lab in a 50 mL conical tube containing Ad-DF+++. Store the tube on ice until the start of isolation.</li>
	<li>Thaw a bottle of basement membrane matrix on ice or overnight at 4 &#176;C.</li>
	<li>Transfer the resected tissue to a 10 cm sterile Petri dish. Examine the tissue macroscopically and make a note if it appears morphologically fatty, vascularized, or necrotic. Additionally, record the size and shape of the tissue. Ideally, take a picture of the tissue with a ruler in view (Figure 2).</li>
	<li>Mince the tissue into very small pieces (~1 mm3) with a sterile no. 10 scalpel, and transfer it into a 50 mL conical tube.</li>
	<li>Add 10 mL of 2 mg/mL collagenase IV solution and seal the tube with a transparent film. Place the tube on an orbital shaker at 37 &#176;C at 140 rpm for 30-90 min in an angled position (~30&#176; angle).</li>
	<li>During the incubation, place an aliquot of complete medium to pre-warm in a 37 &#176;C bead/water bath.</li>
	<li>Every 15 min, resuspend the tissue by mixing up and down vigorously using a 5 mL sterile serological pipette. Pre-coat the pipette with 0.5% BSA solution to prevent tissue loss caused by the sample sticking to the pipette. Monitor dissociation over time by observing the conical tube under the microscope at a 5x or higher magnification.</li>
	<li>Once the tissue is dissociated, centrifuge at 400 x g for 5 min. Aspirate the supernatant, add 10 mL of Ad-DF+++, and centrifuge at 400 x g for 5 min.</li>
	<li>Discard the supernatant carefully. Tissue pellets can occasionally be loose, so be careful when aspirating. Repeat the step once more.</li>
	<li>If the tissue pellet is partially red, add 2 mL of red blood cell lysis buffer and incubate for 5 min at room temperature. Do not incubate for longer, as this will significantly reduce cell viability.</li>
	<li>Add 10 mL of Ad-DF+++ to the tube, centrifuge at 400 x g for 5 min, and discard the supernatant. Resuspend the pellet in 50-300 &#181;L of undiluted, cold basement membrane matrix and mix by pipetting carefully to avoid forming bubbles. 
	NOTE: The volume of the basement membrane matrix added depends on the pellet size. If unsure, plate a small dome of ~10 &#181;L from the resuspended pellet and observe the confluency under the microscope. Adjust by adding more basement membrane matrix if needed. Refer to Figure 3 (day 1 image) for a reference seeding density.</li>
	<li>Plate 300 &#181;L of basement membrane matrix dome containing organoids in each well of a pre-warmed, labeled, 6-well tissue culture plate. Refer to Table 3 for recommended basement membrane matrix and medium volumes if using different plates.
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td>Number of wells per plate</td>
				<td>Basement Membrane Matrix per dome (&#181;L)</td>
				<td>Medium per well (&#181;L)</td>
			</tr>
			<tr>
				<td>6</td>
				<td>300</td>
				<td>3000</td>
			</tr>
			<tr>
				<td>12</td>
				<td>100</td>
				<td>1000</td>
			</tr>
			<tr>
				<td>24</td>
				<td>50</td>
				<td>500</td>
			</tr>
			<tr>
				<td>48</td>
				<td>25</td>
				<td>250</td>
			</tr>
			<tr>
				<td>96</td>
				<td>10 (suspension instead of dome)</td>
				<td>100</td>
			</tr>
		</tbody>
	</table>
	 
	Table 3: Amount of basement membrane matrix and growth medium recommended per well based on plate size.</li>
	<li>Leave the plate undisturbed in the hood for 5 min and then carefully place in the incubator at 37 &#176;C for 20-30 min for the basement membrane matrix dome to fully solidify.</li>
	<li>Add 3 mL of pre-warmed complete medium dropwise to each well of a 6-well plate and place in the incubator at 37 &#176;C and 5% CO2. Take images of the organoids using a 5x objective on an inverted brightfield microscope.</li>
	<li>Replenish the medium every 4-8 days based on the growth of organoids. Carefully aspirate the old medium without disturbing the dome, and add fresh, pre-warmed medium dropwise. 
	NOTE: The protocol is the same for establishing breast cancer organoids derived from the resected tumor and for normal organoids derived from resected healthy breast tissue (either from adjacent and normal breast tissue of the same patient or healthy breast tissue obtained from a reductive mammoplasty surgery).</li>
</ol>
3. Passaging and expanding patient-derived breast organoids in culture
<ol>
	<li>Thaw a bottle of basement membrane matrix on ice or overnight at 4 &#176;C.</li>
	<li>Lift the basement membrane matrix dome into the medium in the well using a cell scraper or a 1 mL pipette tip.</li>
	<li>Pre-coat the pipette tip with 0.5% BSA solution and then transfer the floating dome of the organoids with medium to a 15 mL or 50 mL conical tube, depending on the number of wells being harvested. For more than two wells containing 300 &#181;L of basement membrane matrix domes, use a 50 mL conical tube. Add 1x DPBS to increase the volume to at least 5 mL.</li>
	<li>Spin the tubes at 400 x g for 5 min. The basement membrane matrix with organoids forms a layer at the bottom. Carefully aspirate to remove the supernatant.</li>
	<li>Add 5-20 mL of 1x DPBS, depending on the number of wells pooled in a tube. Pre-coat a sterile disposable pipette with 0.5% BSA and mix the organoid-basement membrane matrix pellet in DPBS uniformly by pipetting up and down.</li>
	<li>Spin the tubes at 400 x g for 5 min. Carefully aspirate and discard the supernatant.</li>
	<li>Add cell dissociation reagent at three times the volume of the basement membrane matrix to the tube. Using a 0.5% BSA-coated pipette tip, resuspend the organoids in the cell dissociation reagent.</li>
	<li>Place the tube on an orbital shaker at 37 &#176;C at 140 rpm for 8-15 min in an angled position. Monitor the tube by observing it under the microscope every 5 min to ensure the organoids are broken down into smaller clusters.</li>
	<li>Add basal medium (i.e., Ad-DF+++) at a volume equal to or greater than the cell dissociation reagent, and pipette to mix the organoids. Spin at 400 x g for 5 min at RT to obtain an organoid pellet.</li>
	<li>If the pellet contains organoids still embedded within the undissolved basement membrane matrix, repeat steps 3.7-3.9 for an additional 5-8 min of trypsinization.</li>
	<li>Once a white organoid pellet with no undissolved basement membrane matrix is obtained, discard the supernatant, and add 1 mL of Ad-DF+++ to resuspend the pellet by pipetting. Then add more Ad-DF+++ up to 10 mL.</li>
	<li>Spin at 400 x g for 5 min at RT for the wash step. Aspirate and discard the supernatant.</li>
	<li>Add the required amount of basement membrane matrix to the digested organoids based on the appropriate split ratio (this will vary widely for each organoid line based on growth rate). Refer to Figure 3 (day 1 image) for an example of seeding density. Mix by gently pipetting up and down to avoid creating bubbles. Immediately place on ice.</li>
	<li>Open and label a pre-warmed 6-well plate. It is recommended to plate a 10-20 &#181;L dome in the corner of a well to observe the confluency under the microscope. If the organoids are confluent, more basement membrane matrix can be added to increase the split ratio.</li>
	<li>Plate 300 &#181;L domes of organoids resuspended in basement membrane matrix. Make sure to keep the tube on ice while plating multiple wells so that the basement membrane matrix remains in solution.</li>
	<li>Leave the plate undisturbed in the hood for 5 min and then carefully place in a 37 &#176;C incubator with 5% CO2 without disturbing the domes.</li>
	<li>After 20-30 min, the domes solidify. Add 3 mL of pre-warmed complete medium to each well and place the plate back in the incubator. Add fresh complete medium every 5-7 days.&#160;Perform routine testing of the cultures for detecting mycoplasma contamination. 
	NOTE: The protocol for the culture of breast tumors and normal organoids is the same. However, in our experience, normal organoids tend to grow well up to passage 6-8 before slowing down. It is advisable to make as many early passage stocks as possible for future research.</li>
</ol>
4. Freezing patient-derived breast organoids
<ol>
	<li>Passage confluent wells of organoids to remove any basement membrane matrix, as stated in steps 3.1-3.12.</li>
	<li>Resuspend the pellet of organoids in cell freezing medium (thawed overnight at 4 &#186;C) with 1 mL of medium per 200-300 &#181;L of basement membrane matrix volume before passaging. Perform a cell count before freezing for reference. Aliquot a small volume (at least 100 &#181;L) of cells to undergo further trypsinization, if needed, to get single cells, and then count them using a cell counter machine.</li>
	<li>Transfer 1 mL of cells into 2 mL cryovials labeled with organoid line number, date, and passage number. Be sure to label the tubes with a permanent marker or use cryolabels.</li>
	<li>Move the vials to a cell freezing container and transfer the container to a -80 &#176;C freezer. After incubation overnight, the vials are ready to be moved to a liquid nitrogen storage freezer for long-term storage.</li>
</ol>
5. Thawing patient-derived breast organoids
<ol>
	<li>Thaw a bottle of basement membrane matrix on ice or overnight at 4 &#176;C. Pre-warm Ad-DF+++ medium at 37 &#176;C. Aliquot 9 mL of pre-warmed medium into 15 mL conical tubes for each frozen vial.</li>
	<li>Rapidly thaw the frozen vial of organoids by placing it in a 37 &#176;C bead bath. Spray 70% ethanol and transfer the tube to the biosafety cabinet.</li>
	<li>Rinse the pipette tip with 0.5% BSA solution to avoid organoid loss. Mix the thawed organoids gently by pipetting up and down to mix any settled organoids in the tube.</li>
	<li>Gently transfer 1 mL of frozen organoids to 9 mL of pre-warmed Ad-DF+++ in a dropwise manner. Spin the cells at 400 x g for 5 min and then carefully discard the supernatant.</li>
	<li>Wash the pellet by adding 10 mL of fresh pre-warmed Ad-DF+++ to the organoid pellet, and mix gently with a pipette pre-coated with 0.5% BSA to resuspend the pellet. Spin the cells at 400 x g for 5 min and carefully discard the supernatant.</li>
	<li>Place the organoid pellet on ice and remove any remnant Ad-DF+++ carefully using a smaller volume pipette tip. Resuspend the pellet in 300 &#181;L of basement membrane matrix and plate in a pre-warmed 6-well plate. Place the plate in a 37 &#176;C incubator with 5% CO2. 
	NOTE: It is recommended to plate a small 10 &#181;L dome from the resuspended pellet in a corner of the well to discern the confluency under the microscope. If the organoids look confluent, dilute by adding 300 &#181;L of basement membrane matrix to plate into two wells of a 6-well plate.</li>
	<li>After a 20-30 min incubation, add 3 mL of pre-warmed complete medium to the solidified dome.</li>
</ol>

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E., 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+Sensitivity+Correlates+with+Therapeutic+Response+in+Patients+with+Pancreatic+Cancer.">Organoid Sensitivity Correlates with Therapeutic Response in Patients with Pancreatic Cancer.</a> Clinical Cancer Research. 28 (4), 708-718 (2022).</li><li>Ganesh, 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=A+rectal+cancer+organoid+platform+to+study+individual+responses+to+chemoradiation.">A rectal cancer organoid platform to study individual responses to chemoradiation.</a> Nature Medicine. 25 (10), 1607-1614 (2019).</li><li>Yao, 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=Patient-Derived+Organoids+Predict+Chemoradiation+Responses+of+Locally+Advanced+Rectal+Cancer.">Patient-Derived Organoids Predict Chemoradiation Responses of Locally Advanced Rectal Cancer.</a> Cell Stem Cell. 26 (1), 17-26 (2020).</li><li>. HCMI Catalog Available from: <a target="_blank" href="https://hcmi-searchable-catalog.ni.nih.gov/">https://hcmi-searchable-catalog.ni.nih.gov/</a> (2022)</li><li>. Search ATCC Available from: <a target="_blank" href="https://www.atcc.org/search#q=hcm&amp;sort=relevancy&amp;numberOfResults=24">https://www.atcc.org/search#q=hcm&amp;sort=relevancy&amp;numberOfResults=24</a> (2022)</li><li>Rosenbluth, J. 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=Organoid+cultures+from+normal+and+cancer-prone+human+breast+tissues+preserve+complex+epithelial+lineages.">Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages.</a> Nature Communications. 11 (1), (2020).</li><li>Pal, 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=Endocrine+Therapy-Resistant+Breast+Cancer+Cells+Are+More+Sensitive+to+Ceramide+Kinase+Inhibition+and+Elevated+Ceramide+Levels+Than+Therapy-Sensitive+Breast+Cancer+Cells.">Endocrine Therapy-Resistant Breast Cancer Cells Are More Sensitive to Ceramide Kinase Inhibition and Elevated Ceramide Levels Than Therapy-Sensitive Breast Cancer Cells.</a> Cancers. 14 (10), 2380 (2022).</li><li>Ding, 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=Single+cell+heterogeneity+and+evolution+of+breast+cancer+bone+metastasis+and+organoids+reveals+therapeutic+targets+for+precision+medicine.">Single cell heterogeneity and evolution of breast cancer bone metastasis and organoids reveals therapeutic targets for precision medicine.</a> Annals of Oncology. , (2022).</li><li>Sudhan, D. 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=Hyperactivation+of+TORC1+Drives+Resistance+to+the+Pan-HER+Tyrosine+Kinase+Inhibitor+Neratinib+in+HER2-Mutant+Cancers.">Hyperactivation of TORC1 Drives Resistance to the Pan-HER Tyrosine Kinase Inhibitor Neratinib in HER2-Mutant Cancers.</a> Cancer Cell. 37 (2), 183-199 (2020).</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>Neal, J. 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=Organoid+Modeling+of+the+Tumor+Immune+Microenvironment.">Organoid Modeling of the Tumor Immune Microenvironment.</a> Cell. 175 (7), 1972-1988 (2018).</li><li>Tsai, 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=Development+of+primary+human+pancreatic+cancer+organoids,+matched+stromal+and+immune+cells+and+3D+tumor+microenvironment+models.">Development of primary human pancreatic cancer organoids, matched stromal and immune cells and 3D tumor microenvironment models.</a> BMC Cancer. 18 (1), 1-13 (2018).</li><li>&#214;hlund, 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=Distinct+populations+of+inflammatory+fibroblasts+and+myofibroblasts+in+pancreatic+cancer.">Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer.</a> Journal of Experimental Medicine. 214 (3), 579-596 (2017).</li><li>Liu, 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=Cancer-Associated+Fibroblasts+Provide+a+Stromal+Niche+for+Liver+Cancer+Organoids+That+Confers+Trophic+Effects+and+Therapy+Resistance.">Cancer-Associated Fibroblasts Provide a Stromal Niche for Liver Cancer Organoids That Confers Trophic Effects and Therapy Resistance.</a> Cellular and Molecular Gastroenterology and Hepatology. 11 (2), 407-431 (2021).</li><li>Puschhof, 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=Intestinal+organoid+cocultures+with+microbes.">Intestinal organoid cocultures with microbes.</a> Nature Protocols. 16 (10), 4633-4649 (2021).</li><li>Puschhof, J., Pleguezuelos-Manzano, C., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+and+organs-on-chips:+Insights+into+human+gut-microbe+interactions.">Organoids and organs-on-chips: Insights into human gut-microbe interactions.</a> Cell Host &amp; Microbe. 29 (6), 867-878 (2021).</li><li>Chan, I. S., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+Co-culture+Methods+to+Capture+Cancer+Cell&#8211;Natural+Killer+Cell+Interactions.">Organoid Co-culture Methods to Capture Cancer Cell&#8211;Natural Killer Cell Interactions.</a> Methods in Molecular Biology. 2463, 235-250 (2022).</li><li>Chatterjee, 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=Paracrine+Crosstalk+between+Fibroblasts+and+ER++Breast+Cancer+Cells+Creates+an+IL1&#946;-Enriched+Niche+that+Promotes+Tumor+Growth.">Paracrine Crosstalk between Fibroblasts and ER+ Breast Cancer Cells Creates an IL1&#946;-Enriched Niche that Promotes Tumor Growth.</a> iScience. 19, 388 (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. 172 (1-2), 373-386 (2018).</li><li>. HUB Organoids: Patient in the lab Available from: <a target="_blank" href="https://www.huborganoids.nl">https://www.huborganoids.nl</a> (2022)</li><li>Dekkers, J. 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=Long-term+culture,+genetic+manipulation+and+xenotransplantation+of+human+normal+and+breast+cancer+organoids.">Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids.</a> Nature Protocols. 16 (4), 1936-1965 (2021).</li><li>Guillen, K. 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=A+human+breast+cancer-derived+xenograft+and+organoid+platform+for+drug+discovery+and+precision+oncology.">A human breast cancer-derived xenograft and organoid platform for drug discovery and precision oncology.</a> Nature Cancer. 3 (2), 232 (2022).</li><li>. PDX Portal Available from: <a target="_blank" href="https://pdxportal.research.bcm.edu/pdxportal/;jsessionid=3rrpefh3qlisqgbbq4vywfywc1dvn2vwaedbnizs.pdxportal?dswid=8217">https://pdxportal.research.bcm.edu/pdxportal/;jsessionid=3rrpefh3qlisqgbbq4vywfywc1dvn2vwaedbnizs.pdxportal?dswid=8217</a> (2022)</li><li>Veeman, M. T., Slusarski, D. C., Kaykas, A., Louie, S. H., Moon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Prickle,+a+Modulator+of+Noncanonical+Wnt/Fz+Signaling,+Regulates+Gastrulation+Movements.">Zebrafish Prickle, a Modulator of Noncanonical Wnt/Fz Signaling, Regulates Gastrulation Movements.</a> Current Biology. 13 (8), 680-685 (2003).</li><li>Chen, X., Prywes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-Induced+Expression+of+the+cdc25A+Gene+by+Relief+of+E2F-Mediated+Repression+.">Serum-Induced Expression of the cdc25A Gene by Relief of E2F-Mediated Repression .</a> Molecular and Cellular Biology. 19 (7), 4695-4702 (1999).</li><li>Sflomos, G., 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=Atlas+of+lobular+breast+cancer+models:+Challenges+and+strategic+directions.">Atlas of lobular breast cancer models: Challenges and strategic directions.</a> Cancers. 13 (21), 5396 (2021).</li><li>Sharick, J. 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The authors declare no conflicts of interest.

Our lab has successfully employed the above protocols to establish organoids from na&#239;ve tumor resections or scrapings. We have also utilized this protocol to develop normal organoids from breast tissue obtained via reductive mammoplasties or from cancer patients&#39; adjacent or distal normal breast tissue. About 30%-40% of the resected primary tumors resulted in successful long-term (&#62;passage 8) tumor organoid cultures. The tumor organoid lines that tapered off after a few passages either had an outgro...

Breast cancer (BC) is the most commonly occurring malignancy in females, with 287,850 new cases estimated to be diagnosed in the United States in 20221. Despite the recent advances in early detection with annual screenings, targeted therapies, and a better understanding of genetic predisposition, it prevails to be the second leading cause of cancer deaths in females in the United States, with &#62;40,000 deaths attributed to breast cancer annually1. Breast cancer is currently classified into multiple subtypes based on histopathological and molecular evaluation of the primary tumor. Better subtype stratification has improved patient outcomes with subtype-specific treatment options2. For instance, the identification of HER2 as a proto-oncogene3 has led to the development of Trastuzumab, which has made this highly aggressive subtype manageable in most patients4. Further research into the genetics and transcriptomics of this complex disease in a patient-specific manner will aid in developing and predicting better patient-specific personalized treatment regimens2,5. Patient-derived organoids (PDOs) are a promising new model to gain insights into cancer at the molecular level, identifying novel targets or biomarkers and designing new treatment strategies6,7,8.
PDOs are multicellular, three-dimensional (3D) structures derived from freshly resected primary tissue samples8,9. They are grown three-dimensionally by being embedded in a hydrogel matrix, typically composed of a combination of extracellular matrix (ECM) proteins, and hence can be used to study tumor cell-ECM interactions. PDOs represent patient diversity and recapitulate cellular heterogeneity and genetic features of the tumor10,11,12. Being in vitro models, they allow for genetic manipulation and high-throughput drug screens13,14,15. Further, PDOs can be plausibly utilized to evaluate patient drug sensitivity and treatment strategies in parallel to the clinic and help predict patient outcomes16,17,18. Besides chemotherapy, certain organoid models have also been used to examine individual patient responses to chemoradiation19,20. Given the promising applicability of PDOs for research and clinical use, the National Cancer Institute has initiated an international consortium, The Human Cancer Models Initiative (HCMI)21, to generate and provide these tumor-derived novel cancer models. Many of the organoid models of various cancer types developed through the HCMI are available via the American Type Culture Collection (ATCC)22.
Normal breast organoids have been shown to be comprised of different epithelial cell populations present in the mammary gland11,23 and thus serve as great models to study basic biological processes, to analyze driver mutations causing tumorigenesis, and for cancer cell-of-origin lineage studies6,15. Breast tumor organoid models have been used to identify novel targets that are encouraging prospects for developing new therapies, particularly for resistant tumors24,25,26. Using patient-derived xenograft (PDX) and matched PDX-derived organoid (PDxO) models of treatment-resistant breast tumors, Guillen et al. showed that organoids are powerful models for precision medicine, which can be leveraged to evaluate drug responses and direct therapy decisions parallelly28. Furthermore, the development of new co-culture methods for culturing PDOs with various immune cells27,28,29, fibroblasts30,31 and microbes32,33 presents an opportunity to study the impact of the tumor microenvironment on cancer progression. While many such co-culture methods are actively being established for PDOs derived from pancreatic or colorectal tumors, similar established co-culture methods for breast PDOs have only been reported for natural killer cells34 and fibroblasts35.
The first biobank of &#62;100 patient-derived organoids representing different breast cancer subtypes was developed by the Hans Clevers group36,37. As part of this effort, the Clevers group also developed the first complex culture medium for breast organoid growth, which is currently widely used36. A follow-up study provided a comprehensive account of the establishment and culture of breast PDOs and patient-derived organoid xenografts (PDOXs)38. The Welm lab developed a large collection of BC PDX models and PDxOs that are cultured in a comparatively simpler growth medium containing fetal bovine serum (FBS) and fewer growth factors39,40. We have independently developed and characterized a large array of na&#239;ve patient-derived breast cancer organoid models11, and participated in developing BC PDO models as part of the HCMI initiative21. Here, we aim to provide a practical guide detailing the methodology employed by us in generating patient-derived breast organoid model systems.

<table><tbody><tr><td>15 mL conical tubes</td><td>VWR</td><td>525-1068</td><td></td></tr><tr><td>175 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask</td><td>VWR (Corning)</td><td>29185-308</td><td></td></tr><tr><td>37 &amp;deg;C bead bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>50 mL conical tubes</td><td>VWR</td><td>525-1077</td><td></td></tr><tr><td>50 mL vacuum filtration system (0.22 &amp;micro;m Filter)</td><td>Millipore Sigma</td><td>SCGP00525</td><td>SCGP00525</td></tr><tr><td>500 mL Rapid-Flow Filter Unit, 0.2 &amp;micro;m aPES membrane, 75 mm diameter</td><td>Nalgene</td><td>566-0020</td><td></td></tr><tr><td>6-well culture plates&amp;nbsp;</td><td>Greiner Cellstar</td><td>82050-842</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask</td><td>VWR (Corning)</td><td>29185-304</td><td></td></tr><tr><td>96-well opaque plates</td><td>Corning</td><td>353296</td><td>For CTG assay</td></tr><tr><td>A83-01</td><td>Tocris</td><td>2939</td><td></td></tr><tr><td>Advanced DMEM/F12</td><td>Gibco</td><td>12634-010</td><td></td></tr><tr><td>B-27 supplement</td><td>Life Technologies</td><td>12587010</td><td></td></tr><tr><td>BioTek&amp;nbsp;Synergy H4 Hybrid Microplate Reader</td><td>Fisher Scientific (Agilent)</td><td></td><td>For dual luciferase assay and CTG assay</td></tr><tr><td>BSA fraction V (7.5%)</td><td>Thermo Fisher</td><td>15260037</td><td></td></tr><tr><td>Cell Titer-Glo (CTG) Reagent</td><td>Promega</td><td>G9683</td><td>luminescent cell viability assay</td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>5804</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum</td><td>Millipore Sigma</td><td>C5138</td><td>Type IV</td></tr><tr><td>Cryolabels</td><td>Amazon</td><td>DTCR-1000</td><td>Direct Thermal Cryo-Tags, White, 1.05 x 0.5&quot;</td></tr><tr><td>Cryovials&amp;nbsp;</td><td>Simport Scientific Inc.</td><td>T311-1</td><td></td></tr><tr><td>Countess 3 Automated Cell Counter</td><td>Thermo Fisher</td><td>AMQAX2000</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Thermo Fisher (Gibco)</td><td>11995040</td><td></td></tr><tr><td>Dual Luciferase Reporter Assay System</td><td>Promega</td><td>E1910</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (1X)</td><td>Gibco</td><td>14190-144</td><td>DPBS</td></tr><tr><td>Epidermal growth factor (hEGF)</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>FGF-10 (human)</td><td>Peprotech</td><td>100-26</td><td></td></tr><tr><td>FGF-7/KGF (human)</td><td>Peprotech</td><td>100-19</td><td></td></tr><tr><td>GlutaMax</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>HEK293T cells</td><td>ATCC</td><td>CRL-3216&amp;nbsp;</td><td>For TOPFlash Assay</td></tr><tr><td>HEK293T-HA-Rspondin1-Fc cells</td><td>R&amp;amp;D Systems</td><td>3710-001-01</td><td>Cultrex HA-R-Spondin1-Fc 293T Cells</td></tr><tr><td>HEPES</td><td>Life Technologies</td><td>15630-080</td><td></td></tr><tr><td>Heregulin&amp;beta;-1 (human)</td><td>Peprotech</td><td>100-03</td><td></td></tr><tr><td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (~10 mg/mL protein concentration)</td><td>Corning</td><td>356231</td><td>Phenol-red free, LDEV-free; basement membrane matrix</td></tr><tr><td>Mr. Frosty Cell Freezing Container</td><td>Thermo Fisher</td><td>5100-0001</td><td></td></tr><tr><td>Mycoplasma detection kit</td><td>Lonza</td><td>LT07-418</td><td></td></tr><tr><td>N-acetyl-l-cysteine</td><td>Millipore Sigma</td><td>A9165</td><td></td></tr><tr><td>Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membranes</td><td>Thermo Fisher</td><td>166-0045&amp;nbsp;</td><td></td></tr><tr><td>Nicotinamide</td><td>Millipore Sigma</td><td>N0636</td><td></td></tr><tr><td>Noggin (human)</td><td>Peprotech</td><td>120-10C</td><td></td></tr><tr><td>P1000, P200, P10 pipettes with tips</td><td></td><td></td><td></td></tr><tr><td>p38 MAPK inhibitor (p38i) SB 202190</td><td>Millipore Sigma</td><td>S7067</td><td></td></tr><tr><td>Parafilm</td><td></td><td></td><td>transparent film</td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>Plasmid1: pRL-SV40P</td><td>Addgene</td><td>27163</td><td></td></tr><tr><td>Plasmid2: M51 Super 8x FOPFlash</td><td>Addgene</td><td>12457</td><td></td></tr><tr><td>Plasmid3: M50 Super 8x TOPFlash</td><td>Addgene</td><td>12456</td><td></td></tr><tr><td>pluriStrainer 200 &amp;micro;m</td><td>pluriSelect</td><td>43-50200-01</td><td></td></tr><tr><td>Primocin</td><td>Invivogen</td><td>ANT-PM-1</td><td></td></tr><tr><td>Recovery Cell Culture Freezing Medium</td><td>Thermo Fisher (Gibco)</td><td>12648-010</td><td>cell freezing medium</td></tr><tr><td>Red Blood Cell lysis buffer</td><td>Millipore Sigma</td><td>11814389001</td><td></td></tr><tr><td>R-spondin conditioned media</td><td>In-house or commercial from Peprotech</td><td>120-38</td><td></td></tr><tr><td>Scalpel (No.10)</td><td>Sklar Instruments</td><td>Jun-10</td><td></td></tr><tr><td>Shaker (Incu-shaker Mini)</td><td>Benchmark</td><td>H1001-M</td><td></td></tr><tr><td>TGF-&amp;beta; receptor inhibitor A 83-01</td><td>Tocris</td><td>2939</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>TrypLE Express Enzyme (1X), phenol red</td><td>Life Technologies</td><td>12605028</td><td>cell dissociation reagent</td></tr><tr><td>X-tremeGENE 9 DNA transfection reagent</td><td>Millipore Sigma</td><td>6365779001</td><td></td></tr><tr><td>Y-27632 Dihydrochloride (RhoKi)</td><td>Abmole Bioscience</td><td>Y-27632</td><td></td></tr><tr><td>Zeocin</td><td>Thermo Fisher</td><td>R25001</td><td></td></tr></tbody></table>

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.

We have established a biobank of patient-derived breast tumor organoids comprising various subtypes11. Additionally, we have established multiple normal breast organoid lines derived from reductive mammoplasty tissue samples or adjacent/distal normal breast from BC patients using the approach outlined in Figure 1.
The various patient-derived breast tumor organoid lines differ in their morphology (Figure 2) and ...

Watch this Scientific Journal Video about Establishment and Culture of Patient-Derived Breast Organoids at JoVE.com

Establishment and Culture of Patient-Derived Breast Organoids

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 ...

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4.4K Views.  Cold Spring Harbor. 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. 

Video: Establishment and Culture of Patient-Derived Breast Organoids

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

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 ...

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 ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

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 Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Developmental Biology

Standardizing Ovarian Cancer Organoid Biobanking

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

Author Spotlight: Advancing Personalized Medicine in Ovarian Cancer 

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...