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.
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Method Article
Establishment and Culture of Patient-Derived Breast Organoids
In This Article
Summary
Abstract
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.
Introduction
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 >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 impro....
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Protocol
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/org....
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Results
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 .......
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Discussion
Our lab has successfully employed the above protocols to establish organoids from naïve 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' adjacent or distal normal breast tissue. About 30%-40% of the resected primary tumors resulted in successful long-term (>passage 8) tumor organoid cultures. The tumor organoid lines that tapered off after a few passages either had an outgro.......
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Disclosures
The authors declare no conflicts of interest.
Acknowledgements
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 th....
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 15 mL conical tubes | VWR | 525-1068 | |
| 175 cm2 tissue culture flask | VWR (Corning) | 29185-308 | |
| 37 °C bead bath | |||
| 37 °C CO2 incubator | |||
| 50 mL conical tubes | VWR | 525-1077 | |
| 50 mL vacuum filtration system (0.22 µm Filter) | Millipore Sigma | SCGP00525 | SCGP00525 |
| 500 mL Rapid-Flow Filter Unit, 0.2 µm aPES membrane, 75 mm diameter | Nalgene | 566-0020 | |
| 6-well culture plates | Greiner Cellstar | 82050-842 | |
| 75 cm2 tissue culture flask | VWR (Corning) | 29185-304 | |
| 96-well opaque plates | Corning | 353296 | For CTG assay |
| A83-01 | Tocris | 2939 | |
| Advanced DMEM/F12 | Gibco | 12634-010 | |
| B-27 supplement | Life Technologies | 12587010 | |
| BioTek Synergy H4 Hybrid Microplate Reader | Fisher Scientific (Agilent) | For dual luciferase assay and CTG assay | |
| BSA fraction V (7.5%) | Thermo Fisher | 15260037 | |
| Cell Titer-Glo (CTG) Reagent | Promega | G9683 | luminescent cell viability assay |
| Centrifuge | Eppendorf | 5804 | |
| Collagenase from Clostridium histolyticum | Millipore Sigma | C5138 | Type IV |
| Cryolabels | Amazon | DTCR-1000 | Direct Thermal Cryo-Tags, White, 1.05 x 0.5" |
| Cryovials | Simport Scientific Inc. | T311-1 | |
| Countess 3 Automated Cell Counter | Thermo Fisher | AMQAX2000 | |
| DMEM, high glucose, pyruvate | Thermo Fisher (Gibco) | 11995040 | |
| Dual Luciferase Reporter Assay System | Promega | E1910 | |
| Dulbecco’s Phosphate Buffered Saline (1X) | Gibco | 14190-144 | DPBS |
| Epidermal growth factor (hEGF) | Peprotech | AF-100-15 | |
| Fetal Bovine Serum (FBS) | Corning | 35-010-CV | |
| FGF-10 (human) | Peprotech | 100-26 | |
| FGF-7/KGF (human) | Peprotech | 100-19 | |
| GlutaMax | Life Technologies | 35050061 | |
| HEK293T cells | ATCC | CRL-3216 | For TOPFlash Assay |
| HEK293T-HA-Rspondin1-Fc cells | R&D Systems | 3710-001-01 | Cultrex HA-R-Spondin1-Fc 293T Cells |
| HEPES | Life Technologies | 15630-080 | |
| Heregulinβ-1 (human) | Peprotech | 100-03 | |
| Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (~10 mg/mL protein concentration) | Corning | 356231 | Phenol-red free, LDEV-free; basement membrane matrix |
| Mr. Frosty Cell Freezing Container | Thermo Fisher | 5100-0001 | |
| Mycoplasma detection kit | Lonza | LT07-418 | |
| N-acetyl-l-cysteine | Millipore Sigma | A9165 | |
| Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membranes | Thermo Fisher | 166-0045 | |
| Nicotinamide | Millipore Sigma | N0636 | |
| Noggin (human) | Peprotech | 120-10C | |
| P1000, P200, P10 pipettes with tips | |||
| p38 MAPK inhibitor (p38i) SB 202190 | Millipore Sigma | S7067 | |
| Parafilm | transparent film | ||
| Penicillin-Streptomycin | Life Technologies | 15140122 | |
| Plasmid1: pRL-SV40P | Addgene | 27163 | |
| Plasmid2: M51 Super 8x FOPFlash | Addgene | 12457 | |
| Plasmid3: M50 Super 8x TOPFlash | Addgene | 12456 | |
| pluriStrainer 200 µm | pluriSelect | 43-50200-01 | |
| Primocin | Invivogen | ANT-PM-1 | |
| Recovery Cell Culture Freezing Medium | Thermo Fisher (Gibco) | 12648-010 | cell freezing medium |
| Red Blood Cell lysis buffer | Millipore Sigma | 11814389001 | |
| R-spondin conditioned media | In-house or commercial from Peprotech | 120-38 | |
| Scalpel (No.10) | Sklar Instruments | Jun-10 | |
| Shaker (Incu-shaker Mini) | Benchmark | H1001-M | |
| TGF-β receptor inhibitor A 83-01 | Tocris | 2939 | |
| Trypan Blue Stain (0.4%) | Gibco | 15250-061 | |
| TrypLE Express Enzyme (1X), phenol red | Life Technologies | 12605028 | cell dissociation reagent |
| X-tremeGENE 9 DNA transfection reagent | Millipore Sigma | 6365779001 | |
| Y-27632 Dihydrochloride (RhoKi) | Abmole Bioscience | Y-27632 | |
| Zeocin | Thermo Fisher | R25001 |
References
- Siegel, R. L., Miller, K. D., Fuchs, H. E., Jemal, A. Cancer statistics, 2022. CA: A Cancer Journal for Clinicians. 72 (1), 7-33 (2022).
- Greenwalt, I., Zaza, N., Das, S., Li, B. D. Precision Medicine and Targeted Therapies in Breast Cancer. Surgical Oncology Clinics of N....
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