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A novel inducible cell lineage tracing system for simultaneous fluorescence cell labeling and single-cell clonal reconstruction

ABSTRACT: A novel inducible cell lineage tracing system for simultaneous fluorescence cell labeling and single-cell clonal reconstruction

PROVIDER: PRJNA945461 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR23881569_1.fastq.gz	Fastqsanger.gz
	SRR23881569_2.fastq.gz	Fastqsanger.gz
	SRR23881570_1.fastq.gz	Fastqsanger.gz
	SRR23881570_2.fastq.gz	Fastqsanger.gz
	SRR23881571_1.fastq.gz	Fastqsanger.gz

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A novel inducible cell lineage tracing system for simultaneous fluorescence cell labeling and single-cell clonal reconstruction

Project description:In vivo clonal analysis utilizing recombinase-activated fluorescence cell labeling has become an essential method for visualizing stem cell activities and cell lineage formation. Recently, the simultaneous acquisition of cell lineage information and single-cell transcriptome data has further advanced our understanding of clonal cell evolution during development, tissue homeostasis, and disease progression. Here we present a novel inducible cell lineage tracing system, called TiTracer, which simultaneously activates fluorescence cell labeling and cellular barcode generation by transiently expressing template independent terminal transferase (TdT) and I-SceI homing endonuclease. We applied this system in HEK293T cells and primary mouse keratinocytes and performed single-cell RNA-seq for barcode recovery and clonal reconstruction.

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Project description:Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor, and surgical resection is a key part of the standard-of-care. In fluorescence-guided surgery (FGS), fluorophores are used to differentiate tumor tissue from surrounding normal brain. The heme synthesis pathway converts 5-aminolevulinic acid (5-ALA), a fluorogenic substrate, to the fluorophore protoporphyrin IX (PpIX). The resulting fluorescence is thought to be specific to transformed glioma cells, but this specificity has not been examined at single cell level. Here, we performed paired single cell imaging and RNA sequencing of individual cells (SCOPE-seq2) on human GBM surgical specimens with visible PpIX fluorescence from patients who received 5-ALA prior to surgery. SCOPE-seq2 allows us to simultaneously measure PpIX fluorescence by imaging and unambiguously identify transformed glioma cells from single-cell RNA-seq (scRNA-seq). We observed that 5-ALA treatment results in labeling that is not specific to transformed tumor cells. In cell culture experiments, we further demonstrated untransformed cells can be labeled by 5-ALA directly or by PpIX secreted from surrounding transformed cells. In acute slice cultures from mouse glioma models, we showed that 5-ALA preferably labels GBM tumor tissue over non-neoplastic brain tissue at bulk level, and that this contrast is not due to blood-brain-barrier disruption. Taken together, our findings support the use of 5-ALA as an indicator of GBM tissue, but not as a specific marker of transformed glioma cells.

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