Search bioRxiv⌕ Search

Biology subjects

Beykou, M.

Publications and source records attributed to Beykou, M..

3 recordsLinked to original sources

Biocompatibility characterisation of CMOS-based Lab-on-Chip electrochemical sensors for in vitro cancer cell culture applications

Lab-on-Chip electrochemical sensors, such as Ion-Sensitive Field-Effect Transistors (ISFETs), are being developed for use in point-of-care diagnostics, such as pH detection of tumour microenvironments, due to their integration with standard Complementary Metal Oxide Semiconductor technology. With this approach, the passivation of the CMOS process is used as a sensing layer to minimise post-processing, and Silicon Nitride (Si3N4) is the most common material at the microchip surface. ISFETs have the potential to be used for cell-based assays however, there is a poor understanding of the biocompatibility of microchip surfaces. Here, we quantitatively evaluated cell adhesion, morphogenesis, proliferation and mechano-responsiveness of both normal and cancer cells cultured on a Si3N4, sensor surface. We demonstrate that both normal and cancer cell adhesion decreased on Si3N4. Activation of the mechano-responsive transcription regulators, YAP/TAZ, are significantly decreased in cancer cells on Si3N4 in comparison to standard cell culture plastic, whilst proliferation marker, Ki67, expression markedly increased. Non-tumorigenic cells on chip showed less sensitivity to culture on Si3N4 than cancer cells. Treatment with extracellular matrix components increased cell adhesion in normal and cancer cell cultures, surpassing the adhesiveness of plastic alone. Moreover, poly-l-ornithine and laminin treatment restored YAP/TAZ levels in both non-tumorigenic and cancer cells to levels comparable to those observed on plastic. Thus, engineering the electrochemical sensor surface with treatments will provide a more physiologically relevant environment for future cell-based assay development on chip.

cancer biology↗

An Nav1.5-dependent feedback mechanism driving glycolytic acidification in breast cancer metastasis

Solid tumours have abnormally high intracellular [Na+]. The activity of various Na+ channels may underlie this Na+ accumulation. Voltage-gated Na+ channels (VGSCs) have been shown to be functionally active in cancer cell lines, where they promote invasion. However, the mechanisms involved, and clinical relevance, are incompletely understood. Here, we show that protein expression of the Nav1.5 VGSC subtype strongly correlates with increased metastasis and shortened cancer-specific survival in breast cancer patients. In addition, VGSCs are functionally active in patient-derived breast tumour cells, cell lines, and cancer-associated fibroblasts. Knock down of Nav1.5 in a mouse model of breast cancer suppresses expression of invasion-regulating genes. Nav1.5 activity increases glycolysis in breast cancer cells, likely by up-regulating activity of the Na+/K+ ATPase, thus promoting H+ production and extracellular acidification. The pH of murine xenograft tumours is lower at the periphery than in the core, in regions of higher proliferation and lower apoptosis. In turn, acidic extracellular pH elevates persistent Na+ influx through Nav1.5 into breast cancer cells. Together, these findings show positive feedback between extracellular acidification and movement of Na+ into cancer cells which can facilitate invasion. These results highlight the clinical significance of Nav1.5 activity as a potentiator of breast cancer metastasis and provide further evidence supporting the use of VGSC inhibitors in cancer treatment.

cancer biology↗

Structure, function and variants analysis of the androgen-regulated TMPRSS2, a drug target candidate for COVID-19 infection

SARS-CoV-2 is a novel virus causing mainly respiratory, but also gastrointestinal symptoms. Elucidating the molecular processes underlying SARS-CoV-2 infection, and how the genetic background of an individual is responsible for the variability in clinical presentation and severity of COVID-19 is essential in understanding this disease. Cell infection by the SARS-CoV-2 virus requires binding of its Spike (S) protein to the ACE2 cell surface protein and priming of the S by the serine protease TMPRSS2. One may expect that genetic variants leading to a defective TMPRSS2 protein can affect SARS-CoV-2 ability to infect cells. We used a range of bioinformatics methods to estimate the prevalence and pathogenicity of TMPRSS2 genetic variants in the human population, and assess whether TMPRSS2 and ACE2 are co-expressed in the intestine, similarly to what is observed in lungs. We generated a 3D structural model of the TMPRSS2 extracellular domain using the prediction server Phyre and studied 378 naturally-occurring TMPRSS2 variants reported in the GnomAD database. One common variant, p.V160M (rs12329760), is predicted damaging by both SIFT and PolyPhen2 and has a MAF of 0.25. Valine 160 is a highly conserved residue within the SRCS domain. The SRCS is found in proteins involved in host defence, such as CD5 and CD6, but its role in TMPRSS2 remains unknown. 84 rare variants (53 missense and 31 leading to a prematurely truncated protein, cumulative minor allele frequency (MAF) 7.34x10-4) cause structural destabilization and possibly protein misfolding, and are also predicted damaging by SIFT and PolyPhen2 prediction tools. Moreover, we extracted gene expression data from the human protein atlas and showed that both ACE2 and TMPRSS2 are expressed in the small intestine, duodenum and colon, as well as the kidneys and gallbladder. The implications of our study are that: i. TMPRSS2 variants, in particular p.V160M with a MAF of 0.25, should be investigated as a possible marker of disease severity and prognosis in COVID-19 and ii. in vitro validation of the co-expression of TMPRSS2 and ACE2 in gastro-intestinal is warranted.

bioinformatics↗