Single cell chromatin accessibility profiling of human metastatic tumor cells

Metastasis is responsible for the vast majority of cancer deaths, yet the regulatory programs that enable metastatic tumor cells (MTCs) to survive, adapt, and expand in distant organs remain incompletely understood. Brain metastasis (BrM) is particularly lethal, marked by limited therapeutic options and profound resistance to existing treatments. Although chromatin accessibility orchestrates transcriptional states and cellular plasticity, its contribution to metastatic colonization has been largely unexplored.Here, we present the most comprehensive single-cell chromatin accessibility atlas of human BrM MTCs to date, spanning 15 metastases originating from diverse primary carcinomas. Following immune-cell depletion, nuclei were profiled using 10x Genomics single-cell ATAC-seq and analyzed with Cell Ranger ATAC, Signac, and Seurat, integrating inferred gene expression from published BrM datasets. We reconstructed gene regulatory networks (GRNs) with Pando to connect transcription factor activity, DNA accessibility, and downstream gene regulation.Across 49,907 high-quality single cells and 147,139 shared accessible peaks, we uncovered three dominant regulomes: two aligned with known inflammatory and proliferative programs, and a third non-proliferative, developmentally biased state not previously characterized in BrM or any other distant metastasis. We performed mechanistic validation of key markers from these modules, complemented by protein-level validation in human specimens. We also identified 22,242 conserved cis-regulatory elements (CREs), with 91% mapping to known human enhancers-yet with previously unknown implications for metastatic fitness. Notably, we discovered abundant CREs located in non-coding regions linked to developmental and mesodermal transcription factors, highlighting a core regulatory architecture underlying metastatic adaptation. This work provides an expansive reference map of the cis-regulatory landscape of human BrM and reveals previously unappreciated developmental regulatory circuits in MTCs, offering new avenues for therapeutic targeting in metastatic cancer.