PO.ET08.01 · 实验与分子治疗
微卫星不稳定性作为结直肠癌放化疗敏感性的决定因素：整合临床、影像组学与三维类器官分析
Microsatellite instability as a determinant of chemoradiotherapy sensitivity in colorectal cancer: Integrated clinical, radiomic, and 3D organoid-based analyses
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摘要 Abstract
中文摘要
背景：微卫星不稳定性（MSI）是一个影响免疫治疗结局的既定生物标志物，并可能影响放化疗（CRT）后的DNA损伤应答。虽然临床研究报道MSI高（MSI-H）结直肠肿瘤的CRT结局良好，但差异放射敏感性的生物学基础仍未完全阐明。为填补这一空白，我们将临床-影像组学分析与使用来源于MSI-H和微卫星稳定（MSS）结直肠癌（CRC）细胞系的三维类器官模型的功能性分析相整合。
方法：分析了87例接受新辅助CRT的直肠癌患者的临床数据。MSI状态通过PCR以及MLH1、MSH2、MSH6和PMS2的免疫组织化学确定。从CRT前的CT图像提取影像组学特征，并训练一个机器学习模型对MSI状态进行分类。为进行生物学验证，将4株MSI-H细胞系（HCT116、HCT15、RKO、DLD-1）和5株MSS细胞系（HT29、CACO2、SW480、COLO205、COLO320DM）进行分级照射。将细胞包埋于70% Matrigel或BME中以生成三维类器官。使用基于Calcein-AM的活性面积分析、基于ATP的三维CellTiter-Glo检测以及在0-8 Gy范围内的曲线下面积（AUC）量化来评估放射应答。
结果：MSI肿瘤仅见于应答者组（18.52%），而在非应答者中缺失（P<0.05）。应答者的病理降期（T0-T2）显著更高（88.89% vs. 38.33%，P<0.001），淋巴结清除率增加（N0；92.59% vs. 61.67%，P=0.003）。在三维类器官模型中，MSI-H细胞系表现出比MSS细胞系明显更强的放射敏感性。MSI-H类器官在Calcein-AM和ATP三维检测中均表现出更强的生长抑制和更低的活性，导致放射应答AUC值降低。相比之下，MSS类器官在照射后维持较高的活性和代谢活性，表明相对的放射抗性。
结论：MSI-H结直肠癌在临床结局、影像组学特征和基于三维类器官的功能性检测中一致地表现出更高的放化疗敏感性。在MSI-H类器官中观察到的增强放射敏感性提示DNA错配修复缺陷可能促进对放射诱导损伤的易感性增加，尽管确切的分子通路尚未完全明确。这些整合的发现支持将MSI检测与基于类器官的功能性分析相结合以完善患者分层并指导结直肠癌个体化CRT方法的潜在价值。需要在更大规模和种族多样化的队列中进行验证以确认其普适性。
查看英文原文 English abstract
Background: Microsatellite instability (MSI) is an established biomarker affecting immunotherapy outcomes and may influence DNA damage responses following chemoradiotherapy (CRT). While clinical studies have reported favorable CRT outcomes in MSI-high (MSI-H) colorectal tumors, the biological basis for differential radiosensitivity remains incompletely understood. To address this gap, we integrated clinical-radiomic analyses with functional assays using 3D organoid models derived from MSI-H and microsatellite-stable (MSS) colorectal cancer (CRC) cell lines.
Methods: Clinical data from 87 rectal cancer patients receiving neoadjuvant CRT were analyzed. MSI status was determined by PCR and immunohistochemistry for MLH1, MSH2, MSH6, and PMS2. Radiomic features were extracted from pre-CRT CT images, and a machine learning model was trained to classify MSI status. For biological validation, 4 MSI-H (HCT116, HCT15, RKO, DLD-1) and 5 MSS cell lines (HT29, CACO2, SW480, COLO205, COLO320DM) were subjected to graded irradiation. Cells were embedded in 70% Matrigel or BME to generate 3D organoids. Radiation responses were evaluated using Calcein-AM-based viability area analysis, ATP-based 3D CellTiter-Glo assays, and area-under-curve (AUC) quantification across 0-8 Gy.
Results: MSI tumors were exclusively found in the responder group (18.52%) and absent in non-responders (P<0.05). Pathological downstaging (T0-T2) was significantly higher in responders (88.89% vs. 38.33%, P<0.001), with increased nodal clearance (N0; 92.59% vs. 61.67%, P=0.003). In 3D organoid models, MSI-H cell lines displayed markedly greater radiosensitivity than MSS lines. MSI-H organoids showed stronger growth inhibition and lower viability in both Calcein-AM and ATP 3D assays, resulting in reduced radiation-response AUC values. In contrast, MSS organoids maintained higher viability and metabolic activity after irradiation, indicating relative radioresistance.
Conclusion: MSI-H colorectal cancer demonstrated consistently higher chemoradiotherapy sensitivity across clinical outcomes, radiomic signatures, and 3D organoid-based functional assays. The enhanced radiosensitivity observed in MSI-H organoids suggests that deficiencies in DNA mismatch repair may contribute to increased susceptibility to radiation-induced damage, although the precise molecular pathways have not yet been fully defined. These integrated findings support the potential value of combining MSI testing with organoid-based functional profiling to refine patient stratification and guide personalized CRT approaches in colorectal cancer. Validation in larger and ethnically diverse cohorts will be necessary to confirm generalizability.
利益披露 Disclosure
H. Hong, None.