In-situ direct single-cell visualization of 3D genome architecture in ER+ and HER2+ breast cancer cell lines using PaintScape TM system

Breast cancer (BC) is highly heterogeneous, and classification based on ER, PR, and HER2 expression alone does not fully capture its clinical diversity or therapeutic vulnerabilities. In particular, the aggressive HER2 positive (HER2+) and estrogen receptor positive (ER+) subtypes are driven by complex genomic and cellular dynamics-including epithelial to mesenchymal transition (EMT), chromosomal instability, and 3D genome reorganization that contribute to metastasis, therapy resistance, and poor prognosis. Bulk studies such as WGS and Hi-C have revealed key genomic features in BC sub-types such as focal amplifications (e.g. CCND1-HER2 co-amplification in HER2+ BC; Chr 20q alterations in ER+ BC), A/B compartment switching, TAD boundary disruptions and associated oncogenic rewiring. However, these approaches remain ex-situ and bulk-based obscuring the spatial and cellular heterogeneity and fail to capture the full spectrum of structural and functional interactions within individual cells which is critical to understanding disease progression. To overcome these limitations, in-situ single-cell technologies capable of resolving dynamic 3D genome changes are essential for uncovering resistance mechanisms and enabling precision therapies tailored to the unique genomic landscape of each tumor cell. Here we present the PaintScape™ system enabling simultaneous in-situ visualization of the 3D genome structure in single BC cells. Over 1,000 targets relevant in several important cancer pathways including Cell Cycle and Apoptosis, Transcriptional Regulation and Chromatin Structure are visualized across all chromosomes using our OncoPaint™ Oncogenic Pathways Panel in ER+ MCF7 and HER2+ HCC 1954 cell lines. We identified common and cell line specific unique 3D genome alterations between MCF7, HCC 1954 and other breast cell lines, and relate these to patterns of genomic rearrangement inferred from patient samples. In ER+ MCF 7, we identified distributions in copy gain/loss of specific chromosome arms e.g. Chr 20q, individual targets including regions of clustered breakpoints, unique ecDNA and distal estrogen response elements (DEREs), at the single cell, and sub-population level. In HER2+ HCC 1954, we identified focal amplification of Chr 8q and directly visualized ecDNA amplification, rearrangement and nuclear localization of key oncogenes such as MYC and CCND1. We simultaneously show A/B compartment switching and TAD boundary disruption of key oncogenic regions in MCF7 and HCC 1954 in-situ, showing consistency with up/down regulation of genes between the cell lines. The PaintScape system enables simultaneous in-situ 3D genome mapping within single cells revealing structural differences linked to gene dysregulation across BC sub-types, offering an integrated view of how genome architecture influences cellular processes during disease progression.