Establishment and Culture of Patient-Derived Breast Organoids

Disha Aggarwal; Suzanne Russo; Payal Naik; Sonam Bhatia; David L. Spector

doi:10.3791/64889

JoVE Journal > Cancer Research

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Cancer Research

患者来源乳腺类器官的建立和培养

Published: February 17, 2023

doi:

10.3791/64889

Disha Aggarwal², Suzanne Russo, Payal Naik, Sonam Bhatia, David L. Spector²

¹Cold Spring Harbor Laboratory,Cold Spring Harbor, ²Genetics Graduate Program,Stony Brook University

Summary

这里提供了一个详细的方案，用于从患者来源的乳腺肿瘤切除术或正常乳腺组织中建立人乳腺类器官。该协议为培养、冷冻和解冻人类患者来源的乳腺类器官提供了全面的分步说明。

Abstract

乳腺癌是一种复杂的疾病，已分为几种不同的组织学和分子亚型。我们实验室开发的患者来源乳腺肿瘤类器官由多个肿瘤来源的细胞群的混合物组成，因此比已建立的2D癌细胞系更近似于肿瘤细胞多样性和环境。类器官是一种理想的体外模型，允许细胞 – 细胞外基质相互作用，已知在细胞 – 细胞相互作用和癌症进展中起重要作用。患者来源的类器官也比小鼠模型具有优势，因为它们是人类来源的。此外，它们已被证明可以概括患者肿瘤的基因组、转录组和代谢异质性;因此，它们能够代表肿瘤的复杂性以及患者的多样性。因此，他们准备为靶标发现和验证以及药物敏感性测定提供更准确的见解。在该协议中，我们详细演示了如何从切除的乳腺肿瘤（癌症类器官）或还原性乳房整形术衍生的乳腺组织（正常类器官）中建立患者来源的乳腺类器官。接下来是3D类器官培养，扩增，传代，冷冻以及患者来源的乳腺类器官培养物的解冻的全面说明。

Introduction

乳腺癌（BC）是女性中最常见的恶性肿瘤，据估计，2022 年美国将诊断出 287，850 例新病例¹.尽管最近在早期发现方面取得了进展，包括年度筛查、靶向治疗和更好地了解遗传易感性，但它仍然是美国女性癌症死亡的第二大原因>，每年有 40，000 人死于乳腺癌¹。乳腺癌目前根据原发肿瘤的组织病理学和分子学评估分为多种亚型。更好的亚型分层通过亚型特异性治疗方案改善了患者的预后².例如，将HER2鉴定为原癌基因³导致了曲妥珠单抗的发展，这使得这种高度侵袭性的亚型在大多数患者中易于控制⁴。以患者特异性方式进一步研究这种复杂疾病的遗传学和转录组学将有助于开发和预测更好的患者特异性个性化治疗方案²^，⁵。患者来源类器官（PDO）是一种很有前途的新模型，可以在分子水平上深入了解癌症，确定新的靶点或生物标志物并设计新的治疗策略⁶，⁷^，⁸。

PDO是源自新鲜切除的原代组织样本的多细胞三维（3D）结构⁸^，⁹。它们通过嵌入水凝胶基质（通常由细胞外基质（ECM）蛋白的组合组成）进行三维生长，因此可用于研究肿瘤细胞 – ECM相互作用。PDO代表患者的多样性，并概括了肿瘤的细胞异质性和遗传特征¹⁰，¹¹^，¹²。作为体外模型，它们允许基因操作和高通量药物筛选¹³，¹⁴^，¹⁵。此外，PDO可以合理地用于评估患者的药物敏感性和与临床平行的治疗策略，并帮助预测患者结果¹⁶，¹⁷^，¹⁸。除化疗外，某些类器官模型也已用于检查个体患者对放化疗的反应¹⁹^，²⁰。鉴于PDO在研究和临床应用中的可喜应用性，美国国家癌症研究所发起了一个国际联盟，即人类癌症模型倡议（HCMI）²¹，以生成和提供这些肿瘤衍生的新型癌症模型。通过HCMI开发的各种癌症类型的许多类器官模型可通过美国类型培养物保藏（ATCC）^获得22。

正常的乳腺类器官已被证明由乳腺中存在的不同上皮细胞群组成¹¹，²³，因此可以作为研究基本生物学过程，分析导致肿瘤发生的驱动突变以及癌细胞起源谱系研究的重要模型⁶^，¹⁵.乳腺肿瘤类器官模型已被用于识别新的靶点，这些靶点正在鼓励开发新疗法的前景，特别是对于耐药肿瘤²⁴，²⁵^，²⁶。Guillen等人使用患者来源的异种移植物（PDX）和匹配的PDX衍生类器官（PDxO）模型对难治性乳腺肿瘤进行研究，表明类器官是精准医学的强大模型，可用于评估药物反应和平行指导治疗决策²⁸。此外，开发用于培养具有各种免疫细胞²⁷，²⁸，²⁹，成纤维细胞^30，31和微生物³²^，³³的PDO的新共培养方法为研究肿瘤微环境对癌症进展的影响提供了机会。虽然许多此类共培养方法正在积极用于胰腺或结直肠肿瘤的PDO，但类似的乳腺PDO共培养方法仅在自然杀伤细胞³⁴和成纤维细胞³⁵上报道。

代表不同乳腺癌亚型的>100个患者来源类器官的第一个生物库由Hans Clevers小组³⁶^，³⁷开发。作为这项工作的一部分，Clevers小组还开发了第一个用于乳腺类器官生长的复杂培养基，目前广泛使用³⁶。一项后续研究全面介绍了乳腺PDO和患者来源的类器官异种移植物（PDOX）的建立和培养情况³⁸。Welm实验室开发了大量BC PDX模型和PDxO，这些模型和PDxO在含有胎牛血清（FBS）和较少生长因子³⁹^，⁴⁰的相对简单的生长培养基中培养。我们独立开发和表征了大量幼稚的患者来源的乳腺癌类器官模型¹¹，并参与了BC PDO模型的开发，作为HCMI计划²¹的一部分。在这里，我们旨在提供一份实用指南，详细说明我们在生成患者衍生的乳腺类器官模型系统时采用的方法。

Protocol

根据机构审查委员会协议IRB-03-012和IRB 20-0150，并在患者的书面知情同意下，从Northwell Health获得了乳腺癌患者的肿瘤切除以及远端和邻近正常组织。注意：经生物安全委员会批准，以下提到的所有程序均在指定用于患者样本的哺乳动物组织培养BSL2室中进行。所有程序应按照安全规程执行，以保持生物安全柜中的无菌条件。除非另有说明，否则每个离心步骤均在室温（RT）下进?…

Representative Results

我们已经建立了一个患者来源的乳腺肿瘤类器官的生物库，包括各种亚型11。此外，我们使用图1中概述的方法建立了多个来自还原性乳房整形组织样本或BC患者的相邻/远端正常乳房的正常类器官系。各种患者来源的乳腺肿瘤类器官系在其形态（图2）和生长速率（图3）上有所不同。正常的?…

Discussion

我们的实验室已成功采用上述方案从幼稚的肿瘤切除或刮片中建立类器官。我们还利用该协议从通过复位乳房成形术获得的乳腺组织或癌症患者的邻近或远端正常乳腺组织中开发正常类器官。约30%-40%的切除原发性肿瘤导致长期（>传代8）肿瘤类器官培养成功。在几次传代后逐渐变细的肿瘤类器官系要么具有正常类器官或基质细胞的生长，要么主要由非增殖性肿瘤细胞组成。进一步评估和?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们要感谢Spector实验室的成员在整个工作过程中进行的批判性讨论。我们感谢Norman Sachs和Hans Clevers（荷兰Hubrecht研究所）最初为我们提供他们的类器官培养方案。我们感谢CSHL癌症中心组织学和显微镜共享资源提供的服务和技术专长（NCI 2P3OCA45508）。我们感谢高青博士在组织学样本制备方面的帮助。我们感谢Karen Kostroff博士（Northwell Health）为患者提供肿瘤样本的支持。我们也感谢Northwell Health Biobank团队为样本采集所做的努力，我们感谢患者及其家人捐赠组织用于研究。这项研究得到了CSHL / Northwell Health（D.L.S.），NCI 5P01CA013106-Project 3（D.L.S.）和Leidos Biomedical HHSN26100008（David Tuveson和D.L.S.）的支持。

Materials

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	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

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Aggarwal, D., Russo, S., Naik, P., Bhatia, S., Spector, D. L. Establishment and Culture of Patient-Derived Breast Organoids. J. Vis. Exp. (192), e64889, doi:10.3791/64889 (2023).