PO.TB10.18 · 肿瘤生物学
通过序贯高多重免疫荧光解码体内模拟的3D人类胰腺CAF/ECM单元的表型和功能状态
Decoding phenotypic and functional states of in vivo -mimetic 3D human pancreatic CAF/ECM units through sequential high-plex IF
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：胰腺导管腺癌（PDAC）的特点是广泛的促结缔组织增生反应，癌症相关成纤维细胞（CAF）主导肿瘤微环境（TME）并驱动富含胶原的细胞外基质（ECM）沉积。虽然驻留的胰腺成纤维细胞发挥天然的肿瘤限制功能，但CAF/ECM单元采用肿瘤促进和肿瘤抑制两种表型，关键性地塑造肿瘤进展、治疗反应和患者结局。定义CAF/ECM表型特征的患者间异质性并理解其在治疗反应中的功能转变，对于预测临床结局、预测治疗反应以及识别新的TME靶向策略至关重要。
方法：我们开发了一种芯片上的人类PDAC TME，将新鲜组织来源的3D CAF/ECM单元整合到玻璃载玻片上的定制微流控腔室中培养。在COMET™平台上进行的高多重序贯免疫荧光（seqIF™）标准化了约20种间充质生物标志物，以区分肿瘤支持型和肿瘤限制型CAF表型。自动化图像分析（HORIZON™软件）量化了单细胞、亚细胞和细胞外标志物的表达模式。
结果：CAF激活状态干预揭示了经典TGFβ信号的显著改变、Ki67阳性细胞增殖的减少，以及ECM结构和组成的变化，反映了基质功能状态的转变。这一整合平台有效解析了CAF/ECM单元的异质性，并追踪了TME对治疗扰动的适应性反应。
结论：这一技术进步提供了一个稳健的中通量框架，用于剖析CAF异质性并探索PDAC（及其他癌症）中的基质生物学。与传统方法相比，该平台能够从最少的起始材料进行全面分析，同时大幅减少抗体消耗和处理时间，为在复杂TME中进行功能筛选和评估治疗干预建立了可扩展的基础。
查看英文原文 English abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia, with cancer-associated fibroblasts (CAFs) dominating the tumor microenvironment (TME) and driving collagen-rich extracellular matrix (ECM) deposition. While resident pancreatic fibroblasts exert natural tumor-restrictive functions, CAF/ECM units adopt both tumor-promoting and tumor-suppressive phenotypes, critically shaping tumor progression, therapeutic response, and patient outcomes. Defining inter-patient heterogeneity in CAF/ECM phenotypic traits and understanding their functional shifts in response to therapy is essential for predicting clinical outcomes, predicting therapy responses, and identifying new TME-targeting strategies.
Methods: We developed a human PDAC TME-within-a-chip integrating fresh tissue-derived 3D CAF/ECM units cultured in customized microfluidic chambers on glass slides. High-plex sequential immunofluorescence (seqIF™) on the COMET™ platform standardized ~20 mesenchymal biomarkers to distinguish tumor-supportive from tumor-restrictive CAF phenotypes. Automated image analysis (HORIZON™ software) quantified single-cell, subcellular, and extracellular marker expression patterns.
Results: CAF activation-state interventions revealed marked alterations in canonical TGFbeta signaling, reduced Ki67-positive cell proliferation, and changes in ECM architecture and composition, reflecting shifts in stromal functional states. This integrated platform effectively resolved CAF/ECM unit heterogeneity and tracked TME adaptations in response to therapeutic perturbation.
Conclusions: This technological advancement provides a robust, medium-throughput framework for dissecting CAF heterogeneity and exploring stromal biology in PDAC (and other cancers). The platform enables comprehensive profiling from minimal starting material while substantially reducing antibody consumption and processing time compared to conventional methods, establishing a scalable foundation for functional screening and evaluating therapeutic interventions in complex TMEs.
利益披露 Disclosure
M. Dmitrieva,
Lunaphore ).
E. Cukierman,
Lunaphore ).
J. Franco-Barraza,
Lunaphore ).