PO.TB04.07 · 肿瘤生物学
开发患者来源的胰腺癌类器官-CAF共培养模型以研究肿瘤微环境对放疗反应的影响
Development of a patient-derived pancreatic cancer organoid-CAF coculture model to study tumor microenvironment influence on radiotherapy response
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：放疗（RT）是胰腺导管腺癌（PDAC）各疾病阶段的基础治疗方式。然而，由治疗耐药驱动的局部复发仍是改善患者预后的关键问题。虽然肿瘤细胞内在因素导致耐药，但有证据表明肿瘤微环境（TME），尤其是癌症相关成纤维细胞（CAF），通过不同机制发挥关键作用。目前的患者来源类器官（PDO）模型无法真实再现CAF-肿瘤相互作用及其对RT反应的影响，限制了其转化相关性。我们开发了一种共培养系统，以更好地模拟这些相互作用并实现具有生理相关性的放射生物学研究。
方法：将PDAC的PDO与原代CAF在超低吸附板中以3D悬浮方式共培养，添加300ug/mL的Matrigel。使用H&E、Masson三色染色、PAS-阿尔辛蓝染色以及针对vimentin、pan-cytokeratin、phalloidin和DAPI的免疫荧光（IF）对所得聚集体的结构完整性和组成进行表征。为检验该模型用于RT研究的效用，对共培养物施加单次8 Gy的辐射剂量。在治疗后12天，使用H&E、Ki-67和p16的免疫组织化学、IF（gamma-H2AX）以及calcein-AM/碘化丙啶活力测定评估放射生物学效应。
结果：所建立的方案生成了具有有序上皮-基质分区的聚集体，经IF和组织学分析证实。在单次8 Gy辐射剂量后，共培养物在治疗后前12天内表现出聚集体尺寸减小和细胞密度下降，与未治疗对照组相比生长动力学显著受损。辐照样品的组织学分析显示基质完整性破坏和细胞碎裂增加。升高的gamma-H2AX焦点表明DNA双链断裂，而Ki-67减少和p16染色增加则反映了增殖减少和衰老增加。
结论：本研究建立了患者来源的PDAC类器官与CAF的直接共培养模型，为在分区的肿瘤-基质环境中表征RT反应提供了平台。我们的研究结果揭示了分区特异性反应和差异化的生长动力学，凸显了整合的肿瘤-基质模型在研究RT效应和推进更具生理相关性的临床前方法方面的效用。
资助：ANID/FONDECYT基金#1241269和1221253，ANID/FONDAP基金#152220002。
查看英文原文 English abstract
Introduction: Radiotherapy (RT) is a fundamental treatment modality for pancreatic ductal adenocarcinoma (PDAC) at all disease stages. However, local relapse, driven by treatment resistance, remains a critical issue for improving patient outcomes. While tumor cell-intrinsic factors contribute to resistance, evidence indicates that the tumor microenvironment (TME), particularly cancer-associated fibroblasts (CAFs), plays a crucial role through different mechanisms. Current patient-derived organoid (PDO) models fail to authentically recapitulate CAF-tumor interactions and their influence on RT response, limiting their translational relevance. We developed a coculture system to better mimic these interactions and enable physiologically relevant radiobiological studies.
Methods: PDOs of PDAC and primary CAFs were cocultured in 3D suspension in ultra-low attachment plates with 300ug/mL of Matrigel. The structural integrity and composition of the resulting aggregates were characterized using H&E, Masson's trichrome, PAS-Alcian blue staining, and immunofluorescence (IF) for vimentin, pan-cytokeratin, phalloidin, and DAPI. To test the utility of this model for RT studies, cocultures were exposed to a single radiation dose of 8 Gy. Radiobiological effects were evaluated 12 days post-treatment using H&E, immunohistochemistry for Ki-67 and p16, IF (gamma-H2AX), and calcein-AM/propidium iodide viability assay.
Results: The established protocol generated aggregates with organized epithelial-stromal compartmentalization, confirmed by IF and histological analysis. Following a single 8 Gy radiation dose, cocultures exhibited reduced aggregate size and decreased cellular density within the first 12 days post-treatment, with significantly impaired growth kinetics compared to untreated controls. Histological analysis of irradiated samples showed disrupted matrix integrity and increased cellular fragmentation. Elevated gamma-H2AX foci indicated DNA double-strand breaks, while reduced Ki-67 and increased p16 staining reflected decreased proliferation and increased senescence.
Conclusion: This work establishes a direct coculture model of patient-derived PDAC organoids with CAFs, providing a platform to characterize RT response in a compartmentalized tumor-stroma context. Our findings reveal compartment-specific responses and differential growth dynamics, highlighting the utility of integrated tumor-stromal models for investigating the effects of RT and advancing toward more physiologically relevant preclinical approaches.
Funding: ANID/FONDECYT Grants #1241269 and 1221253, ANID/FONDAP Grant #152220002.
利益披露 Disclosure
D. Muñoz-Salazar, None..
C. Bizama, None..
P. Caprile, None..
F. Cabrera, None..
A. Castillo, None..
V. Manriquez, None..
M. Medina, None..
P. Munoz-Schuffenegger, None..
J. Roa, None..
P. Garcia, None.