Abstract B037: ClonMapper Duo: An scRNAseq compatible lineage tracing method to track cell-cell fusion at single-cell resolution

Abstract

Abstract Spontaneous cell-cell fusion in cancer can generate genetic heterogeneity and often leads to more pathological phenotypes. While the consequences of cell-cell fusion in cancer continue to be revealed, few tools exist to determine the mechanisms and cell states which promote cell-cell fusion. To meet this need, we engineered ClonMapper Duo, a two-color, two-index expressed barcoding system which allows tracking of cell-cell fusion using live-cell imaging and lineage tracing of fusion cells in scRNAseq. The ClonMapper Duo system was created by modifying the ClonMapper DNA barcoding system to express indexed barcodes on either H2B-GFP or H2B-mCherry. Briefly, 8 unique 5-nucleotide sequences were designed by maximizing Hamming distance and 4 sequences were assigned to GFP and mCherry each. The index sequences were placed adjacent to a 15N random nucleotide in the expressed barcode region of the construct. Low-diversity GFP and mCherry barcoded cell libraries were created through lentiviral transduction of HCC1806, MDA-MB-231, and MCF-7 cells. Diversity and evenness of each library was confirmed via targeted barcode sequencing. For each cell line, homotypic GFP and mCherry barcoded cells were cocultured together for 1 week. A parallel culture was monitored using live-cell imaging to measure the rate of spontaneous cell-cell fusion and the probability of different fates (e.g. death, hybrid formation, senescence) for each cell line. Spontaneously fused cells identified by expression of both GFP and mCherry were enriched by FACS. Enriched fusion cells were expanded in parallel with the parental cocultures until ~1e5 fusion cells or 1 week was reached. Fusion and parental populations for each cell line were multiplexed for scRNAseq using the 10X Genomics 3’ CellPlex Kit. FACS enriched multibarcoded fusion cells were further bioinformatically distinguished from cell multiplets and the barcodes of spontaneously fused cells were traced to their single-barcoded cell lineage of origin. From this data set we ask: 1. Are certain cell lineages more primed for fusion than others, i.e. does a more fusogenic cell phenotype exist or is fusion random? 2. Is there a convergent state of post-fusion cells or does fusion tend to diversify phenotypes? Here, we will present the first results from this two-color, two-index live-cell imaging and scRNAseq lineage tracing system toward understanding cancer cell-cell fusion at a single cell resolution. Combining lineage traced transcriptomic data from scRNAseq with fusion rates and outcomes from live-cell imaging using ClonMapper Duo provides an unprecedented look into the causes and consequences of spontaneous cell-cell fusion in cancer. Citation Format: Andrea L. Gardner, Lan Zheng, Daylin Morgan, Kennedy Howland, Amy Brock. ClonMapper Duo: An scRNAseq compatible lineage tracing method to track cell-cell fusion at single-cell resolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B037.