As an important signaling molecule, hydrogen peroxide (H2O2) secreted externally by the cells influences cell migration, immunity generation, and cellular communications. Herein, we have developed a ...microfluidic approach with droplets in combination with Au nanoclusters for the sensitive detection of H2O2 secreted by a single cell. Isolated in the ultrasmall volume (4.2 nL) of a microdroplet, single-cell secreted H2O2 can initiate dramatic fluorescence changes of horseradish peroxidase–Au nanoclusters. We have demonstrated an ultrahigh sensitivity (200–400 attomole H2O2 directly measured from a single cell) with good specificity. It offers a useful research tool to study the cell-to-cell differences of H2O2 secretion at the single-cell level.
Circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT) may exhibit more aggressive features than epithelial CTCs and are more frequently observed during disease ...progression. Therefore, detection and characterization of both epithelial and mesenchymal CTCs in cancer patients are urgently needed to allow for a better understanding of the metastatic process and more effective treatment. Here we describe a method for detection and isolation of viable epithelial and mesenchymal CTCs from peripheral blood of breast cancer patients. The method is based on density gradient centrifugation, multiplex immunofluorescent staining, and negative anti-CD45 selection. Cells obtained after the procedure are suitable for genomic or transcriptomic profiling, and they can also be isolated by micromanipulation for single-cell analysis.
Molecular typing has revolutionized epidemiological studies of infectious diseases, including those of a mycobacterial etiology. With the advent of fingerprinting techniques, many traditional ...concepts regarding transmission, infectivity, or pathogenicity of mycobacterial bacilli have been revisited, and their conventional interpretations have been challenged. Since the mid-1990s, when the first typing methods were introduced, a plethora of other modalities have been proposed. So-called molecular epidemiology has become an essential subdiscipline of modern mycobacteriology. It serves as a resource for understanding the key issues in the epidemiology of tuberculosis and other mycobacterial diseases. Among these issues are disclosing sources of infection, quantifying recent transmission, identifying transmission links, discerning reinfection from relapse, tracking the geographic distribution and clonal expansion of specific strains, and exploring the genetic mechanisms underlying specific phenotypic traits, including virulence, organ tropism, transmissibility, or drug resistance. Since genotyping continues to unravel the biology of mycobacteria, it offers enormous promise in the fight against and prevention of the diseases caused by these pathogens. In this review, molecular typing methods for Mycobacterium tuberculosis and nontuberculous mycobacteria elaborated over the last 2 decades are summarized. The relevance of these methods to the epidemiological investigation, diagnosis, evolution, and control of mycobacterial diseases is discussed.
Tumor dissemination is one of the most-investigated steps of tumor progression, which in recent decades led to the rapid development of liquid biopsy aiming to analyze circulating tumor cells (CTCs), ...extracellular vesicles (EVs), and circulating nucleic acids in order to precisely diagnose and monitor cancer patients. Flow cytometry was considered as a method to detect CTCs; however, due to the lack of verification of the investigated cells’ identity, this method failed to reach clinical utility. Meanwhile, imaging flow cytometry combining the sensitivity and high throughput of flow cytometry and image-based detailed analysis through a high-resolution microscope might open a new avenue in CTC technologies and provide an open-platform system alternative to CellSearch®, which is still the only gold standard in this field. Hereby, we shortly review the studies on the usage of flow cytometry in CTC identification and present our own representative images of CTCs envisioned by imaging flow cytometry providing rationale that this novel technology might be a good tool for studying tumor dissemination, and, if combined with a high CTC yield enrichment method, could upgrade CTC-based diagnostics.
Cancer-associated fibroblasts (CAFs) have been shown to support tumor development in a variety of cancers. Different markers were applied to classify CAFs in order to elucidate their impact on tumor ...progression. However, the exact mechanism by which CAFs enhance cancer development and metastasis is yet unknown.
Alpha-smooth muscle actin (α-SMA) was examined immunohistochemically in intratumoral CAFs of nonmetastatic breast cancers and correlated with clinicopathological data. Four CAF cell lines were isolated from patients with luminal breast cancer (lumBC) and classified according to the presence of α-SMA protein. Conditioned medium (CM) from CAF cultures was used to assess the influence of CAFs on lumBC cell lines: MCF7 and T47D cells using Matrigel 3D culture assay. To identify potential factors accounting for promotion of tumor growth by α-SMA
CAFs, nCounter PanCancer Immune Profiling Panel (NanoString) was used.
In luminal breast cancer, presence of intratumoral CAFs expressing high level of α-SMA (13% of lumBC group) correlated with poor prognosis (p = 0.019). In in vitro conditions, conditioned medium obtained from primary cultures of α-SMA-positive CAFs isolated from luminal tumors was observed to enhance growth of lumBC cell line colonies in 3D Matrigel, in contrast to CM derived from α-SMA-negative CAFs. Multigene expression analysis indicated that osteopontin (OPN) was overexpressed in α-SMA-positive CAFs in both clinical samples and in vitro models. OPN expression was associated with higher percentage of Ki67-positive cells in clinical material (p = 0.012), while OPN blocking in α-SMA-positive CAF-derived CM attenuated growth of lumBC cell line colonies in 3D Matrigel.
Our findings demonstrate that α-SMA-positive CAFs might enhance tumor growth via secretion of OPN.
Circulating tumor cells (CTCs) mediate dissemination of solid tumors and can be an early sign of disease progression. Moreover, they show a great potential in terms of non-invasive, longitudinal ...monitoring of cancer patients. CTCs have been extensively studied in breast cancer (BC) and were shown to present a significant phenotypic plasticity connected with initiation of epithelial-mesenchymal transition (EMT). Apart from conferring malignant properties, EMT affects CTCs recovery rate, making a significant portion of CTCs from patients’ samples undetected. Wider application of methods and markers designed to isolate and identify mesenchymal CTCs is required to expand our knowledge about the clinical impact of mesenchymal CTCs. Therefore, here we provide a comprehensive review of clinical significance of mesenchymal CTCs in BC together with statistical analysis of previously published data, in which we assessed the suitability of a number of methods/markers used for isolation of CTCs with different EMT phenotypes, both in in vitro spike-in tests with BC cell lines, as well as clinical samples. Results of spiked-in cell lines indicate that, in general, methods not based on epithelial enrichment only, capture mesenchymal CTCs much more efficiently that CellSearch
®
(golden standard in CTCs detection), but at the same time are not much inferior to Cell Search
®
, though large variation in recovery rates of added cells among the methods is observed. In clinical samples, where additional CTCs detection markers are needed, positive epithelial-based CTCs enrichment was the most efficient in isolating CTCs with mesenchymal features from non-metastatic BC patients. From the marker side, PI3K and VIM were contributing the most to detection of CTCs with mesenchymal features (in comparison to SNAIL) in non-metastatic and metastatic BC patients, respectively. However, additional data are needed for more robust identification of markers for efficient detection of CTCs with mesenchymal features.
Circulating tumor cells (CTCs) are tumor cells that separate from the solid tumor and enter the bloodstream, which can cause metastasis. Detection and enumeration of CTCs show promising potential as ...a predictor for prognosis in cancer patients. Furthermore, single-cells sequencing is a technique that provides genetic information from individual cells and allows to classify them precisely and reliably. Sequencing data typically comprises thousands of gene expression reads per cell, which artificial intelligence algorithms can accurately analyze. This work presents machine-learning-based classifiers that differentiate CTCs from peripheral blood mononuclear cells (PBMCs) based on single cell RNA sequencing data. We developed four tree-based models and we trained and tested them on a dataset consisting of Smart-Seq2 sequenced data from primary tumor sections of breast cancer patients and PBMCs and on a public dataset with manually annotated CTC expression profiles from 34 metastatic breast patients, including triple-negative breast cancer. Our best models achieved about 95% balanced accuracy on the CTC test set on per cell basis, correctly detecting 133 out of 138 CTCs and CTC-PBMC clusters. Considering the non-invasive character of the liquid biopsy examination and our accurate results, we can conclude that our work has potential application value.
•Aggressive biology of squamous cell lung cancer (SCC) is mediated by miRNA.•MiR-192 and miR-662 enhance invasiveness and anchorage independent growth.•Resistance to etoposide but not to cisplatin is ...imparted by miR-192 and miR-662.•MiR-192 and miR-662 enhance expression of EMT- and WNT-activating genes.•MiR-192 and miR-662 down-regulate the expression of immunogenic genes.
Overexpression of miR-192, miR-192* and miR-662 was previously found to correlate with poor prognosis of early-stage squamous cell lung cancer (SCC) patients. In this study, we investigated the relevance of these miRNAs to cancer cell biology and chemoresistance.
MiRNA expression profile was analysed in 10 non-small cell lung cancer (NSCLC) cell lines using RT-qPCR. H520 and H1703 cells were transfected with miRNA inhibitors (anti-miR-192, -192* and -662) for functional studies. Chemoresistance to cisplatin and etoposide was evaluated using MTT colorimetric assay. H520 cells were subjected to 3D soft-agar colony formation assay and H1703 cells to wound healing assay. Whole transcriptome analysis was used to assess the effect of miR-192 and miR-662 inhibition on gene expression.
SCC cell lines, H520 and H1703, differed in miRNA expression and phenotypic features. MiR-192 and miR-662 inhibition decreased clonogenicity and motility of SCC cells. MiR-192 and miR-662 inhibition sensitized SCC cells to etoposide but not to cisplatin. Whole transcriptome analysis revealed genes regulated by miR-192 and miR-662 in SCC, relevant to maintaining chemoresistance, invasiveness, epithelial-mesenchymal transition (EMT) and immune evasion.
We showed for the first time that miR-192 and miR-662 have functional role in SCC cells. Our findings suggest that targeting these miRNAs may impact both chemoresistance and invasiveness of SCC, and add to the evidence linking these aspects of tumour biology. Overexpression of miR-192 and miR-662 might be useful as a marker of resistance to etoposide.
The occurrence of either regional or distant metastases is an indicator of poor prognosis for cancer patients. The mechanism of their formation has not yet been fully uncovered, which limits the ...possibility of developing new therapeutic strategies. Nevertheless, the discovery of circulating tumor cells (CTCs), which are responsible for tumor dissemination, and cancer stem cells (CSCs), required for tumor growth maintenance, shed light on the metastatic cascade. It seems that CTCs and CSCs are not necessarily separate populations of cancer cells, as CTCs generated in the process of epithelial-mesenchymal transition (EMT) can bear features characteristic of CSCs. This article describes the mechanisms of CTC and CSC formation and characterizes their molecular hallmarks. Moreover, we present different types of EMT occurring in physiological and pathological conditions, and we demonstrate its crucial role in providing CTCs with a CSC phenotype. The article delineates molecular changes acquired by cancer cells undergoing EMT that facilitate metastasis formation. Deeper understanding of those processes is of fundamental importance for the development of new strategies of early cancer detection and effective cancer treatment approaches that will be translated into clinical practice.
Skin Microbiome in Prurigo Nodularis Tutka, Klaudia; Żychowska, Magdalena; Żaczek, Anna ...
International journal of molecular sciences,
04/2023, Letnik:
24, Številka:
8
Journal Article
Recenzirano
Odprti dostop
Prurigo nodularis (PN) is a chronic condition characterized by the presence of nodular lesions accompanied by intense pruritus. The disease has been linked to several infectious factors, but data on ...the direct presence of microorganisms in the lesions of PN are scarce. The aim of this study was to evaluate the diversity and composition of the bacterial microbiome in PN lesions by targeting the region V3-V4 of 16S rRNA. Skin swabs were obtained from active nodules in 24 patients with PN, inflammatory patches of 14 patients with atopic dermatitis (AD) and corresponding skin areas of 9 healthy volunteers (HV). After DNA extraction, the V3-V4 region of the bacterial 16S rRNA gene was amplified. Sequencing was performed using the Illumina platform on the MiSeq instrument. Operational taxonomic units (OTU) were identified. The identification of taxa was carried out using the Silva v.138 database. There was no statistically significant difference in the alpha-diversity (intra-sample diversity) between the PN, AD and HV groups. The beta-diversity (inter-sample diversity) showed statistically significant differences between the three groups on a global level and in paired analyses.
was significantly more abundant in samples from PN and AD patients than in controls. The difference was maintained across all taxonomic levels. The PN microbiome is highly similar to that of AD. It remains unclear whether the disturbed composition of the microbiome and the domination of
in PN lesions may be the trigger factor of pruritus and lead to the development of cutaneous changes or is a secondary phenomenon. Our preliminary results support the theory that the composition of the skin microbiome in PN is altered and justify further research on the role of the microbiome in this debilitating condition.
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