PO.TB10.06 · 肿瘤生物学
用于可视化肿瘤微环境中蛋白质相互作用的整合空间多组学流程
Integrated spatial multiomics pipeline for visualizing protein interactions in the tumor microenvironment
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
蛋白质-蛋白质相互作用是单个细胞与邻近细胞或细胞外基质进行通讯以调节组织环境的众多机制之一。使用空间平台可视化这些相互作用，可以在相关空间域内验证与疾病病理学相关的已知相互作用，尤其是在癌症中。我们开发了 ProximityScope™ 检测法，可与 RNAscope™ Multiomic LS 检测法配合使用，通过在单一组织切片上同时可视化蛋白质和 RNA 来检测蛋白质相互作用及其对分子通路的影响。该工作流程在 Leica Biosystems 的 BOND RX 染色平台上完全自动化。该检测法检测一个蛋白质-蛋白质相互作用，并可在同一切片上同时与五个 RNA/蛋白质靶标相结合。在此，我们通过染色 FFPE 人扁桃体组织样本以检测 PD1-PDL1 相互作用以及免疫细胞标志物（如 PDCD1 RNA、CD68 RNA、CD4 蛋白、CD8 蛋白和 PanCK 蛋白）来展示一个完整的端到端工作流程。使用 Leica Biosystems 的 Aperio FL 120 采集图像。使用 Indica Labs 的 HALO® 图像分析软件，我们进行了 RNA 和蛋白质定量以及细胞表型分析。成功可视化了免疫细胞与肿瘤细胞之间的 PD-1/PD-L1 相互作用。T 细胞通过 CD8 或 CD4 蛋白检测识别，巨噬细胞通过 CD68 RNA 识别，而肿瘤细胞通过 PanCK 蛋白染色识别。相互作用表现为两个细胞之间或细胞表面的点状小点或点簇。HALO 软件识别了相邻细胞之间的 PD-1/PD-L1 相互作用。本研究证明了拥有端到端空间解决方案对于靶标蛋白质相互作用的染色、成像和定量以评估蛋白质功能的重要性。ProximityScope 检测法有潜力研究广泛的蛋白质相互作用，以评估信号通路并获得生物学洞察、评估治疗成效，或检测可作为患者分层生物标志物的 PD-1/PD-L1 及相关相互作用。仅供研究使用。不用于诊断程序。
查看英文原文 English abstract
Protein-protein interaction is one of the many mechanisms where individual cells communicate with nearby cells or extracellular matrix to modulate the tissue environment. Visualizing these interactions using a spatial platform can validate known interactions implicated in disease pathology within relevant spatial domains, especially in cancer. We developed the ProximityScope™ assay that can be used with the RNAscope™ Multiomic LS assay to detect protein interactions and their impact on molecular pathways by simultaneously visualizing proteins and RNA on a single tissue section. This workflow is fully automated on Leica Biosystems' BOND RX staining platform. The assay detects one protein-protein interaction and can be combined with five RNA/protein targets simultaneously on the same section. Here, we demonstrate a complete end-to-end workflow by staining FFPE human tonsil tissue samples to detect PD1-PDL1 interactions along with immune cell markers such as PDCD1 RNA, CD68 RNA, CD4 protein, CD8 protein, and PanCK protein. Images were acquired using the Aperio FL 120 by Leica Biosystems.[AD1] Using the HALO® image analysis software from Indica Labs, we performed RNA and protein quantification as well as cell phenotyping. PD-1/PD-L1 interactions were successfully visualized between immune cells and tumor cells. T cells were identified by either CD8 or CD4 protein detection, macrophages were identified with CD68 RNA, while tumor cells were identified by PanCK protein staining. Interactions appear as punctate dots or dot clusters between two cells or on the cell surface. HALO software identified the PD-1/PD-L1 interaction between adjacent cells. This study demonstrates the importance of having an end-to-end spatial solution for staining, imaging, and quantification of target protein interactions to evaluate protein function. The ProximityScope assay has the potential to study a broad range of protein interactions to evaluate signaling pathways and gain biological insights, assess therapeutic success, or detect PD-1/PD-L1 and related interactions that can serve as biomarkers for patient stratification.For Research Use Only. Not for use in diagnostic procedures.
利益披露 Disclosure
A. Dikshit,
ACD, a Bio-techne brand Employment.
J. Brownlee,
ACD, a Bio-techne brand Employment.
G. Kim,
ACD, a Bio-Techne brand Employment.
S. Deshpande,
ACD, a Bio-Techne brand Employment.
D. Robirds,
Leica Biosystems Employment.
A. Hellebust,
Indica labs Employment.