肺癌类器官模型：构建、表征与药物筛选

以下为英文全文（English full text）

Abstract

Lung cancer remains the leading cause of cancer-related mortality worldwide. Patient-derived lung cancer organoids (LCOs) have emerged as powerful preclinical models that preserve the histopathological features, mutational profiles, and drug response patterns of the original tumors. This article provides a detailed technical protocol for lung cancer organoid construction, including tissue processing, culture media composition, and quality control measures. We discuss characterization strategies encompassing histopathology, immunostaining, genomic profiling, and functional assays. Finally, we review drug screening applications, including high-throughput compound testing, targeted therapy evaluation, and combination regimen optimization, with attention to clinical predictive accuracy.

1. Introduction

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases and includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma subtypes. The genomic landscape of lung adenocarcinoma is characterized by recurrent mutations in EGFR, KRAS, TP53, and ALK rearrangements, among others. Small cell lung cancer (SCLC) is characterized by rapid growth and early metastasis. Despite advances in targeted therapies and immunotherapy, drug resistance remains a major clinical challenge.

Patient-derived lung cancer organoids (LCOs) offer a unique platform for studying lung cancer biology and predicting therapeutic responses. LCOs can be established from surgically resected tissues, biopsy specimens, and even malignant pleural effusions. They maintain the morphological and genetic characteristics of the parental tumors and can be expanded for high-throughput drug screening [1].

2. Lung Cancer Organoid Construction Protocol

2.1 Tissue Collection and Processing

Fresh tumor tissue should be obtained immediately after surgical resection or biopsy. The sample is placed in ice-cold Advanced DMEM/F12 supplemented with 1% penicillin-streptomycin and 10 mM HEPES. Tissue viability declines significantly after 24 hours; therefore, processing should occur within 4–6 hours of collection when possible.

The tissue is washed three times in ice-cold PBS with antibiotics to remove blood and debris. Using sterile forceps and a scalpel, the specimen is minced into 1–2 mm³ fragments in a tissue culture dish. The minced tissue is transferred to a 15 mL conical tube containing digestion medium: Advanced DMEM/F12 supplemented with 1 mg/mL collagenase I, 0.5 mg/mL dispase II, and 10 μM Y-27632 (ROCK inhibitor). The tube is placed on a rocker at 37°C for 30–60 minutes, with periodic assessment of digestion progress under a microscope. The digestion is terminated by adding 5 volumes of ice-cold washing medium (Advanced DMEM/F12 with 10% FBS) [2].

The digested slurry is centrifuged at 300 × g for 5 minutes at 4°C. The supernatant is removed, and the pellet is resuspended in 1 mL TrypLE Express containing 10 μM Y-27632. The tube is incubated at 37°C for 10 minutes with gentle agitation. After centrifugation, the cell pellet is washed twice with washing medium.

2.2 Embedding and Initial Culture

The final cell pellet is resuspended in basement membrane matrix (such as Matrigel or GBiowit Organoid Matrix) at 4°C. The matrix concentration should be 80–100% for optimal mechanical support. The cell-matrix suspension is plated as 40–50 μL droplets in the center of pre-warmed 24-well plates. The plates are placed in a 37°C, 5% CO2 humidified incubator for 15–20 minutes to allow the matrix to solidify.

After polymerization, 500 μL of lung organoid growth medium (L-OGM) is gently added to each well. The medium is changed every 3–4 days. Organoid formation is typically observed within 3–7 days [3].

2.3 Lung Organoid Growth Medium Composition

The base medium is Advanced DMEM/F12 supplemented with:

• 1× B-27 supplement (minus vitamin A)
• 1× N-2 supplement
• 1.25 mM N-acetylcysteine
• 10 mM nicotinamide
• 10 nM human recombinant gastrin I
• 50 ng/mL human recombinant EGF
• 100 ng/mL human recombinant FGF10
• 100 ng/mL human recombinant FGF7
• 1 μM A83-01 (TGF-β receptor inhibitor)
• 10 μM SB202190 (p38 MAPK inhibitor)
• 10 μM Y-27632 (ROCK inhibitor) — added for the first 3 days only
• 1× GlutaMAX
• 100 μg/mL Primocin

Note: Wnt3a and R-spondin 1 are typically omitted for lung cancer organoids, as lung adenocarcinoma cells often exhibit Wnt pathway activation and are Wnt-independent. However, for normal lung organoids, these factors are essential [4].

2.4 Success Rates and Troubleshooting

The success rate of lung cancer organoid establishment varies depending on tumor type and tissue quality. Resected tumor specimens typically achieve 70–80% establishment success, while small biopsy specimens have lower rates of 40–60%. SCLC organoids generally establish more readily than squamous cell carcinoma organoids. Poor tissue quality, excessive necrosis, and bacterial contamination are the primary causes of establishment failure. Pre-washing tissues with antibiotic-supplemented medium and using Primocin in culture medium significantly reduces contamination risk [5].

3. Characterization Strategies

3.1 Histopathological Assessment

Organoids should be compared directly with the parental tumor tissue. Matched formalin-fixed paraffin-embedded (FFPE) sections of the original tumor and the derived organoids are stained with hematoxylin and eosin (H&E). Lung adenocarcinoma organoids typically form solid or acinar structures with glandular architecture, recapitulating the histology of the primary tumor. SCLC organoids form smaller, more compact spheroids with scant cytoplasm and nuclear molding, similar to the small cell morphology in vivo [6].

3.2 Immunohistochemistry and Immunofluorescence

Key markers for lung cancer organoid characterization include:

• Thyroid transcription factor-1 (TTF-1/NKX2-1): positive in lung adenocarcinoma
• p63: positive in squamous cell carcinoma
• Chromogranin A and synaptophysin: positive in neuroendocrine/SCLC
• Pan-cytokeratin (AE1/AE3): epithelial marker
• Ki-67: proliferation index
• E-cadherin: epithelial integrity

For immunofluorescence, organoids are fixed in 4% paraformaldehyde for 30 minutes, permeabilized with 0.5% Triton X-100 for 15 minutes, blocked with 5% BSA, and incubated with primary antibodies overnight at 4°C. Secondary antibodies conjugated to Alexa Fluor dyes are used for detection. Confocal microscopy is recommended for high-resolution 3D imaging [7].

3.3 Genomic Profiling

Whole-exome sequencing (WES) or targeted next-generation sequencing (NGS) panels should be performed on both the parental tumor and the derived organoids to confirm genetic concordance. Key lung cancer driver mutations to assess include EGFR, KRAS, BRAF, PIK3CA, TP53, and ALK rearrangements. Studies have shown that organoids maintain the mutational profile of the original tumors with >95% concordance for common driver mutations [8].

RNA sequencing can be used to assess transcriptomic fidelity and identify the expression of therapeutically relevant targets, such as PD-L1 for immunotherapy assessment.

4. Drug Screening Applications

4.1 High-Throughput Compound Screening

Once organoid lines are established and expanded, they can be dissociated into single cells or small clusters and plated in 96-well or 384-well plates for drug screening. The general protocol is:

1. Harvest organoids from Matrigel using Cell Recovery Solution or mechanical dissociation.
2. Digest with TrypLE Express for 5–10 minutes to generate single-cell suspensions.
3. Count cells and resuspend in Matrigel (1:1 ratio) at 1,000–3,000 cells per 20 μL for 384-well plates.
4. Plate cells and allow Matrigel to polymerize at 37°C for 15 minutes.
5. Overlay with 30 μL of culture medium.
6. Culture for 48 hours before drug addition.
7. Add drugs at 6-point or 10-point dilution series using liquid handling robotics.
8. Incubate for 72–96 hours.
9. Assess viability using CellTiter-Glo 3D (ATP-based luminescence assay) or CellTiter-Blue (resazurin reduction assay).
10. Calculate IC50 values using GraphPad Prism or similar software [9].

4.2 Targeted Therapy Evaluation

Lung cancer organoids have been extensively validated for predicting responses to EGFR tyrosine kinase inhibitors (TKIs). Studies have shown that EGFR-mutant LCOs (exon 19 deletions, L858R) are sensitive to erlotinib, gefitinib, and osimertinib, while EGFR wild-type LCOs are resistant. The sensitivity of LCO-based drug screening in predicting clinical responses to osimertinib was reported at 86.7% (13/15 patients) [10].

For ALK-rearranged LCOs, crizotinib and alectinib sensitivity correlates with clinical response. KRAS-mutant LCOs are generally resistant to EGFR inhibitors, consistent with clinical experience, but show differential responses to downstream pathway inhibitors such as MEK and ERK inhibitors [11].

4.3 Combination Regimen Optimization

Lung cancer organoids are particularly valuable for testing combination therapies. Chemotherapy doublets (e.g., cisplatin + pemetrexed) and targeted therapy combinations (e.g., EGFR TKI + MET inhibitor for acquired resistance) can be evaluated in parallel. Organoids-on-chip platforms enable automated, continuous perfusion of multiple drugs at varying concentrations, reducing manual handling and improving reproducibility [12].

5. Clinical Predictive Accuracy and Future Directions

Multiple studies have validated the clinical predictive accuracy of LCO-based drug screening. A prospective study demonstrated that PDO drug sensitivity results achieved an overall positive predictive accuracy of 75–80% and a negative predictive accuracy exceeding 90% [13]. Clinical trials are currently evaluating the utility of LCO-guided treatment selection in metastatic NSCLC.

Future directions include the integration of immune cells into LCO models to assess immunotherapy responses, the use of lung organoids-on-chip to model mechanical ventilation and airflow effects, and the incorporation of patient-matched cancer-associated fibroblasts (CAFs) to recapitulate the tumor microenvironment. Air-liquid interface (ALI) cultures of lung organoids enable the polarization of airway epithelium and the study of mucociliary clearance, providing additional physiological relevance for infection and drug delivery studies [14].

Conclusion

Lung cancer organoids represent a robust, clinically relevant platform for modeling lung cancer biology and predicting therapeutic responses. With establishment success rates exceeding 70% for resected specimens and drug screening protocols compatible with high-throughput automation, LCOs are positioned to become standard tools in both translational research and personalized oncology.

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