Head and neck squamous cell carcinomas (HNSCC) belong among severe and highly complex malignant diseases showing a high level of heterogeneity and consequently also a variance in therapeutic response, regardless of clinical stage. Our study implies that the progression of HNSCC may be supported by cancer-associated fibroblasts (CAFs) in the tumour microenvironment (TME) and the heterogeneity of this disease may lie in the level of cooperation between CAFs and epithelial cancer cells, as communication between CAFs and epithelial cancer cells seems to be a key factor for the sustained growth of the tumour mass. In this study, we investigated how CAFs derived from tumours of different mRNA subtypes influence the proliferation of cancer cells and their metabolic and biomechanical reprogramming. We also investigated the clinicopathological significance of the expression of these metabolism-related genes in tissue samples of HNSCC patients to identify a possible gene signature typical for HNSCC progression. We found that the right kind of cooperation between cancer cells and CAFs is needed for tumour growth and progression, and only specific mRNA subtypes can support the growth of primary cancer cells or metastases. Specifically, during coculture, cancer cell colony supporting effect and effect of CAFs on cell stiffness of cancer cells are driven by the mRNA subtype of the tumour from which the CAFs are derived. The degree of colony-forming support is reflected in cancer cell glycolysis levels and lactate shuttle-related transporters.

• Keywords
• HNSCC, cancer, cancer-associated fibroblasts, cell stiffness, tumour microenvironment,
• Publication type
• Journal Article MeSH

Cell viscoelastic properties are affected by the cell cycle, differentiation, and pathological processes such as malignant transformation. Therefore, evaluation of the mechanical properties of the cells proved to be an approach to obtaining information on the functional state of the cells. Most of the currently used methods for cell mechanophenotyping are limited by low robustness or the need for highly expert operation. In this paper, the system and method for viscoelasticity measurement using shear stress induction by fluid flow is described and tested. Quantitative phase imaging (QPI) is used for image acquisition because this technique enables one to quantify optical path length delays introduced by the sample, thus providing a label-free objective measure of morphology and dynamics. Viscosity and elasticity determination were refined using a new approach based on the linear system model and parametric deconvolution. The proposed method allows high-throughput measurements during live-cell experiments and even through a time lapse, whereby we demonstrated the possibility of simultaneous extraction of shear modulus, viscosity, cell morphology, and QPI-derived cell parameters such as circularity or cell mass. Additionally, the proposed method provides a simple approach to measure cell refractive index with the same setup, which is required for reliable cell height measurement with QPI, an essential parameter for viscoelasticity calculation. Reliability of the proposed viscoelasticity measurement system was tested in several experiments including cell types of different Young/shear modulus and treatment with cytochalasin D or docetaxel, and an agreement with atomic force microscopy was observed. The applicability of the proposed approach was also confirmed by a time-lapse experiment with cytochalasin D washout, whereby an increase of stiffness corresponded to actin repolymerization in time.

• MeSH
• Cytochalasin D MeSH
• Elastic Modulus MeSH
• Neoplasms * MeSH
• Elasticity MeSH
• Reproducibility of Results MeSH
• Viscosity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytochalasin D MeSH

The incidence of death caused by cancer has been increasing worldwide. The growth of cancer cells is not the main problem. The majority of deaths are due to invasion and metastasis, where cancer cells actively spread from primary tumors. Our inbred rat model of spontaneous metastasis revealed dynamic phenotype changes in vitro correlating with the metastatic potential in vivo and led to a discovery of a metastasis suppressor, protein 4.1B, which affects their 2D motility on flat substrates. Subsequently, others confirmed 4.1B as metastasis suppressor using knock-out mice and patient data suggesting mechanism involving apoptosis. There is evidence that 2D motility may be differentially controlled to the 3D situation. Here we show that 4.1B affects cell motility in an invasion assay similarly to the 2D system, further supporting our original hypothesis that the role of 4.1B as metastasis suppressor is primarily mediated by its effect on motility. This is encouraging for the validity of the 2D analysis, and we propose Quantitative Phase Imaging with incoherent light source for rapid and accurate testing of cancer cell motility and growth to be of interest for personalized cancer treatment as illustrated in experiments measuring responses of human adenocarcinoma cells to selected chemotherapeutic drugs.

• MeSH
• Neoplasm Invasiveness * MeSH
• Rats MeSH
• Humans MeSH
• Microscopy methods   MeSH
• Cell Line, Tumor MeSH
• Cell Movement * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

In this paper, a novel U-Net-based method for robust adherent cell segmentation for quantitative phase microscopy image is designed and optimised. We designed and evaluated four specific post-processing pipelines. To increase the transferability to different cell types, non-deep learning transfer with adjustable parameters is used in the post-processing step. Additionally, we proposed a self-supervised pretraining technique using nonlabelled data, which is trained to reconstruct multiple image distortions and improved the segmentation performance from 0.67 to 0.70 of object-wise intersection over union. Moreover, we publish a new dataset of manually labelled images suitable for this task together with the unlabelled data for self-supervised pretraining.

• Publication type
• Journal Article MeSH

Emerging flaviviruses are causative agents of severe and life-threatening diseases, against which no approved therapies are available. Among the nucleoside analogues, which represent a promising group of potentially therapeutic compounds, fluorine-substituted nucleosides are characterized by unique structural and functional properties. Despite having first been synthesized almost 5 decades ago, they still offer new therapeutic opportunities as inhibitors of essential viral or cellular enzymes active in nucleic acid replication/transcription or nucleoside/nucleotide metabolism. Here, we report evaluation of the antiflaviviral activity of 28 nucleoside analogues, each modified with a fluoro substituent at different positions of the ribose ring and/or heterocyclic nucleobase. Our antiviral screening revealed that 3'-deoxy-3'-fluoroadenosine exerted a low-micromolar antiviral effect against tick-borne encephalitis virus (TBEV), Zika virus, and West Nile virus (WNV) (EC50 values from 1.1 ± 0.1 μM to 4.7 ± 1.5 μM), which was manifested in host cell lines of neural and extraneural origin. The compound did not display any measurable cytotoxicity up to concentrations of 25 μM but had an observable cytostatic effect, resulting in suppression of cell proliferation at concentrations of >12.5 μM. Novel approaches based on quantitative phase imaging using holographic microscopy were developed for advanced characterization of antiviral and cytotoxic profiles of 3'-deoxy-3'-fluoroadenosine in vitro In addition to its antiviral activity in cell cultures, 3'-deoxy-3'-fluoroadenosine was active in vivo in mouse models of TBEV and WNV infection. Our results demonstrate that fluoro-modified nucleosides represent a group of bioactive molecules with excellent potential to serve as prospective broad-spectrum antivirals in antiviral research and drug development.

• Keywords
• 3′-deoxy-3′-fluoroadenosine, antiviral activity, cytotoxicity, flavivirus, mouse model, nucleoside analogue, tick-borne encephalitis virus,
• MeSH
• Antiviral Agents pharmacology   MeSH
• Deoxyadenosines pharmacology   MeSH
• Zika Virus Infection * MeSH
• Mice MeSH
• Prospective Studies MeSH
• Virus Replication MeSH
• Zika Virus * MeSH
• Encephalitis Viruses, Tick-Borne * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3'-fluoro-3'-deoxyadenosine MeSH Browser
• Antiviral Agents MeSH
• Deoxyadenosines MeSH