PaintScape™在完整新鲜冷冻胶质母细胞瘤组织的天然肿瘤微环境中实现单细胞空间3D基因组结构的多组学原位直接可视化

The 3D organization of genome structure, which plays a critical role in regulating gene expression and cellular function, is often restructured in many different cancers including Glioblastoma (GBM). Characterized by extreme molecular and phenotypic diversity, GBM is the most aggressive and lethal form of brain cancer and often show clonal and sub-clonal variations across genomic, epigenomic, transcriptomic, and proteomic signaling networks within distinct tumor microenvironments. Structural aberrations such as copy number variations (CNVs), structural variations (SVs), and topological changes rewire the 3D genome allowing aberrant activation of oncogenes, repression of tumor suppressors and dysregulation in gene expression. Current methods such as WGS or Hi-C provide bulk-level understanding of dysregulation but lack single-cell spatial resolution and do not provide insight into spatial distribution and association between molecular subclones and microenvironment in intact tissue. Since tumor heterogeneity drives therapy resistance and hinders successful GBM treatment, understanding the molecular mechanism in a heterogenous spatial context demands state-of-the-art in-situ single cell and spatial technologies that can provide deeper insight into early detection and disease progression. In this study, we present a novel multiomic jebFISH TM protocol on the PaintScape TM platform that can be used to analyze and directly visualize 3D chromatin architecture of fresh frozen GBM cells in different tissue microenvironments such as perinecrotic, perivascular, infiltrative, at single-cell, sub-population and population level. Using the ChromoPaint™ PanChromo MPX Panel, which visualizes over 400 loci across all chromosomes, we show disruption in 3D folding of individual chromosomes such as re-organization of chromosome territory, differential p-q arm interaction, intra and inter-chromosomal proximity at single cell and sub-population level in intact GBM tissue. By combining jebFISH with multiplexed immunofluorescence proteomic detection of hypoxia and microvasculature, we show how different tissue microenvironment e.g. hypoxic vs normoxic regions within the same GBM tissue section have different 3D genome aberrations and structural organization in the cancer cell sub-populations. We characterize chromosomal instability, CNVs and SVs of different GBM cancer cell and immune cell states at sub-chromosomal level in-situ within the tissue. The PaintScape system will enhance understanding of glioblastoma disease progression by providing deeper insight into the 3D genomic heterogeneity of single cancer cells and sub-populations with unique spatial context within intact tissue microenvironments, potentially guiding better cancer treatment design in the future.