Biology subjects

Cheow, L. F.

Publications and source records attributed to Cheow, L. F..

7 recordsLinked to original sources

bioRxiv2026

DynaKiller-Scan: Multimodal characterization of single-cell killing dynamics via combinatorial encoding of target lysis

Cytotoxic lymphocytes are crucial for eradicating infected or cancerous cells. To comprehensively characterize their killing capacity at single-cell level is challenging. Here we introduce DynaKiller-Scan, a high-throughput platform utilizing DNA barcodes to simultaneously interrogate the killing dynamics and molecular underpinnings of individual effector cells. DynaKiller-Scan reveals variable kinetics in serial killing between CD4+ and CD8+ CAR-T killers. The highly heterogeneous killing behaviors are associated with distinct molecular signatures orchestrating cell activation, secretion, migration, and metabolic fitness. While rapid killing involves cells with stronger type-I cytokine secretion, persistent killing is accompanied with more of type-II cytokine response. Furthermore, gene expression patterns closely linked to serial-killing were identified. DynaKiller-Scan could be a valuable tool for improving the development of precision immunotherapy.

immunology↗

bioRxiv2024

Microfluidic-enabled production of DNA barcoded APC library (MEDAL) for high throughput T cell epitope screening

Screening for peptide fragments that can be displayed on antigen-presenting cells is an essential step in vaccine development. The current approach for this process is slow and costly as it involves separately pulsing cells with chemically synthesized peptides. We present Microfluidic-Enabled production of DNA-barcoded APC Library (MEDAL), a high throughput microfluidic droplet platform for parallel production of DNA-barcoded APCs loaded with enzymatically synthesized peptides. Droplets containing peptides and their encoding DNA are produced from microfluidic PCR-IVTT reaction. APCs presenting both peptides and DNA barcodes are obtained by injecting cells into these droplets. Up to 9,000 different APCs can be produced and screened within a 10-hour workflow. This approach allowed us to identify peptide sequences that bind to APCs expressing H-2Kb MHC class 1 molecule with next-generation sequencing of DNA barcodes.

immunology↗

bioRxiv2024

Automated high-throughput profiling of single-cell total transcriptome with scComplete-seq

Detecting the complete portrait of the transcriptome is essential to understanding the roles of both polyadenylated and non-polyadenylated RNA species. However, current efforts to investigate the heterogeneity of the total cellular transcriptome in single cells are limited by the lack of an automated, high-throughput assay that can be carried out on existing platforms. To address this issue, we developed scComplete-seq, a method that can easily augment existing high-throughput droplet-based single-cell mRNA sequencing to provide additional information on the non-polyadenylated transcriptome. Using scComplete-seq, we have successfully detected long and short non-polyadenylated RNAs at single-cell resolution, including cell-cycle-specific histone RNAs, cell-type-specific short non-coding RNA, as well as enhancer RNAs in cancer cells and PBMCs. By applying scComplete-seq, we have identified changes in both coding and non-coding transcriptome in PBMCs during different stimulations. Measuring the enhancer RNA expression also revealed the activation of specific biological processes and the transcription factors regulating such changes.

genomics↗

bioRxiv2024

Deciphering the function of intrinsic and genomics-driven epigenetic heterogeneity in head and neck cancer progression with single-nucleus CUT&RUN

Interrogating regulatory epigenetic alterations during tumor progression at the resolution of single cells has remained an understudied area of research. Here we developed a highly sensitive single-nucleus CUT&RUN (snCUT&RUN) assay to profile histone modifications in isogenic primary, metastatic, and cisplatin-resistant head and neck squamous cell carcinoma (HNSCC) patient-derived tumor cell lines. We find that the epigenome can be involved in diverse modes to contribute towards HNSCC progression. First, we demonstrate that gene expression changes during HNSCC progression can be co-modulated by alterations in both copy number and chromatin activity, driving epigenetic rewiring of cell-states. Furthermore, intratumour epigenetic heterogeneity (ITeH) may predispose sub-clonal populations within the primary tumour to adapt to selective pressures and foster the acquisition of malignant characteristics. In conclusion, snCUT&RUN serves as a valuable addition to the existing toolkit of single-cell epigenomic assays and can be used to dissect the functionality of the epigenome during cancer progression.

cancer biology↗

bioRxiv2021

Time-resolved assessment of single-cell protein secretion by sequencing

Secreted proteins play critical roles in cellular communication and functional orchestration. Methods enabling concurrent measurement of cellular protein secretion, phenotypes and transcriptomes are still unavailable. Here, we describe time-resolved assessment of protein secretion from single cells by sequencing (TRAPS-seq). Released proteins are trapped onto cell surface via affinity matrices, and the captured analytes together with phenotypic markers can be probed by oligonucleotide-barcoded antibodies and simultaneously sequenced with transcriptomes. We used TRAPS-seq to interrogate secretion dynamics of pleiotropic cytokines (IFN-{gamma}, IL-2 and TNF-) of early activated human T lymphocytes, unraveling limited correlation between cytokine secretion and its transcript abundance with regard to timing and strength. We found that early central memory T cells with CD45RA expression (TCMRA) are the most effective responders in multiple cytokine secretion, and polyfunctionality involves unique yet dynamic combinations of gene signatures over time. TRAPS-seq presents a useful tool for cellular indexing of secretions, phenotypes, and transcriptomes at single-cell resolution.

immunology↗

bioRxiv2021

Heatrich-BS enables efficient CpG enrichment and highly scalable cell-free DNA methylation profiling

Genome wide analysis of cell-free DNA (cfDNA) methylation profile has been shown to be a promising approach for sensitive and specific multi-cancer detection. However, scaling these assays for clinical translation is impractical due to the high cost of whole genome bisulfite sequencing. We showed that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbored the majority of cancer-specific methylation signature. Here, we report on the simple but effective Heat enrichment of CpG-rich regions for Bisulfite Sequencing (Heatrich-BS) platform that enables focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable high accuracy and sensitivity in tumor burden estimation and quantitative monitoring of colorectal patient response to treatment, at much reduced sequencing requirement. Heatrich-BS holds great potential for highly scalable screening and regular monitoring of cancer using liquid biopsy.

genomics↗

bioRxiv2020

Label-free lymphocytes reconstitution using side scatter for optimal T cell manufacturing

Lymphocyte biology research commonly involves purification of lymphocyte subpopulations by fluorescence-activated cell sorting (FACS) or immunomagnetic separation (IMS), both of which typically rely on antibody labeling of validated cell markers. Methods enabling label-free segregation of lymphocyte subpopulations would be invaluable with regard to less-perturbation, simplicity and cost-effectiveness. Here, we introduce TRuST, a label-free approach for T cell reconstitution using side-scatter (SSC). TRuST-sorted SSClow cells enrich for CD4+ T and naive T cells, while SSChigh cells enrich for CD8+ T, NK and differentiated T cells. Enrichment purity can be improved by computational gate design. SSClow cells have superior expansion capacity and generate more central memory precursors with naive-resembling cytokine responses. Moreover, we find that both T cell differentiation status and CD4/CD8 T ratio in the starting cellular material are critical attributes predicting T cell product quality and quantity. TRuST presents an effective and reliable technique for label-free lymphocytes selection and reconstitution.

immunology↗