van Steenderen, D. | bioRxiv Science

bioRxiv2021

Functional single cell selection and annotated profiling of dynamically changing cancer cells

A method connecting single cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, would be transformative for single cell and cancer biology. Here, we present fSCS: functional single cell selection. This technology combines a custom-built ultrawide field-of-view optical screening microscope, fast automated image analysis and a new photolabeling method, phototagging, using a newly synthesized visible-light-photoactivatable dye. Using fSCS, we screen, selectively photolabel and isolate cells of interest from large heterogeneous populations based on functional dynamics like fast migration, morphological variation, small molecule uptake or cell division. We combined fSCS with single cell RNA sequencing for functionally annotated transcriptomic profiling of fast migrating and spindle-shaped MCF10A cells with or without TGF{beta} induction. We identified critical genes and pathways driving aggressive migration as well as mesenchymal-like morphology that could not be detected with state-of-the-art single cell transcriptomic analysis. fSCS provides a crucial upstream selection paradigm for single cell sequencing independent of biomarkers, allows enrichment of rare cells and can facilitate the identification and understanding of molecular mechanisms underlying functional phenotypes.

bioengineering↗

bioRxiv2019

Quantification of DNA methylation using methylation-sensitive restriction enzymes and multiplex digital PCR

Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one well-studied aspect of epigenetic regulation, but is challenging to quantify accurately. In this study, we introduce a digital approach for the absolute quantification of the amount, density and allele-specificity of DNA methylation. Combining the efficiency of methylation-sensitive restriction enzymes with the quantitative power of digital PCR, DNA methylation is measured accurately without the need to treat the DNA samples with sodium bisulphite. Moreover, as the combination of PCR amplicon and restriction enzyme is flexible, the context and density of DNA methylation can be taken into account. Additionally, by extending the experimental setup to a multiplex digital PCR, methylation markers may be analysed together with physically linked genetic markers to determine the allele-specificity of the methylation. In-silico simulations demonstrated the mathematical validity of the experimental setup. Next the approach was validated in a variety of healthy and malignant reference samples in the context of RASSF1A promotor methylation. RASSF1A is an established tumour suppressor gene, that is aberrantly methylated in many human cancers. A dilution series of well-characterized reference samples cross-validated the sensitivity and dynamic range of the approach. Compared to conventional PCR based methods, digital PCR provides a more accurate and more sensitive approach to quantify DNA methylation. As no sodium bisulphite conversion is needed, also analysis of minute amounts of DNA could be carried out efficiently.

genomics↗