PO.CL01.14 · 临床研究
Direct-Seq™实现在FFPE组织中以亚细胞分辨率对IgH和TCRbeta转录本进行空间分辨的原位测序
Direct-Seq™ enables spatially resolved in situ sequencing of IgH and TCRbeta transcripts in FFPE tissue at subcellular resolution
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
空间多组学正在变革我们对肿瘤微环境、免疫反应和疾病机制的理解。然而，在以单细胞和亚细胞分辨率原位测序高度可变的转录本区域方面仍存在关键空白，而这对于在天然组织背景下绘制B细胞和T细胞克隆型至关重要。
在此，我们展示了Direct-Seq™，一种建立在G4X™原位多组学平台之上的新型原位测序方法，能够对RNA可变区进行高分辨率分析。Direct-Seq采用靶向IgH和TCRbeta转录本中多样化CDR3结构域两侧V区和J区的探针，从而实现对J区和CDR3区的测序。该工作流程整合了原位逆转录、扩增和合成测序化学。
我们将Direct-Seq应用于5 µm的FFPE扁桃体和肾细胞癌（RCC）切片以及10 µm的新鲜冷冻扁桃体切片。在FFPE扁桃体中最多可分析9%的B细胞，在新鲜冷冻组织中可分析约20%的B细胞和T细胞。检测到广泛的CDR3多样性，携带高度相似CDR3序列的克隆扩增B细胞定位于生发中心内，展示了Direct-Seq的高分辨率克隆分型能力。
在肾细胞癌（RCC）中，九张连续的FFPE肾脏切片揭示了富集于肿瘤区域周围的克隆扩增B细胞群体以及具有持续扩增的T细胞克隆。一个占主导地位的T细胞克隆在连续切片中被一致检出，凸显了该方法的稳健性及其在空间上追踪克隆群体的能力。
Direct-Seq在同一切片上与多重蛋白检测相结合，证实了转录本身份与蛋白表型之间的空间一致性。
综上所述，这些结果确立了Direct-Seq作为一种高分辨率、可扩展的方法，用于在FFPE和新鲜冷冻组织中进行空间免疫组库绘制，从而能够对克隆性、免疫结构和转化免疫学进行详细研究。
查看英文原文 English abstract
Spatial multi-omics is transforming our understanding of the tumor microenvironment, immune responses, and disease mechanisms. A critical gap remains in the ability to sequence highly variable transcript regions in situ at single-cell and subcellular resolution, essential for mapping B- and T-cell clonotypes in their native tissue context.
Here, we present Direct-Seq™, a novel in situ sequencing approach built on the G4X™ in situ multiomic platform, enabling high-resolution profiling of RNA variable regions. Direct-Seq employs probes targeting the V and J regions flanking the diverse CDR3 domain of IgH and TCRbeta transcripts, allowing sequencing of both the J and CDR3 regions. The workflow integrates in situ reverse transcription, amplification, and sequencing-by-synthesis chemistry.
We applied Direct-Seq to 5-µm FFPE tonsil and renal cell carcinoma (RCC) sections and 10-µm fresh frozen tonsil sections. Up to 9% of B cells were profiled in FFPE tonsil, and ~20% of B and T cells in fresh frozen tissue. Extensive CDR3 diversity was detected, with clonally expanded B cells carrying highly similar CDR3 sequences localized within germinal centers, demonstrating the high-resolution clonotyping capacity of Direct-Seq.
In Renal Cell Carcinoma (RCC), nine serial FFPE kidney sections revealed clonally expanded B-cell populations enriched around tumor regions and T-cell clones with persistent expansion. One dominant T-cell clone was consistently detected across serial sections, highlighting the method's robustness and ability to track clonal populations spatially.
Direct-Seq was combined with multiplexed protein detection on the same sections, confirming spatial concordance between transcript identity and protein phenotype.
Together, these results establish Direct-Seq as a high-resolution, scalable approach for spatial immune repertoire mapping in both FFPE and fresh frozen tissue, enabling detailed studies of clonality, immune architecture, and translational immunology.
利益披露 Disclosure
M. Lawson, None..
A. Tsue, None..
N. Ing, None.