Abstract 789: PaintScapeTM enables in situ, single cell spatial multiomic visualization of 3D genome organization in fresh frozen colorectal carcinoma tissue in spatially resolved tissue microenvironments.

Abstract Intratumoral heterogeneity (ITH) continues to be one of the major challenges in successful cancer treatments causing poor prognosis, therapeutic resistance, metastasis and relapses across many human cancer types. This heterogeneity largely originates from the complex tumor immune microenvironment, where cancer and immune cells engage in intricate signaling crosstalk, shaped by clonal and sub-clonal variations in genomic, transcriptomic, and proteomic networks across cancer and immune cells. In recent years, disruptions in 3D genome architecture, including CNVs, SVs, and topological changes, have been shown to drive oncogene activation, tumor suppressor silencing, and gene expression dysregulation in many different cancers. While methods like WGS and Hi-C offer bulk-level insights into genomic dysregulation, they disrupt native tissue architecture and lack the single cell nuclear examination, limiting the ability to analyze spatial relationships and organizational features essential for understanding cellular context and heterogeneity. Given its emerging role in therapeutic resistance and poor prognosis, understanding the molecular mechanism behind high ITH demands advanced single-cell and spatial technologies for early detection and disease monitoring. Here, we present a novel multiomic jebFISHTM protocol on the PaintScapeTM platform that can be used to characterize ITH of 3D chromatin architecture of fresh frozen normal human colon tissue and colorectal cancer (CRC) samples in different tissue microenvironments at single cell, sub-population and population level. Using the ChromoPaint™ PanChromo MPX Panel, which visualizes over 400 genomic loci across all chromosomes per nucleus, we show chromosomal instability including CNVs such as gain in Chr20q, loss in Chr17p, copy loss of Chr 14, SVs such as Chr13-Chr20q translocation and complex rearrangement of Chr13 in different colon cancer cell sub-populations in situ within the tissue. These genomic aberrations were specific to histologically cancerous sections and were not detected in the normal region of the tissue. By combining the jebFISH protocol with multiplexed immunofluorescence, we characterize 3D genome organization of cancer cell sub-populations with unique cell states along with associated proteomic signatures e.g. Pan-CK+ CDH1- and Pan-CK+ CDH1+ subpopulations in different tissue microenvironments. We also visualized genome structure in different tumor-immune microenvironments, e.g. M2 macrophage rich vs CD3+ T-cells rich regions within the same CRC tissue section. In-situ multiomic capability of the PaintScape platform enhances understanding of colon cancer progression and reveals 3D genomic heterogeneity of single cells and sub-populations in their spatial context in native tissue microenvironments. Citation Format: Shyamtanu Chattoraj, Pamela Flatley, Stacy Elliott, David Castillo, Kenny Chung, Doug Werner, Jude Dunne, Huy Nguyen. PaintScapeTM enables in situ, single cell spatial multiomic visualization of 3D genome organization in fresh frozen colorectal carcinoma tissue in spatially resolved tissue microenvironments abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 789.