Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.10.28.762523

Gene context shapes spatial organization and transcriptional heterogeneity of extrachromosomal DNA in cancer cells

Frank, L.·Marinopoulou, G.·Medema, R. H.
View original source ↗Export metadata ↓

Abstract

Extrachromosomal DNA (ecDNA) promotes tumor adaptability by making oncogenic driver genes variably abundant, spatially mobile, and heterogeneously expressed. Transcriptional hyperactivation and spatial mobility of ecDNA-amplified genes is associated with aberrant regulatory states that could create targetable vulnerabilities in ecDNA-driven cancers. However, how gene context shapes their nuclear positioning and aberrant transcription remains unclear. To explore spatio-transcriptional behavior across ecDNA-amplified genes, we developed FLAGx, a multimodal imaging method that quantitatively resolves transcriptional activity and nuclear position of individual ecDNA genes in situ. Surprisingly, we find that gene context affects subnuclear distribution and transcriptional deregulation of ecDNA. Further, ecDNA clustering, cell cycle regulation, and drug responses depend on gene context, while transcriptional shutdown in mitosis persists across ecDNA species, arguing against pervasive mitotic transcription as a common feature. These findings show that driver gene context shapes ecDNA behavior and establish FLAGx as a useful approach to separate gene-specific from shared ecDNA features.

Explore related subjects

cancer biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Frank, L., Marinopoulou, G., Medema, R. H.. 2026-10-29. Gene context shapes spatial organization and transcriptional heterogeneity of extrachromosomal DNA in cancer cells. https://doi.org/10.64898/2026.10.28.762523

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

C19 trityl and silyl andrographolide analogs exhibit in vitro anticancer activity in murine cancer models

Andrographolide, a labdane diterpenoid isolated from A. paniculata has been investigated for activity in a broad panel of biological contexts, including its anti-inflammatory, antiviral, and anticancer properties. Previously, A-ring analogs of andrographolide bearing silyl and trityl ethers at C19 have demonstrated improved in vitro anticancer activity in human cancer cell lines, with putative activity through the Wnt1/{beta}-catenin signaling axis. In order to investigate the activity of this series of C19 andrographolide analogs, alongside a cyclic A-ring acetonide analog, as anticancer agents in murine cancer cell models, we evaluated antiproliferative activity of these compounds in B16F10 melanoma, CT26 colorectal adenocarcinoma, Neuro2a neuroblastoma, and 4T1 mammary ductal carcinoma cell lines, and found that most compounds exhibited potent micromolar level anticancer activity. Among these, all trityl ethers exhibited nearly indistinguishable IC50 values of 4-20 M, and generally exhibited greater potency compared to C19 silyl ether analogs. This anticancer activity was further validated through apoptosis flow cytometry, wherein we concluded that our compounds induced apoptosis to a greater extent than the parent compound, andrographolide. Further, we examine the role of modulation of canonical Wnt1/{beta}-catenin signaling in a series of co-delivery assays with CHIR-99021, a selective GSK-3{beta} kinase inhibitor, and MSAB, a selective {beta}-catenin degrader. Finally, we demonstrate that a selection of silyl ether analogs of andrographolide elicit cell cycle arrest by flow cytometry in a manner that is decoupled to in vitro potency trends. Altogether, we report the first in vitro evaluation of trityl and silyl ether analogs of andrographolide within murine adherent cancer cells.

cancer biology↗
bioRxiv2026

Identification of TM4SF1 as a novel tumor-associated antigen in biliary tract cancers targetable by CAR-T cell therapy

Background & Aims: Biliary tract cancers (BTCs) are a rare set of genetically heterogenous and aggressive malignancies associated with delayed presentation and poor prognosis. Thus, there is an unmet clinical need for the development of novel therapeutic strategies. Identification of uniquely expressed cell-surface, tumor-associated antigens (TAAs) holds promise in epithelial tumors more generally, as they can serve as ligands for a variety of therapeutics, including CAR-T cell therapy. However, TAA identification in BTCs has been limited by expression in normal liver tissue. Here, we aim to characterize the cell-surface protein transmembrane 4 L six family member 1 (TM4SF1) as a potential BTC TAA targetable by CAR-T therapy. Methods: TM4SF1 expression in BTC relative to normal hepatic/biliary tissue was characterized at the mRNA level by analyzing TCGA datasets and at the protein level by immunohistochemistry of archival primary BTC tissue. Anti-TM4SF1 CAR-T cells were evaluated through in vitro coculture assays against human-derived BTC cell lines and in a cell line-derived xenograft (CDX) model. Results: We find TM4SF1 is significantly upregulated in BTC relative to normal hepatic/biliary tissue and across BTC anatomical subtypes at the mRNA and protein level. To validate TM4SF1 as a targetable TAA in BTC, we show anti-TM4SF1 CAR-T cells demonstrate robust growth inhibition of BTC cell lines in vitro and significant anti-tumor activity against a CDX in vivo. Conclusions: Together, our data provides support for TM4SF1 as a promising TAA in BTCs that can be used for the rational development of targeted therapeutic modalities, including CAR-T cells, in a patient population with significant unmet need.

cancer biology↗
bioRxiv2026

Evolution of immune infiltration and immune triad formation across early melanoma progression

Tumor-infiltrating lymphocytes (TIL) in melanoma are a clinically important prognostic marker and are routinely used to inform patient care. However, when immune recognition of transformed melanocytes begins and how it evolves during early melanoma progression is not well understood. Using spatial transcriptomics and a unique cohort of patient-matched lesions representing multiple stages of early evolution, we investigate T cells, dendritic cells (DCs) and multi-cell immune "triads" during melanoma initiation. We find that T cell and DC density, triad density, and triad proximity to melanoma cells are elevated in regions of Stage I invasive melanoma as compared to Stage 0 in situ lesions and nevi. Further, we find that triad abundance correlates with both T cell and DC density, but is not predicted by clustering patterns of these cell types over larger spatial scales. We conclude that immune recognition of melanoma and engagement with the lesion significantly increases with progression from Stage 0 to Stage I disease. Further, using nevi that are depth-matched in the dermis to Stage I invasive lesions, we demonstrate this increased immune recognition is not a consequence of dermal invasion alone. This points to a paradigm where immune engagement of invasive melanoma reflects recognition of melanoma cell-intrinsic changes that emerge in Stage I disease.

cancer biology↗