PO.CL01.13 · 临床研究
使用 Ultivue InSituPlex 与无蛋白酶 RNAscope 混合工作流对实体瘤中抗体药物偶联物标志物和 RNA 生物标志物进行快速检测
Rapid antibody drug conjugate markers and RNA biomarkers in solid tumors using a hybrid Ultivue InSituPlex and Protease-Free RNAscope Workflow
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：肿瘤细胞、免疫浸润细胞与周围基质之间的空间关系——由蛋白表达和转录活性共同表征——是决定治疗反应和疾病预后的关键因素。传统的空间图谱分析方法往往需要连续切片，从而损害精确的细胞共定位，或采用会降解靶标完整性的严苛处理步骤。目前迫切需要稳健、高通量的单张切片方法，以高保真度共同检测蛋白和 RNA。
方法：我们开发并验证了一种新颖、精简的混合工作流，将 Ultivue 的高度多重化 InSituPlex 技术（蛋白检测）与 Advanced Cell Diagnostics（ACD）新开发的无蛋白酶 RNAscope™ 原位杂交（ISH）工作流相结合。这种配对至关重要：无蛋白酶 ISH 步骤可防止稳健的下游多重免疫荧光（mIF）蛋白染色所需的关键细胞表面表位发生降解。我们快速组装了一个全面的检测组合，通过 InSituPlex 纳入抗体药物偶联物的关键免疫肿瘤学蛋白标志物（如 HER2），并通过 ISH 共同检测相关转录靶标（如细胞因子 mRNA、治疗靶标转录本）。该检测应用于非小细胞肺癌（NSCLC）、三阴性乳腺癌（TNBC）和胃癌（GC）的 FFPE 组织切片，以证明其广泛适用性。
结果：我们成功在单张组织切片内、在全部三种异质性癌症类型中实现了多达 5 种蛋白标志物和 3 种 RNA 转录本的同步高分辨率共定位。无蛋白酶预处理保持了最佳的组织和表位形态，使蛋白和 RNA 通道均获得优异的信噪比。关键的是，这种组合的多组学读出能够对细胞群体进行精确的空间表型分析（如共表达特定细胞因子 mRNA 的 CD8+ T 细胞），并评估由蛋白/转录联合特征所定义的细胞之间的空间邻近性。Ultivue 检测的模块化特性使抗体组合能够快速组装和替换，从而实现高速的检测优化。
结论：这种创新的 Ultivue InSituPlex 与无蛋白酶 ACD ISH 联合检测提供了一个强大的单张切片平台，用于空间分辨的多组学分析。它克服了传统连续染色的技术局限，提供了一种高通量、高保真的工具，可快速部署用于复杂的生物标志物验证、详细的肿瘤微环境表征，以及改善转化肿瘤学和临床试验中的患者分层策略。
查看英文原文 English abstract
Introduction: The spatial relationships between tumor cells, immune infiltrates, and the surrounding stroma-characterized by both protein expression and transcriptional activity-are critical determinants of therapeutic response and disease prognosis. Traditional spatial profiling approaches often require serial sections, compromising exact cellular co-localization, or utilize harsh processing steps that degrade target integrity. There is an urgent need for robust, high-throughput, single-slide methods to co-detect protein and RNA with high fidelity.
Methods: We developed and validated a novel, streamlined hybrid workflow combining Ultivue's highly multiplexed InSituPlex technology (protein detection) with Advanced Cell Diagnostics' (ACD) newly developed protease-free capability with RNAscopeTM in situ hybridization (ISH) workflow. This pairing is crucial: the protease-free ISH step prevents the degradation of critical cell surface epitopes required for robust downstream multiplex immunofluorescence (mIF) protein staining. A comprehensive panel was rapidly assembled, including key immuno-oncology protein markers for antibody drug conjugates (ex. HER2) via InSituPlex, co-detected with relevant transcriptional targets (e.g., cytokine mRNA, therapeutic target transcripts) via ISH. The assay was applied to FFPE tissue sections from Non-Small Cell Lung Cancer (NSCLC), Triple-Negative Breast Cancer (TNBC), and Gastric Cancer (GC) to demonstrate broad applicability.
Results: We successfully achieved simultaneous, high-resolution co-localization of up to 5 protein markers and 3 RNA transcripts within a single tissue section across all three heterogeneous cancer types. The protease-free pre-treatment maintained optimal tissue and epitope morphology, enabling exceptional signal-to-noise ratios for both protein and RNA channels. Critically, this combined multi-omic readout allowed for the precise spatial phenotyping of cell populations (e.g., CD8+ T cells co-expressing specific cytokine mRNA) and the assessment of spatial proximity between cells defined by combined protein/transcriptional signatures. The modularity of the Ultivue assay allowed for rapid assembly and substitution of antibody panels, enabling high-speed assay optimization.
Conclusion: This innovative, combined Ultivue InSituPlex and protease-free ACD ISH assay provides a powerful, single-slide platform for spatially resolved multi-omic analysis. It overcomes the technical limitations of traditional sequential staining, offering a high-throughput, high-fidelity tool that can be rapidly deployed for complex biomarker validation, detailed tumor microenvironment characterization, and improved patient stratification strategies in translational oncology and clinical trials.
利益披露 Disclosure
P. Savickas, None.