胰腺导管腺癌（PDAC）由于其复杂的生物学和侵袭行为，仍然是一种具有挑战性的疾病，迫切需要有效的治疗策略。为了评估治疗反应，需要基于 PDAC 类器官的临床前模型与准确的实时监测相结合。我们建立了稳定的实时成像类器官/外周血单核细胞 (PBMC) 共培养物，并引入了 OrganoIDNet，一种深度学习网络基于算法，​​能够分析通过活细胞成像获得的小鼠和人类患者来源的 PDAC 类器官的明场图像。我们研究了在有或没有 HLA 匹配 PBMC 的情况下随时间培养的 PDAC 类器官和 PD-L1 抑制剂 Atezolizumab 对化疗吉西他滨的反应。使用 OrganoIDNet 获得的结果通过终点增殖测定 CellTiter-Glo 进行了验证。活细胞成像与 OrganoIDNet 相结合，可以准确地检测类器官随时间推移对吉西他滨的尺寸特异性药物反应，表明较大的类器官更容易产生细胞毒性作用。这种方法还可以区分健康和不健康状态，并测量类器官对治疗反应的偏心率。此外，基于新型类器官/PBMC 三明治共培养物的成像能够对类器官对 Atezolizumab 的反应进行纵向分析，表明添加 Atezolizumab 后，PBMC 以类器官个体方式杀灭肿瘤的效力增强。优化的 PDAC 类器官成像分析OrganoIDNet 代表了一个能够准确检测类器官对标准 PDAC 化疗随时间的反应的平台。此外，类器官/免疫细胞共培养可以监测类器官对免疫治疗的反应，为与 PBMC 共培养环境中的治疗行为提供动态洞察。该设置有望实时评估个体患者来源的 PDAC 类器官的免疫治疗效果。© 2024。作者。

Pancreatic Ductal Adenocarcinoma (PDAC) remains a challenging disease due to its complex biology and aggressive behavior with an urgent need for efficient therapeutic strategies. To assess therapy response, pre-clinical PDAC organoid-based models in combination with accurate real-time monitoring are required.We established stable live-imaging organoid/peripheral blood mononuclear cells (PBMCs) co-cultures and introduced OrganoIDNet, a deep-learning-based algorithm, capable of analyzing bright-field images of murine and human patient-derived PDAC organoids acquired with live-cell imaging. We investigated the response to the chemotherapy gemcitabine in PDAC organoids and the PD-L1 inhibitor Atezolizumab, cultured with or without HLA-matched PBMCs over time. Results obtained with OrganoIDNet were validated with the endpoint proliferation assay CellTiter-Glo.Live cell imaging in combination with OrganoIDNet accurately detected size-specific drug responses of organoids to gemcitabine over time, showing that large organoids were more prone to cytotoxic effects. This approach also allowed distinguishing between healthy and unhealthy status and measuring eccentricity as organoids' reaction to therapy. Furthermore, imaging of a new organoids/PBMCs sandwich-based co-culture enabled longitudinal analysis of organoid responses to Atezolizumab, showing an increased potency of PBMCs tumor-killing in an organoid-individual manner when Atezolizumab was added.Optimized PDAC organoid imaging analyzed by OrganoIDNet represents a platform capable of accurately detecting organoid responses to standard PDAC chemotherapy over time. Moreover, organoid/immune cell co-cultures allow monitoring of organoid responses to immunotherapy, offering dynamic insights into treatment behavior within a co-culture setting with PBMCs. This setup holds promise for real-time assessment of immunotherapeutic effects in individual patient-derived PDAC organoids.© 2024. The Author(s).