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. 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=Metabolic+Heterogeneity+in+Patient+Tumor-Derived+Organoids+by+Primary+Site+and+Drug+Treatment.">Metabolic Heterogeneity in Patient Tumor-Derived Organoids by Primary Site and Drug Treatment.</a> Frontiers in Oncology. 10, 1-17 (2020).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical tubes</td>
			<td>VWR</td>
			<td>525-1068</td>
			<td></td>
		</tr>
		<tr>
			<td>175 cm2 tissue culture flask</td>
			<td>VWR (Corning)</td>
			<td>29185-308</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C bead bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C CO2 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 &#181;m Filter)</td>
			<td>Millipore Sigma</td>
			<td>SCGP00525</td>
			<td>SCGP00525</td>
		</tr>
		<tr>
			<td>500 mL Rapid-Flow Filter Unit, 0.2 &#181;m aPES membrane, 75 mm diameter</td>
			<td>Nalgene</td>
			<td>566-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates&#160;</td>
			<td>Greiner Cellstar</td>
			<td>82050-842</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 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&#160;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&#160;</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&#34;</td>
		</tr>
		<tr>
			<td>Cryovials&#160;</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&#8217;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&#160;</td>
			<td>For TOPFlash Assay</td>
		</tr>
		<tr>
			<td>HEK293T-HA-Rspondin1-Fc cells</td>
			<td>R&#38;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&#946;-1 (human)</td>
			<td>Peprotech</td>
			<td>100-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</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&#160;</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 &#181;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-&#946; 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 ...

establishment-and-culture-of-patient-derived-breast-organoids

Research

JoVE Journal

Cancer Research

4.1K 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

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

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

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

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

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

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 ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

Optimizing HCC Organoid Formation for Target Identification and Drug Development

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

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

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

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

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

Derivation of 3D Glioma Organoids from Chopper-Processed Patient Tumors

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing realistic ex vivo models remain challenging. Patient-derived 3-dimensional organoid (PDO) models offer innovative platforms that capture the phenotypic and molecular heterogeneity of GBM, while preserving key characteristics of the original tumors. However, manual dissection for PDO generation is time-consuming, expensive and can result in a number of irregular and unevenly sized PDOs. This study presents an innovative method for PDO production using an automated tissue chopper. Tumor samples from four GBM and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients were processed manually as well as using the tissue chopper. In the manual approach, the tumor material was dissected using scalpels under microscopic control, while the tissue chopper was employed at three different angles. Following culture on an orbital shaker at 37 &#176;C, morphological changes were evaluated using bright field microscopy, while proliferation (Ki67) and apoptosis (CC3) were assessed by immunofluorescence after 6 weeks. The tissue chopper method reduced almost 70% of the manufacturing time and resulted in a significantly higher PDOs mean count compared to the manually processed tissue from the second week onwards (week 2: 801 vs. 601, P = 0.018; week 3: 1105 vs. 771, P = 0.032; and week 4:1195 vs. 784, P &lt; 0.01). Quality assessment revealed similar rates of tumor-cell apoptosis and proliferation for both manufacturing methods. Therefore, the automated tissue chopper method offers a more efficient approach in terms of time and PDO yield. This method holds promise for drug- or immunotherapy-screening of GBM patients.

Author Spotlight: Enhanced Generation of Patient-Derived 3D Organoids for Glioblastoma and Glioma

This study introduces an automated method to generate patient-derived glioblastoma 3-dimensional organoids utilizing a tissue chopper. The method provides a suitable and effective approach to obtain such organoids for therapeutical testing.

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing ...