Tracking intractable single-cell fusion events via two-color DNA barcoding platform

CMDuo is a DNA barcoding system using two colors to monitor individual cell fusion in cancer cells. It enables detailed tracking and analysis of how cells merge, helping clarify tumor behavior and identify potential treatment targets.

Background

The study of cell-cell fusion is a critical area in cancer research, particularly concerning triple-negative breast cancer (TNBC). Understanding fusion events at the single-cell level is essential for unraveling tumor evolution and intra-tumoral heterogeneity. Accurate tracking and analysis of these fusion events provides valuable insights into how cancer cells interact, evolve, and develop resistance to therapies. This knowledge is pivotal for developing targeted treatments and improving patient outcomes, as fusion events can significantly influence tumor behavior and progression.

However, current methodologies face significant challenges in accurately identifying and tracking cell-cell fusion events. Traditional approaches often underestimate fusion rates because they struggle to distinguish true fusion cells from mere cell doublets without specific markers. Additionally, existing techniques lack the precision needed for single-cell resolution analysis, making it difficult to capture the nuanced transcriptomic changes associated with fusion events. Technical limitations, such as the absence of antibiotic selection, further complicate the process by necessitating multiple rounds of fluorescence-activated cell sorting (FACS). These shortcomings hinder comprehensive understanding and effective investigation of fusion-related mechanisms in cancer progression.

Technology description

CMDuo is a two-color, two-index DNA barcoding platform engineered to monitor cell-cell fusion events at single-cell resolution. It integrates unique barcode sequences with nuclear-localized histone H2B fused to fluorescent proteins GFP or mCherry, facilitating live-cell imaging and accurate cell counting. The system employs distinct index sequences to distinguish authentic fusion events from cell doublets in single-cell RNA sequencing (scRNA-seq) data.

When applied to triple-negative breast cancer cell lines, CMDuo identified varying fusion tendencies among clones, revealed distinct transcriptomic clusters, and highlighted the upregulation of pathways related to actin, actomyosin, chromatin remodeling, and DNA repair. This approach effectively addresses the challenge of identifying fusion cells without the need for antibiotic selection.

CMDuo differentiates itself through its precise dual-color, dual-index barcoding system, which offers unparalleled accuracy in identifying genuine cell fusion events compared to traditional methods. This level of specificity prevents the common issue of conflating fusion events with cell doublets, ensuring more reliable data in scRNA-seq analyses.

Additionally, CMDuo’s ability to reveal detailed transcriptomic landscapes and associated gene signatures provides deeper insights into the molecular mechanisms of cancer progression and tumor heterogeneity. Its application to triple-negative breast cancer underscores its potential in uncovering therapeutic targets and advancing cancer research. The platform's innovative design and robustness make it a valuable tool for studying complex cellular interactions and their implications in disease contexts.

Benefits

Tracks cell-cell fusion events at single-cell resolution
Differentiates true fusion events from cell doublets in single-cell RNA sequencing
Reveals varying fusion propensities among cancer cell clones
Identifies distinct transcriptomic clusters associated with fusion
Enables live-cell imaging and accurate cell counting
Facilitates discovery of fusion-associated therapeutic targets
Enhances understanding of tumor evolution and intratumoral heterogeneity