PO.CH02.02 · 化学
使用HCR™ Gold IF和临床相关抗体克隆进行自动化、灵活的多重免疫荧光肿瘤微环境分析
Automated, flexible multiplex immunofluorescence for tumor microenvironment profiling using HCR™ Gold IF and clinically-relevant antibody clones
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摘要 Abstract
中文摘要
背景：多重免疫荧光（multiplexed immunofluorescence, mIF）是肿瘤学研究的核心，但常常受到固定面板、抗体修饰需求、剧烈剥脱以及有损表位风险的工作流程的限制。由HCR™ HiFi编码器（HiFi Encoders）赋能的HCR™ Gold IF通过提供无酶扩增以及对未修饰的兔IgG和小鼠IgG1一抗的通用兼容性，解决了这些限制。在此，我们评估了一套完整、可互操作的工作流程，该流程结合了在Leica Biosystems的BOND RX染色仪上运行的自动化HCR™ Gold IF、经验证的Abcam抗体，以及使用Indica Lab的HALO平台进行的定量图像分析，用于肿瘤微环境（TME）分析。
方法：来自健康组织和肿瘤的FFPE组织切片在BOND RX染色仪上通过HCR™ Gold IF进行染色。面板包括上皮、增殖、免疫和检查点标志物（如panCK、Ki67、CD3、CD8、CD68、PD-1、PD-L1）。所有一抗均获自Abcam，并在多重组装前经单重实验鉴定合格。切片用全玻片扫描仪成像，并在HALO®平台中进行分析，以定量生物标志物表达并探究细胞群体之间的空间关系。
结果：这一自动化、无蛋白酶的工作流程产生了高信噪比，具有清晰的亚细胞定位和干净的通道分离，跨越各种组织和靶标。与未修饰的Abcam抗体的通用兼容性简化了面板设计并最大限度地缩短了检测开发时间。单重与多重的一致性达到了预定义的验收标准，表明表位掩蔽或通道串扰极小。在各肿瘤队列中，我们观察到与匹配的健康组织相比预期的、可量化的TME变化，包括肿瘤/髓系区室上PD-L1升高，以及肿瘤-基质界面处CD8+和CD68+密度增加。
结论：将由HiFi编码器赋能的HCR™ Gold IF整合到BOND RX染色仪上，并在HALO平台中进行分析，提供了一套自动化、即插即用的mIF解决方案，将卓越的无酶扩增与真正的"自带抗体"灵活性相结合。这一端到端工作流程降低了定制化、高重数TME研究的门槛，保护了组织完整性，并能够标准化地生成空间生物标志物，以助力免疫肿瘤学研究和患者分层。该方法可轻松扩展到更大的面板，以及RNA/蛋白联合检测，以获得更深入的多组学背景。仅供研究使用。不用于诊断程序。
查看英文原文 English abstract
Background: Multiplexed immunofluorescence (mIF) is central to oncology research but is often limited by fixed panels, antibody modification requirements, harsh stripping, and workflows that risk epitope damage. HCR™ Gold IF enabled by HCR™ HiFi Encoders addresses these constraints by providing enzyme-free amplification with universal compatibility to unmodified rabbit IgG and mouse IgG1 primaries. Here, we evaluated a complete, interoperable workflow combining automated HCR™ Gold IF on Leica Biosystems' BOND RX staining instrument, validated Abcam antibodies, and quantitative image analysis using Indica Lab's HALO platform for tumor microenvironment (TME) profiling.
Methods: FFPE tissue sections from healthy tissues and tumors were stained via HCR™ Gold IF on the BOND RX staining instrument. Panels included epithelial, proliferation, immune, and checkpoint markers (e.g., panCK, Ki67, CD3, CD8, CD68, PD-1, PD-L1). All primary antibodies were obtained from Abcam and qualified in a single-plex experiment prior to multiplex assembly. Slides were imaged with whole-slide scanners and analyzed in the HALO® platform to quantify biomarker expression and interrogate spatial relationships between cell populations.
Results: The automated, protease-free workflow generated high signal-to-background with crisp subcellular localization and clean channel separation across tissues and targets. Universal compatibility with unmodified Abcam antibodies streamlined panel design and minimized assay development time. Single-plex to multiplex concordance met predefined acceptance criteria, indicating minimal epitope masking or channel cross-talk. Across tumor cohorts, we observed expected and quantifiable TME shifts versus matched healthy tissues, including elevated PD-L1 on tumor/myeloid compartments and increased CD8+ and CD68+ densities at tumor-stroma interfaces.
Conclusions: Integrating HCR™ Gold IF, powered by the HiFi Encoders, on the BOND RX staining instrument and analyzing in the HALO platform delivers an automated, plug-and-play mIF solution that couples superior, enzyme-free amplification with true bring-your-own-antibody flexibility. This end-to-end workflow lowers barriers to custom, high-plex TME studies, preserves tissue integrity, and enables standardized generation of spatial biomarkers to inform immune-oncology research and patient stratification. The approach is readily extensible to larger panels and to combined RNA/protein co-detection for deeper, multi-omic context. For Research Use Only. Not for use in diagnostic procedures.
利益披露 Disclosure
W. Yang, None..
R. Chen, None..
H. Choi, None..
A. Acharya, None..
W. Howat, None.