中文导读
结直肠癌是全球第三大常见癌症。本文介绍从患者活检样本建立结直肠癌类器官的全流程及其在个体化用药中的价值。
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Abstract
Colorectal cancer (CRC) is the third most common cancer worldwide and a leading cause of cancer-related mortality. Patient-derived colorectal cancer organoids (CRC-PDOs) have emerged as the most clinically validated organoid model system, with established biobanks, standardized protocols, and prospective clinical trials demonstrating their utility in predicting patient responses. This article provides detailed technical guidance on CRC organoid establishment from patient biopsies, expansion protocols, and quality control. We review applications in personalized therapy, including drug sensitivity testing for standard chemotherapy regimens, targeted agents, and emerging immunotherapy strategies.
1. Introduction
Colorectal cancer develops through a well-defined adenoma-to-carcinoma sequence characterized by sequential mutations in APC, KRAS, TP53, and SMAD4. The molecular classification of CRC includes microsatellite-stable (MSS) and microsatellite-instable (MSI) subtypes, with the latter showing better responses to immune checkpoint inhibitors. Despite advances in surgical techniques and systemic therapy, approximately 50% of patients develop metastatic disease, and treatment resistance remains a major challenge.
CRC was the first cancer type for which robust organoid culture protocols were established, and it remains the most extensively validated system. The seminal work by Sato et al. (2011) demonstrated that human colorectal epithelium could be expanded indefinitely as organoids while maintaining the genetic and histological characteristics of the original tissue [1]. This foundational work has been expanded to establish living biobanks comprising hundreds of CRC organoid lines, spanning the full spectrum of disease stages and molecular subtypes [2].
2. CRC Organoid Establishment from Patient Biopsies
2.1 Specimen Collection and Initial Processing
CRC organoids can be established from surgical resections, endoscopic biopsies (2–5 mm), and metastatic lesions (liver, lung, peritoneal). For optimal results, tissue should be transported in ice-cold organoid basal medium (Advanced DMEM/F12 with 1% GlutaMAX, 10 mM HEPES, and 1% penicillin-streptomycin) within 4–6 hours of collection. The addition of 10% FBS during transport can improve viability for longer transport times.
The tissue is washed three times in ice-cold PBS with antibiotics. Using sterile scissors and forceps, the specimen is minced into 1 mm³ fragments in a 35 mm tissue culture dish. The minced tissue is transferred to a 15 mL conical tube containing 8 mL of digestion medium at 37°C: Advanced DMEM/F12 supplemented with 1 mg/mL collagenase II and 0.5 mg/mL dispase [3].
The tube is placed on a rocker at 37°C for 30–60 minutes. Digestion progress is monitored microscopically; crypt-like structures should be visible. The digestion is terminated by adding an equal volume of ice-cold wash medium (Advanced DMEM/F12 with 10% FBS). The mixture is centrifuged at 300 × g for 5 minutes at 4°C. The supernatant is removed, and the pellet is washed twice with wash medium.
2.2 Crypt Isolation and Matrix Embedding
For normal colonic tissue, the washed pellet contains crypts that can be directly embedded in Matrigel. For tumor tissue, further dissociation may be required. The pellet is resuspended in 2 mL TrypLE Express and incubated at 37°C for 5–10 minutes. The digestion is neutralized with wash medium, and the cells are pelleted by centrifugation.
The final pellet is resuspended in cold basement membrane matrix (Matrigel or GBiowit CRC Organoid Matrix) at a density of approximately 200–500 crypts per 50 μL. The 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 incubator for 15–20 minutes to allow polymerization.
2.3 Culture Medium for CRC Organoids
The complete CRC organoid medium (WENR medium) 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
- 10 nM [Leu15]-gastrin
- 100 ng/mL recombinant Noggin (or 10% Noggin-conditioned medium)
- 1 μg/mL recombinant R-spondin 1 (or 10% R-spondin-conditioned medium)
- 50% Wnt3a-conditioned medium (or 100 ng/mL recombinant Wnt3a)
- 10 μM SB202190 (p38 inhibitor)
- 500 nM A83-01 (TGF-β/Activin receptor inhibitor)
- 10 μM Y-27632 (ROCK inhibitor) — for first 3 days only
- 100 μg/mL Primocin
For tumor organoids, Wnt3a and R-spondin 1 can often be omitted after initial establishment, as many CRC tumors have autocrine Wnt pathway activation through APC mutations or RSPO fusions. However, these factors should be retained during the initial establishment phase [4].
2.4 Success Rates and Expansion
CRC organoids have among the highest establishment success rates of any tumor type. Surgical specimens achieve 80–90% success, while endoscopic biopsies achieve 70–80% success. The time from initial plating to first passage is typically 7–14 days. Organoids are passaged when they reach approximately 80% confluence within the Matrigel droplet, typically every 7–10 days.
Passaging involves mechanical disruption of the Matrigel with a P1000 pipette tip, followed by incubation in TrypLE Express at 37°C for 5–10 minutes. The organoids are dissociated into fragments of 10–50 cells by gentle pipetting. The typical split ratio is 1:3 to 1:5. After 3–5 passages, ROCK inhibitor can be omitted from the medium [5].
3. Characterization and Quality Control
3.1 Histological and Immunohistochemical Validation
CRC organoids should be validated by comparing H&E-stained sections with the original tumor. Adenocarcinoma organoids typically form irregular, dense structures with enlarged nuclei and prominent nucleoli. Mucinous adenocarcinoma organoids produce mucin that accumulates in the lumen. Key immunohistochemical markers include CK20 and CDX2 (intestinal differentiation), CK7 (variable), p53 (mutant stabilization pattern), β-catenin (nuclear accumulation in Wnt-activated tumors), and Ki-67 (proliferation index) [6].
3.2 Genomic Concordance Assessment
Targeted NGS panels or whole-exome sequencing should be performed to confirm that organoids maintain the mutational profile of the parental tumor. Key genes to assess include APC, KRAS, BRAF, PIK3CA, TP53, SMAD4, and the mismatch repair genes (MLH1, MSH2, MSH6, PMS2). MSI status can be assessed by PCR-based microsatellite analysis or by immunohistochemistry for mismatch repair proteins. Studies have demonstrated >95% concordance for driver mutations between CRC tumors and derived organoids [7].
4. Personalized Therapy Applications
4.1 Chemotherapy Sensitivity Testing
CRC-PDOs have been extensively validated for predicting responses to standard chemotherapy regimens. In a landmark study, Smabers et al. demonstrated that the sensitivity of PDOs to 5-fluorouracil, irinotecan, and oxaliplatin was significantly correlated with the actual treatment response rates in CRC patients, with correlation coefficients of 0.58, 0.61, and 0.60, respectively [8].
A prospective biobank study of 50 CRC patients with liver metastasis confirmed that PDO sensitivity to FOLFOX and FOLFIRI regimens was associated with clinical response and progression-free survival. Patients with PDOs classified as resistant to oxaliplatin showed significantly shorter progression-free survival compared to sensitive patients (3.3 months vs. 10.9 months) [9].
4.2 Targeted Therapy and Anti-EGFR Response Prediction
Anti-EGFR monoclonal antibodies (cetuximab, panitumumab) are effective only in RAS and BRAF wild-type metastatic CRC. PDOs have been shown to accurately predict anti-EGFR sensitivity based on RAS/RAF mutational status. In a pooled analysis of 13 CRC-PDOs, RAS/RAF wild-type organoids achieved growth arrest in response to panitumumab, while RAS or BRAF mutant organoids showed persistent growth, consistent with clinical resistance mechanisms [10].
4.3 Immunotherapy and Microsatellite Instability
MSI-high CRC organoids exhibit high mutation burden and neoantigen load, and PDO-based co-culture systems with autologous tumor-infiltrating lymphocytes (TILs) have been used to assess immune checkpoint inhibitor responses. A patient-derived immunity-organoid platform demonstrated that REG4 is a predictive biomarker of immune checkpoint inhibitor resistance in MSS CRC, and that REG4 knockout in resistant organoids restored T-cell-mediated sensitivity [11].
4.4 Clinical Translation
The phase II clinical trial (NCT03544255) demonstrated the feasibility of using PDO drug sensitivity testing to guide treatment selection in metastatic CRC patients. The median progression-free survival was 67 days, and the median overall survival was 189 days, with some patients achieving partial remission after conventional chemotherapy failures. This trial provided proof-of-concept for integrating PDO-based screening into clinical decision-making workflows [12].
5. Advanced Applications: Gene Editing and Co-Culture Models
CRISPR-Cas9-mediated gene editing in CRC organoids enables the sequential introduction of oncogenic mutations (APC, KRAS, TP53, SMAD4, PIK3CA) to model adenoma-to-carcinoma progression. Drost et al. (2015) demonstrated that engineered mutations in human intestinal organoids recapitulated the morphological and transcriptomic changes observed in CRC, providing a stepwise model of tumor evolution [13].
Co-culture systems incorporating cancer-associated fibroblasts (CAFs), endothelial cells, and immune cells are being developed to better model the tumor microenvironment. These complex models are expected to improve the predictive accuracy of immunotherapy and anti-stromal agent testing [14].
Conclusion
Colorectal cancer organoids represent the most mature and clinically validated organoid platform. With high establishment success rates, validated drug screening protocols, and prospective clinical evidence supporting their predictive utility, CRC-PDOs are leading the translation of organoid technology into routine clinical practice for personalized oncology.