Biology subjects

Mak, C.

Publications and source records attributed to Mak, C..

3 recordsLinked to original sources

bioRxiv2026

Fluorescent Biosensors for Cryogenic Correlative Light and Electron Microscopy

Cryogenic electron tomography provides nanometer-scale views of subcellular structures but lacks essential physiological context, such as the metabolic state or ion concentration. To bridge this gap, we incorporated fluorescent biosensors into a cryogenic correlative light and electron microscopy workflow, enabling direct measurement of physiological conditions alongside structural data. Here, we focused on establishing and calibrating biosensors for calcium, pH, and osmotic stress at cryogenic conditions through an in vitro workflow in which biosensors were frozen in defined chemical environments and then imaged ratiometrically. We then applied these biosensors across a range of sample types: an osmotic stress sensor in Saccharomyces cerevisiae, a calcium sensor in human U2OS cells, and a pH sensor in the bacterium Halothiobacillus neapolitanus. This approach links localized physiological changes to the 3D structures of macromolecules and their organization in situ.

biophysics↗

bioRxiv2020

Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

BackgroundHigh throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity amongst complex human cancers. scRNA-Seq studies using fresh human surgical tissue is logistically difficult, precludes histopathological triage of samples and limits the ability to perform batch processing. This hinderance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers, and on high throughput scRNA-Seq platforms. ResultsWe show that the viable cryopreservation of human cancers provides high quality single-cell transcriptomes using the Chromium 10X platform. We sequenced a total of [~]120,000 cells from fresh and cryopreserved replicates across three breast cancers, two prostate cancers and a cutaneous melanoma. Importantly, tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We then show that cryopreservation had minimal impacts on results of downstream analyses such as biological pathway enrichment. Further, we demonstrate the advantage of cryopreserving whole-cells for immunophenotyping methods such as CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. ConclusionsOur study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

cancer biology↗

bioRxiv2020

Single-cell analysis reveals diverse stromal subsets associated with immune evasion in triple-negative breast cancer

The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single cell RNA-sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states, the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell-signalling predictions, we provide evidence that stromal-immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.

cancer biology↗