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 ma...

<ol>
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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 ...

Human patient-derived organoids are three-dimensional in vitro model systems that represent both patient diversity and cellular heterogeneity of the tumors. This protocol and video demonstration provide a detailed, hands-on guide to establishing patient-derived breast tumor and normal organoids. Patient-derived breast tumor organoids are exciting new models, but difficult to establish.The comprehensive protocol we provide here should aid in preparing the researchers attempting to develop breast organoids and acquaint them with the expected challenges. Every PDO line derived from a different patient is unique in morphology and growth rate. Unlike two 2D cell line systems, organoids grow better when plated at a high density, allowing for better intercellular interactions.Demonstrating the procedure will be Disha Aggarwal, a graduate student in my laboratory. To begin, thaw a bottle of basement membrane matrix on ice or overnight at four degrees Celsius. Transfer the resected tissue to a 10-centimeter 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 and take a picture of the tissue with a ruler in view. Mince the tissue into small pieces with a sterile number 10 scalpel and transfer it into a 50-milliliter conical tube.Add 10 milliliters of two milligrams per milliliter collagenase IV solution and seal the tube. Place the tube on an orbital shaker at 37 degree Celsius at 140 RPM for 30 to 90 minutes at a 30-degree angle. During the incubation, place an aliquot of complete medium to pre-warm at 37 degrees Celsius in a bead or water bath.Every 15 minutes, resuspend the tissue by mixing up and down vigorously using a five-milliliter, sterile, pre-coated serological pipette. Monitored dissociation over time by observing the tube under the microscope at a 5X or higher magnification. Once the tissue is dissociated, centrifuge at 400 G for five minutes, aspirate the supernatant, and add 10 milliliters of AdDF+Again, centrifuge and carefully aspirate the supernatant, as tissue pellets can occasionally be loose.If the tissue pellet is partially red, add two milliliters of red blood cell lysis buffer and incubate for five minutes at room temperature. After incubation, add 10 milliliters of AdDF+to the tube, centrifuge at 400 G for five minutes, and discard the supernatant. Resuspend the pellet in 50 to 300 microliters of undiluted cold basement membrane matrix and mixed by pipetting carefully to avoid forming bubbles.Using a six-well tissue culture plate that is pre-warmed in the incubator overnight, plate 300 microliters of basement membrane matrix dome containing organoids in each well. Leave the plate undisturbed in the hood for five minutes before placing it at 37 degrees Celsius for 20 to 30 minutes for the basement membrane matrix dome to fully solidify. At the end of incubation, add three milliliters of pre-warmed complete medium dropwise to each well and incubate at 37 degrees Celsius and 5%carbon dioxide.After incubation, capture images of the organoids using a 5X objective on an inverted bright-field microscope. Lift the basement membrane matrix dome into the medium in the well using a cell scraper or a one-milliliter pipette tip. Using a pre-coated pipette tip, transfer the floating dome of the organoids with medium to a 15-or 50-milliliter conical tube, depending on the number of wells being harvested.Then add DPBS to increase the volume to at least five milliliters. Spin the tubes at 400 G for five minutes. The basement membrane matrix with organoids forms a layer at the bottom.After aspirating the supernatant, add 5 to 20 milliliters of DPBS, depending on the number of wells pooled in a tube. Mix the organoid basement membrane matrix pellet in DBPS using a pre-coated sterile disposable pipette. Again, centrifuge and discard the supernatant.Using a coated pipette tip, add cell dissociation reagent at three times the volume of the basement membrane matrix and resuspend the organoids. Place the tube on an orbital shaker at 37 degrees Celsius at 140 RPM for 8 to 15 minutes in an angled position. Monitor the tube by observing it under the microscope every five minutes to ensure the organoids are broken into smaller clusters.Add AdDF+at a volume equal to or greater than the cell dissociation reagent and pipette to mix the organoids. Spin at 400 G for five minutes to obtain an organoid pellet. Once a white organoid pellet with no undissolved basement membrane matrix is obtained, discard the supernatant and resuspend the pellet in one milliliter of AdDF+Make up the volume to 10 milliliters with AdDF+Spin the tube for the wash step and discard the supernatant.Add the required amount of basement membrane matrix to the digested organoids based on the appropriate split ratio. Mix by gently pipetting up and down to avoid creating bubbles and immediately place on ice. Plate 300-microliter domes of organoids resuspended in a basement membrane matrix in a pre-warmed six-well plate.Leave the plate undisturbed in the hood for five minutes before placing it at 37 degrees Celsius with 5%carbon dioxide for 20 to 30 minutes for the domes to solidify. At the end of incubation, and three milliliters of pre-warmed complete medium to each well and place the plate back in the incubator. Add fresh complete medium every five to seven days.The various patient-derived breast tumor organoid lines differ in morphology and growth rate. The normal breast organoids and the few early ductal carcinomas in C2-derived organoids resembled the normal breast structure, with a central lumen surrounded by ductal cells. Organoids derived from invasive lobular carcinoma tend to form loosely-attached grape bunch-like structures.Meanwhile, organoids derived from invasive ductal carcinomas tend to form dense, large, and round organoids. The growth of organoids was measured using a luminescent cell viability assay on days three, six, nine, and 12, with a baseline reading on day one after plating. The bright-field images of the same organoids expanded over time are shown here.Some patient-derived organoid lines have a doubling time of two days, while some take five days. It is important to be patient with particular organoid lines that are slow to establish. In our experience, increasing plating density or filtering debris out promotes the growth of PDOs.Patient-derived organoids, or PDOs, are excellent models for drug screening. They model cell-cell as well as cell-extracellular matrix interactions that are key to studying cancer pathophysiology. Additionally, PDOs can undergo genetic manipulation and can be used to develop xenografts in co-culture systems, making them great models for mechanistic studies.

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

Research

JoVE Journal

Cancer Research

3.8K vistas.  Cold Spring Harbor. Los organoides derivados de pacientes humanos son sistemas modelo tridimensionales in vitro que representan tanto la diversidad del paciente como la heterogeneidad celular de los tumores. Este protocolo y demostración en video proporcionan una guía detallada y práctica para establecer el tumor de mama derivado del paciente y los organoides normales. Los organoides de tumores de mama derivados de pacientes son nuevos modelos emocionantes, pero difíciles de establecer.El protocolo in...