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Tackling Tumour Cell Heterogeneity at the Super-Resolution Level in Human Colorectal Cancer Tissue.

ABSTRACT: Tumour cell heterogeneity, and its early individual diagnosis, is one of the most fundamental problems in cancer diagnosis and therapy. Single molecule localisation microscopy (SMLM) resolves subcellular features but has been limited to cultured cell lines only. Since nuclear chromatin architecture and microRNAs are critical in metastasis, we introduce a first-in-field approach for quantitative SMLM-analysis of chromatin nanostructure in individual cells in resected, routine-pathology colorectal carcinoma (CRC) patient tissue sections. Chromatin density profiles proved to differ for cells in normal and carcinoma colorectal tissues. In tumour sections, nuclear size and chromatin compaction percentages were significantly different in carcinoma versus normal epithelial and other cells of colorectal tissue. SMLM analysis in nuclei from normal colorectal tissue revealed abrupt changes in chromatin density profiles at the nanoscale, features not detected by conventional widefield microscopy. SMLM for microRNAs relevant for metastasis was achieved in colorectal cancer tissue at the nuclear level. Super-resolution microscopy with quantitative image evaluation algorithms provide powerful tools to analyse chromatin nanostructure and microRNAs of individual cells from normal and tumour tissue at the nanoscale. Our new perspectives improve the differential diagnosis of normal and (metastatically relevant) tumour cells at the single-cell level within the heterogeneity of primary tumours of patients.

SUBMITTER: Lang F

PROVIDER: S-EPMC8345115 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Investigating native human cardiac tissue with preserved 3D macro- and microarchitecture is fundamental for clinical and basic research. Unfortunately, the low accessibility of the human myocardium continues to limit scientific progress. To overcome this issue, utilizing atrial appendages of the human heart may become highly beneficial. Atrial appendages are often removed during open-heart surgery and can be preserved ex vivo as living tissue with varying durability depending on the culture method. In this study, we prepared living thin myocardial slices from left atrial appendages that were cultured using an air-liquid interface system for overall 10 days. Metabolic activity of the cultured slices was assessed using a conventional methyl thiazolyl tetrazolium (MTT) assay. To monitor the structural integrity of cardiomyocytes within the tissue, we implemented our recently described super-resolution microscopy approach that allows both qualitative and quantitative in-depth evaluation of sarcomere network based on parameters such as overall sarcomere content, filament size and orientation. Additionally, expression of mRNAs coding for key structural and functional proteins was analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR). Our findings demonstrate highly significant disassembly of contractile apparatus represented by degradation of [Formula: see text]-actinin filaments detected after three days in culture, while metabolic activity was constantly rising and remained high for up to seven days. However, gene expression of crucial cardiac markers strongly decreased after the first day in culture indicating an early destructive response to ex vivo conditions. Therefore, we suggest static cultivation of living myocardial slices derived from left atrial appendage and prepared according to our protocol only for short-termed experiments (e.g. medicinal drug testing), while introduction of electro-mechanical stimulation protocols may offer the possibility for long-term integrity of such constructs.

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Tackling Tumour Cell Heterogeneity at the Super-Resolution Level in Human Colorectal Cancer Tissue.

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