PO.TB10.18 · 肿瘤生物学
用于胰腺壶腹周围腺癌共培养系统开发的PDTO与CAF的建立与表征
Establishment and characterisation of PDTOs and CAFs for the development of a co-culture system in pancreatic periampullary adenocarcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
壶腹周围腺癌（PAC）是一组多样化的恶性肿瘤，起源于胰头（60%）、Vater壶腹（20%）、远端胆总管（10%）和十二指肠（10%）。胰腺肿瘤微环境（TME）是肿瘤进展和治疗耐药的主要驱动因素，其中癌症相关成纤维细胞（CAF）作为核心贡献者日益受到关注。CAF代表TME内的一个异质性群体，通过与肿瘤细胞以及免疫和基质成分的复杂相互作用，主动介导基质重塑。对CAF表型的详细表征对于明确其对PAC病理生物学的影响、并促进针对这一高度侵袭性恶性肿瘤的新型治疗策略设计至关重要。从新鲜获取的原发性PAC标本中分离出患者来源肿瘤类器官（PDTO）和CAF。通过对关键CAF蛋白进行免疫荧光染色和转录组测序来表征CAF亚型。通过基因组测序表征PDTO。用talazoparib和olaparib对PDTO进行药物筛选，以检验HRD突变与对PARP抑制剂敏感性之间的相关性。成功地从PAC标本中衍生并培养了4个PDTO和4个CAF。CAF细胞系显示成纤维细胞标志物alpha-SMA和vimentin阳性表达，并表现出CAF标志物（如PDGFR和FAP）的异质性强度和表达。根据关键基因的RNA表达，它们被分类为抗原呈递型（apCAF）、炎症型（iCAF）和肌成纤维细胞型（myCAF）亚组。myCAF根据CTGF、COL1A1、POSTN和ACTA2基因的表达水平进行分组。选择CD74和HLA基因用于表征apCAF，而iCAF则根据IL-1/6/11、FAP和CXCL1/2基因的表达进行表征。对PDTO的突变谱分析在一例肠型腺癌中鉴定出错义ATM变异c.5558A>T（p.Asp1853Val），该肿瘤对olaparib（1.66 µM）和talazoparib（0.45 µM）敏感。PDTO和CAF可作为体外研究PAC细胞结构和异质性的有用模型。在此基础上，将评估已定义的CAF异质性与免疫抑制型和免疫刺激型特征之间的关联，并将T细胞与免疫治疗药物一并纳入共培养体系。这些模型将为PAC肿瘤-基质-免疫生物学提供宝贵的见解，并为测试靶向TME的策略的高级共培养奠定基础。
查看英文原文 English abstract
Periampullary adenocarcinoma (PAC) constitutes a diverse group of malignancies originating from the pancreatic head (60%), the ampulla of Vater (20%), the distal common bile duct (10%), and the duodenum (10%). The pancreatic tumour microenvironment (TME) is a major driver of tumour progression and therapeutic resistance, with cancer-associated fibroblasts (CAFs) emerging as central contributors. Representing a heterogeneous population within the TME, CAFs actively mediate stromal remodelling through complex interactions with tumour cells as well as with immune and stromal components. Detailed characterisation of CAF phenotypes is essential to define their impact to PAC pathobiology and to facilitate the design of novel therapeutic strategies against this highly aggressive malignancy.Patient-derived tumour organoids (PDTOs) and CAFs were isolated from freshly obtained primary PAC specimens. CAFs subtypes were characterised by immunofluorescence staining for key CAF proteins and transcriptomic sequencing. PDTOs were characterised by genomic sequencing. Drug screening of PDTOs with talazoparib and olaparib was conducted to examine the correlation between an HRD mutation and sensitivity to PARP inhibitors.4 PDTOs and 4 CAFs were successfully derived and cultured from PAC specimens. CAF cell lines showed positive expression of fibroblast markers alpha-SMA and vimentin, and showed heterogeneous intensity and expression of CAF markers such as PDGFR and FAP. They were categorised into antigen-presenting (apCAFs), inflammatory (iCAFs) and myofibroblastic (myCAFs) subgroups based on the RNA expression of key genes. MyCAFs were grouped based on the expression levels of CTGF, COL1A1, POSTN and ACTA2 genes. CD74 and HLA genes were chosen for the characterisation of apCAFs, while iCAFs were characterised based on the expression of IL-1/6/11, FAP and CXCL1/2 genes. Mutational profiling of the PDTOs identified a missense ATM variant c.5558A>T (p.Asp1853Val) in an intestinal-type adenocarcinoma, which was sensitive to olaparib (1.66 µM) and talazoparib (0.45 µM).PDTOs and CAFs serve as a useful model for studying the cellular architecture and heterogeneity of PAC in vitro. Building on this, defined CAF heterogeneity will be assessed for its association with immunosuppressive versus immunostimulatory profiles, and T cells, along with immunotherapeutic agents, will be incorporated into the co-cultures. These models will provide valuable insights into PAC tumour-stroma-immune biology and form the basis for advanced co-cultures to test strategies targeting the TME.
利益披露 Disclosure
P. Sarametidis, None..
J. Stoof, None..
L. Ivers, None..
J. Murphy, None..
N. Swan, None..
L. Benigno, None..
F. Hand, None..
K. C. Conlon, None..
M. Lowery, None.