PO.IM02.07 · 免疫学
克服胰腺导管腺癌免疫耐药：微流控预激的类器官相互作用淋巴细胞在患者来源模型中显示出更优的浸润、细胞毒性和肿瘤抑制作用
Overcoming pancreatic ductal adenocarcinoma immunoresistance: Microfluidic-primed organoid interacting lymphocytes demonstrate superior infiltration, cytotoxicity, and tumor suppression in patient-derived models
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：胰腺导管腺癌（PDAC）由于其免疫抑制性肿瘤微环境（TME）而对免疫治疗持续无反应，该微环境以致密的促结缔组织增生、免疫排斥和T细胞耗竭为特征。5年生存率<13%，亟需新的策略。我们开发了一个平台，利用能够再现PDAC TME的免疫功能健全的患者来源类器官（PTO）来生成和评估肿瘤特异性细胞毒性淋巴细胞。
方法：通过将切除的PDAC组织与自体肿瘤引流淋巴结细胞共培养，建立了免疫功能健全的PTO（iPTO）。通过微流控肿瘤芯片循环对自体PBMC进行预激，生成类器官相互作用淋巴细胞（OIL）。OIL扩增14天，并以5×和10×的效应细胞:靶细胞比例与患者匹配的PTO共培养。使用高分辨率延时显微镜结合自动单细胞追踪、流式细胞术（CD45、CK19、活力）以及96小时内的纵向肿瘤面积测量来量化肿瘤-免疫动态。
结果：流式细胞术显示强劲的免疫浸润，OIL处理培养物中CD45+细胞占26%，而仅肿瘤对照组为0.5%。OIL诱导了93%的肿瘤细胞死亡，而未刺激的PBMC为68%。定量追踪显示，在24小时内OIL向肿瘤细胞迁移靠近6.1像素，而PBMC为0.6像素（P<0.05）。在27小时时，OIL处理的类器官表现出一致的肿瘤面积缩小（-2%至-13%），而PBMC处理的孔则显示出可变的反应，包括肿瘤扩张（+7%）。纵向分析证实活OIL可持续抑制肿瘤，而热灭活的OIL则允许肿瘤扩张15倍，证明了细胞毒性依赖于活力。延时显微镜捕捉到直接杀伤事件、免疫突触形成以及与免疫逃逸一致的肿瘤聚集。这些发现重现了在阑尾肿瘤和间皮瘤模型中的观察结果，即OIL的表现优于肿瘤浸润淋巴细胞。
结论：OIL代表了一种强效的、患者特异性的适应性细胞治疗，能够克服PDAC的免疫抑制性TME。这一免疫功能健全的类器官平台能够对个性化免疫治疗进行定量实时评估，并支持基于OIL的疗法在胰腺癌中的临床转化。
查看英文原文 English abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) remains refractory to immunotherapy due to its immunosuppressive tumor microenvironment (TME), characterized by dense desmoplasia, immune exclusion, and T cell exhaustion. With 5-year survival <13%, novel strategies are urgently needed. We developed a platform to generate and evaluate tumor-specific cytotoxic lymphocytes using immunocompetent patient-derived organoids (PTOs) that recapitulate the PDAC TME.
Methods: Immunocompetent PTOs (iPTOs) were established by co-culturing resected PDAC tissue with autologous tumor-draining lymph node cells. Autologous PBMCs were primed via microfluidic tumor-on-a-chip circulation, generating Organoid Interacting Lymphocytes (OILs). OILs were expanded for 14 days and co-cultured with patient-matched PTOs at 5× and 10× effector:target ratios. Tumor-immune dynamics were quantified using high-resolution time-lapse microscopy with automated single-cell tracking, flow cytometry (CD45, CK19, viability), and longitudinal tumor area measurements over 96 hours.
Results: Flow cytometry demonstrated robust immune infiltration, with CD45+ cells comprising 26% of OIL-treated cultures versus 0.5% in tumor-only controls. OILs induced 93% tumor cell death compared to 68% with unstimulated PBMCs. Quantitative tracking revealed OILs migrated 6.1 pixels closer to tumor cells versus 0.6 pixels for PBMCs over 24 hours (P<0.05). At 27 hours, OIL-treated organoids exhibited consistent tumor area reduction (-2% to -13%), while PBMC-treated wells showed variable responses including tumor expansion (+7%). Longitudinal analysis confirmed sustained suppression with live OILs, whereas heat-killed OILs permitted 15-fold tumor expansion, demonstrating viability-dependent cytotoxicity. Time-lapse microscopy captured direct killing events, immune synapse formation, and tumor clustering consistent with immune evasion. These findings replicate observations in appendiceal and mesothelioma models where OILs outperformed tumor-infiltrating lymphocytes.
Conclusions: OILs represent a potent, patient-specific adaptive cell therapy capable of overcoming PDAC's immunosuppressive TME. This immunocompetent organoid platform enables quantitative real-time assessment of personalized immunotherapies and supports clinical translation of OIL-based therapies for pancreatic cancer.
利益披露 Disclosure
E. Makris, None..
D. Hutchins, None..
T. Liu, None..
A. Hall, None..
L. Miller, None..
S. Soker, None..
K. Votanopoulos, None.