乳腺癌类器官模型：异质性与治疗抵抗

以下为英文全文（English full text）

Abstract

Breast cancer is a heterogeneous disease comprising multiple molecular subtypes with distinct clinical behaviors and therapeutic responses. Patient-derived breast cancer organoids (BC-PDOs) capture this inter- and intra-tumoral heterogeneity, providing a platform for studying subtype-specific drug responses and resistance mechanisms. This article details the technical protocols for establishing breast cancer organoids, strategies for preserving and modeling heterogeneity, and approaches for identifying and overcoming therapeutic resistance through combination strategies and microenvironment modulation.

1. Introduction

Breast cancer is classified into four major intrinsic subtypes based on gene expression profiling: luminal A, luminal B, HER2-enriched, and triple-negative (TNBC). Each subtype has distinct therapeutic implications: luminal tumors are treated with endocrine therapy, HER2-enriched tumors with anti-HER2 agents, and TNBC with chemotherapy. Despite these targeted approaches, therapeutic resistance ultimately develops in most patients, particularly in the metastatic setting.

Breast cancer organoids were first established by Sachs et al. (2018), who created a living biobank of 95 breast cancer organoid lines covering all major subtypes. These organoids recapitulated the histopathology, hormone receptor status, and genomic landscape of the original tumors, including key mutations in PIK3CA, TP53, and BRCA1/2 [1]. This foundational work established breast cancer as a highly tractable organoid system and demonstrated the potential for subtype-specific drug screening.

2. Breast Cancer Organoid Establishment Protocol

2.1 Tissue Processing and Digestion

Breast cancer tissue is obtained from surgical resections or core needle biopsies (14–18 gauge). Fresh tissue is transported in ice-cold organoid basal medium (Advanced DMEM/F12 with 1% P/S, 10 mM HEPES, and 1% GlutaMAX). The tissue is washed three times in ice-cold PBS, minced into 2–3 mm pieces, and digested in 10 mL dissociation medium at 37°C for 1–2 hours with gentle shaking. The dissociation medium contains: Advanced DMEM/F12, 2 mg/mL collagenase III, 100 U/mL hyaluronidase, and 10 μM Y-27632 [2].

The digested mixture is filtered through a 100 μm cell strainer to remove undigested tissue fragments. The filtrate is centrifuged at 300 × g for 5 minutes. The pellet is washed twice in wash medium (Advanced DMEM/F12 with 10% FBS). If red blood cell contamination is visible, an ACK lysis buffer step can be included before the final wash.

For epithelial enrichment, the cell suspension can be incubated with anti-EpCAM microbeads and subjected to magnetic-activated cell sorting (MACS). This step is particularly beneficial for highly fibrotic tumors where stromal cells can outcompete epithelial cells in culture [3].

2.2 Matrix Embedding and Culture Conditions

The final epithelial cell pellet is resuspended in cold basement membrane matrix (Matrigel or GBiowit Breast Cancer Organoid Matrix) at a density of 5,000–20,000 cells per 50 μL. The suspension is plated as 40–50 μL droplets in 24-well plates and polymerized at 37°C for 20 minutes.

The complete breast cancer organoid medium (BOM) consists of:

• Advanced DMEM/F12 (base)
• 1× B-27 supplement (without vitamin A)
• 1× N-2 supplement
• 1.25 mM N-acetylcysteine
• 10 mM nicotinamide
• 50 ng/mL human recombinant EGF
• 20 ng/mL human recombinant FGF7
• 100 ng/mL human recombinant Noggin
• 250 ng/mL R-spondin 1
• 100 ng/mL Wnt3a
• 10 nM [Leu15]-gastrin
• 1 μM A83-01
• 5 μM Y-27632 (first 3 days)
• 10 nM β-estradiol (for hormone receptor-positive tumors)
• 1 μM hydrocortisone
• 5 μM forskolin (optional, improves establishment)
• 100 μg/mL Primocin

Note: Wnt3a, R-spondin 1, and Noggin are required for initial establishment of most breast cancer organoids, even though tumor cells may eventually become less dependent on these factors. The inclusion of β-estradiol improves the growth of ER-positive organoids [4].

2.3 Establishment Success Rates and Expansion

Breast cancer organoid establishment success rates vary by subtype: TNBC achieves the highest rates at 70–80%, HER2-enriched at 60–70%, and luminal subtypes at 50–60%. The lower success rates for luminal tumors are attributed to their slower growth and greater dependence on hormone signaling. The time to first passage ranges from 10–21 days. Passaging is performed every 7–14 days at a 1:3 to 1:5 split ratio using TrypLE Express digestion for 5–10 minutes [5].

3. Modeling Tumor Heterogeneity

3.1 Inter-Patient Heterogeneity

The living biobank approach captures the full spectrum of breast cancer heterogeneity across the patient population. Each PDO line represents a distinct patient tumor with unique combinations of driver mutations, copy number alterations, and gene expression patterns. Multi-omics profiling of PDO biobanks has revealed that organoids faithfully preserve the molecular subtypes of the original tumors, enabling subtype-specific drug screening. For example, HER2-enriched organoids are sensitive to trastuzumab and pertuzumab, while luminal organoids respond to tamoxifen and fulvestrant [6].

3.2 Intra-Tumoral Heterogeneity and Clonal Evolution

Within a single tumor, distinct subclones may have different therapeutic sensitivities. Breast cancer organoids can capture this intra-tumoral heterogeneity, particularly when derived from multi-region biopsies or when single-cell-derived subclones are isolated from the primary culture. Studies using optical metabolic imaging (OMI) have tracked individual organoid responses within a population, revealing subclonal populations that confer differential responses to chemotherapy and targeted agents [7].

Longitudinal modeling of resistance involves treating organoids with escalating doses of targeted agents (e.g., HER2 inhibitors) over weeks to months. This approach recapitulates the clinical acquisition of resistance and has identified mechanisms including HER2 amplification, alternative pathway activation (MET, EGFR), and epithelial-to-mesenchymal transition [8].

4. Therapeutic Resistance Mechanisms and Combination Strategies

4.1 Endocrine Therapy Resistance in Luminal Tumors

Resistance to endocrine therapy (tamoxifen, aromatase inhibitors) in ER-positive breast cancer remains a major clinical challenge. Organoid studies have identified multiple resistance mechanisms, including ESR1 mutations, FGFR1 amplification, and CDK4/6 pathway activation. BC organoids with acquired endocrine resistance show cross-resistance profiles that mirror clinical experience, and combination strategies targeting the resistance mechanism (e.g., CDK4/6 inhibitors + endocrine therapy) can be tested directly in the organoid model [9].

4.2 HER2-Targeted Therapy Resistance

In HER2-enriched breast cancer, resistance to trastuzumab and other HER2 inhibitors can be modeled in organoids through both primary (de novo) and acquired resistance approaches. Mechanisms identified in organoid models include: HER2 extracellular domain shedding, PI3K/AKT pathway activation (PTEN loss, PIK3CA mutations), and HER3 upregulation. Organoid-based screening has identified combination strategies, such as HER2 inhibitors plus PI3K inhibitors, that overcome resistance in preclinical models [10].

4.3 Chemotherapy Resistance in TNBC

TNBC organoids have been used to identify predictors of chemotherapy response and to develop novel combination strategies. Campaner et al. (2020) demonstrated that TNBC organoids resistant to docetaxel showed YAP activation and mechanotransduction upregulation. Importantly, treatment with YAP inhibitors (dasatinib, statins) or mechanosignaling inhibitors restored chemosensitivity in resistant organoids, suggesting a clinically actionable strategy [11].

4.4 DNA Repair Deficiency and PARP Inhibitor Sensitivity

Organoids derived from BRCA1/2-mutant breast cancers exhibit high sensitivity to PARP inhibitors (olaparib, talazoparib), consistent with clinical observations. Isogenic control experiments using CRISPR to correct the BRCA mutation in patient-derived organoids have definitively confirmed that the PARP inhibitor sensitivity is directly caused by the BRCA mutation. This approach provides a powerful framework for validating synthetic lethal targets in breast cancer [12].

5. Integrating the Microenvironment: Stroma and Immune Co-Culture

The tumor microenvironment (TME) plays a critical role in breast cancer progression and therapeutic response. Native TME models can be generated by culturing tumor fragments in air-liquid interface (ALI) systems that preserve endogenous stromal and immune cells for limited periods. Reconstituted TME models involve co-culturing BC organoids with isolated cancer-associated fibroblasts (CAFs), adipocytes, or immune cells in engineered matrices.

Co-culture with CAFs has been shown to promote chemoresistance in TNBC organoids through paracrine signaling involving IL-6 and CCL2. The addition of immune cells, particularly tumor-infiltrating lymphocytes (TILs) and natural killer (NK) cells, enables the assessment of antibody-dependent cellular cytotoxicity (ADCC) and immune checkpoint inhibitor responses [13].

Organoid-on-chip platforms enable spatially defined co-culture systems where tumor organoids are surrounded by stromal zones, allowing the study of CAF subtype heterogeneity and its influence on drug response [14].

Conclusion

Breast cancer organoids capture the inter- and intra-tumoral heterogeneity that defines this disease and influences therapeutic outcomes. By preserving subtype-specific features, enabling longitudinal resistance studies, and supporting microenvironment co-culture, BC-PDOs provide a robust platform for precision oncology research and drug development.

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