Technology
Single nuclei RNA-Sequencing, Spatial Metabolomics, Spatial Transcriptomics and Proteomics
Single nuclei RNA-Sequencing
Single nuclei RNA-Sequencing (snRNA-seq) is used to determine the cell-specific transcriptome of a sample of interest where instead of whole cells, just the nuclei are isolated and sequenced. Given that it’s not necessary to keep the whole cell intact, snRNA-seq is preferred for tissues/samples that are not easily dissociated, like highly fibrotic or tumour samples. Furthermore, unlike single cell RNA-sequencing (scRNA-seq), snRNA-seq is compatible with frozen tissue and allows all the cells of the liver to be profiled avoiding the digestion bias typically associated with scRNA-seq. Following a new protocol adapted from Milteny Biotec, snap-frozen liver samples are homogenized to obtain a single nuclei suspension. The samples are then enriched for nuclei through the conjugation to anti-nuclei magnetic beads and run through an LS column, before being loaded onto a 10X Chromium chip for sequencing.
Spatial Metabolomics
Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is used to identify the spatial distribution of macromolecules, in this case specifically lipids, directly in the tissue of interest, with the use of a Bruker timsTOF fleX MALDI-2 instrument. Normally, the sample is embedded and cryosectioned. A slice of tissue is then covered by a thin layer of matrix and hit by a UV laser; this leads to the release of ions from the lipids present in the tissue, that are analysed based on their time-of-flight. Different lipid species are distinguished according to their m/z ratio and are visualized in a coloured image of the tissue. The use of timsTOF fleX MALDI-2 technology allows the identification of lipids at a near single cell resolution of 10µm.
Proteomics
Our preliminary studies and recent data indicate a strong transcriptional, epigenetic and post-translational dysregulation in particular protein tyrosine phosphatases in human HCC. To address the impact of the dysregulated PTPs we will take advantage of a proteomics approach, recently developed by PIs Gurzov & Wu (patent: PCT/EP2022/058188) and assess the PTP expression/oxidation in human liver resections. We will determine the impact of dysfunctional PTP activity and evaluate the association to the HCC development. We will define the expression/oxidation status of PTPs and global proteomics in human snap-frozen samples. Our patented approach allows us to define 1) the complete list of inactivated PTPs, 2) the degree of oxidation of individual PTPs and 3) the global proteomics profile of the sample. We have preliminary proteome data/targets from human liver samples showing reduced PTP expression in HCC samples but not global differences in the total phosphatome of the healthy liver/steatotic/MASH samples.