Isolation of Viable Epithelial and Mesenchymal Circulating Tumor Cells from Breast Cancer Patients.

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.

Osthole Regulates Secretion of Pro-Inflammatory Cytokines and Expression of TLR2 and NF-κB in Normal Human Keratinocytes and Fibroblasts.

Introduction: Osthole (OST), an active compound isolated from Cnidium monnieri, is used in traditional Chinese medicine to treat a variety of human diseases. Although OST has a good therapeutic effect, the underlying mechanism of its action in inflammatory skin diseases in humans is still unknown. Purpose: The present study aimed to test the hypothesis that OST can be used as an herbal substance that minimizes skin inflammation and barrier dysfunction. In this study, histamine and LPS were used to induce inflammation in skin keratinocytes and fibroblasts to test whether OST can inhibit their responses. Methods: Cell migration was analyzed using a wound healing assay. Changes in cell monolayer integrity were assessed by the measurement of transepithelial electrical resistance. Secretion of IL-1ß, IL-6, IL-8, TNF-α, CCL2/MCP-1, CCL5/RANTES, and COX-2 was measured by ELISA, while expression of TLR2, NF-κB, and COX-2 was analyzed by qPCR. Results: OST decreased the level of IL-1ß, TNF-α, CCL2/MCP-1 and CCL5/RANTES, and expression of TLR2, NF-κB and COX-2 during histamine/LPS-induced inflammation in human keratinocytes and fibroblasts. OST also improved cell migration and cell barrier function. Conclusion: Our results suggest that OST suppresses inflammatory responses via regulation of IL-1ß, TNF-α, CCL2/MCP-1 and CCL5/RANTES secretion, and TLR2, and COX-2 expression.

Breast cancer circulating tumor cells with mesenchymal features-an unreachable target?

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.

Effects of Osthole on Inflammatory Gene Expression and Cytokine Secretion in Histamine-Induced Inflammation in the Caco-2 Cell Line.

Hyperactivity of the immune system in the gastrointestinal tract leads to the development of chronic, inflammation-associated disorders. Such diseases, including inflammatory bowel disease, are not completely curable, but the specific line of treatment may reduce its symptoms. However, the response to treatment varies among patients, creating a necessity to uncover the pathophysiological basis of immune-mediated diseases and apply novel therapeutic strategies. The present study describes the anti-inflammatory properties of osthole during histamine-induced inflammation in the intestinal Caco-2 cell line. Osthole reduced the secretion of cytokines (CKs) and the expression level of inflammation-associated genes, which were increased after a histamine treatment. We have shown that the secretion of pro-inflammatory CKs (IL-1ß, IL-6, IL-8, and TNF-α) during inflammation may be mediated by NFκB, and, after osthole treatment, this signaling pathway was disrupted. Our results suggest a possible role for osthole in the protection against inflammation in the gastrointestinal tract; thus, osthole may be considered as an anti-inflammatory modulator.

Activation of epithelial-mesenchymal transition process during breast cancer progression - the impact of molecular subtype and stromal composition.

Breast cancer (BC) is a heterogeneous disease with different molecular subtypes, which can be defined by oestrogen (ER), progesterone (PR) and human epidermal growth factor (HER2) receptors' status as luminal, HER2+ and triple negative (TNBC). Molecular subtypes also differ in their epithelial-mesenchymal phenotype, which might be related to their aggressiveness, as activation of the epithelial-mesenchymal transition (EMT) is linked with increased ability of cancer cells to survive and metastasize. Nevertheless, the reverse process of mesenchymal-epithelial transition was shown to be required to sustain metastatic colonization. In this study we aimed to analyse activation of the EMT process in primary tumours (PT), which have (N+) or have not (N-) colonized the lymph nodes, as well as the lymph nodes metastases (LNM) themselves in 88 BC patients. We showed that luminal N- PT have the lowest activation of the EMT process (27%), in comparison to N+ PT (48%, p=0.06). On the other hand, TNBC do not show statistically significant EMT activation at the stage before lymph colonization (N-, 83%) and after colonization of the lymph nodes (N+, 63%, p=0.58). TNBC are also the least plastic (unable to change the EMT phenotype) in terms of turning EMT on or off between matched PT and LNM (0% EMT plasticity in TNBC vs 36% plasticity in luminal tumours). Moreover, in TNBC activation of EMT was correlated with increased cell division rate of the PT- in mesenchymal TNBC PT median Ki-67 was 45% in comparison to 10% in epithelial TNBC PT (p=0.002), whereas in PT of luminal subtypes Ki-67 did not differ between epithelial and mesenchymal phenotypes. Profiling of immunotranscriptome of epithelial and mesenchymal luminal BC with Nanostring technology revealed that N- PT with epithelial phenotype were enriched in inflammatory response signatures, whereas N+ mesenchymal cancers showed elevated MHC class II antigen presentation. Overall, activation of EMT changes during cancer progression and metastatic colonization of the lymph nodes depending on the PT molecular subtype and is related to differences in stromal signatures. Activation of EMT is associated with colonizing phenotype in luminal PT and proliferative phenotype of TNBC.

Stability of interleukin-1ß, -4, -6, -8, -10, -13, interferon-γ and tumor necrosis factor-α in human sera after repetitive freeze-thaw cycles and long storage.

INTRODUCTION: Cytokines (CKs) are one of the key components of the molecular network modulating multiple immunological interactions. Within such biological systems, CKs functions are associated with several processes, thus quantification of these analytes in serum samples, as well as a faithful determination of its concentration, are crucial for the translational aspect of many studies. AIM: This study is focused on the evaluation of the effects of storage duration and multiple freeze-thaw cycles on CKs stability. MATERIALS AND METHODS: Serum samples were obtained from 24 healthy participants. Samples were prospectively stored at 4 °C for 1-7 and 30 days, and also underwent multiple freeze-thaw cycles. Afterwards, CK levels were determined by enzyme-linked immunosorbent assay. RESULTS: Among the 8 examined CKs all of them showed significant degradation (determined with the two-way ANOVA and post-hoc test) after 4 days of sample storage at 4 °C. Serum were affected by freezing at -20 °C and thawing, and 2 of CKs (IL-1ß and IL-8) showed significant concentration decrease after following 2 freeze-thaw cycles. It has been also determineded that CKs in serum samples after multiple freeze-thaw cycles had better stability, when samples were stored at -80 °C (compared to storage at -20 °C). CONCLUSIONS: This study demonstrates that long storage at 4 °C, as well as multiple freeze-thaw cycles of serum samples, must be avoided and CK concentrations should be measured immediately after sample collection.

Osthole Inhibits Expression of Genes Associated with Toll-like Receptor 2 Signaling Pathway in an Organotypic 3D Skin Model of Human Epidermis with Atopic Dermatitis.

The Toll-like receptor (TLR) family signature has been linked to the etiopathology of atopic dermatitis (AD), a chronic inflammatory skin disease associated with skin barrier dysfunction and immune system imbalance. We aimed to investigate whether osthole (a plant-derived compound) can inhibit the genetic profile of key genes associated with TLR2 signaling (TIRAP, MyD88, IRAK1, TRAF6, IκBα, NFκB) after stimulation with LPS or histamine in a 3D in vitro model of AD. Overexpression of the aforementioned genes may directly increase the secretion of proinflammatory cytokines (CKs) and chemokines (ChKs), which may exacerbate the symptoms of AD. Relative gene expressions were quantified by qPCR and secretion of CKs and ChKs was evaluated by ELISA assay. LPS and histamine increased the relative expression of genes related to the TLR2 pathway, and osthole successfully reduced it. In summary, our results show that osthole inhibits the expression of genes associated with the TLR signaling pathway in a skin model of AD. Moreover, the secretion of CKs and ChKs after treatment of AD with osthole in a 3D skin model in vitro suggests the potential of osthole as a novel compound for the treatment of AD.

Modulatory Effects of Osthole on Lipopolysaccharides-Induced Inflammation in Caco-2 Cell Monolayer and Co-Cultures with THP-1 and THP-1-Derived Macrophages.

Lipopolysaccharydes (LPS) are responsible for the intestinal inflammatory reaction, as they may disrupt tight junctions and induce cytokines (CKs) secretion. Osthole has a wide spectrum of pharmacological effects, thus its anti-inflammatory potential in the LPS-treated Caco-2 cell line as well as in Caco-2/THP-1 and Caco-2/macrophages co-cultures was investigated. In brief, Caco-2 cells and co-cultures were incubated with LPS to induce an inflammatory reaction, after which osthole (150-450 ng/mL) was applied to reduce this effect. After 24 h, the level of secreted CKs and changes in gene expression were examined. LPS significantly increased the levels of IL-1ß, -6, -8, and TNF-α, while osthole reduced this effect in a concentration-dependent manner, with the most significant decrease when a 450 ng/mL dose was applied (p < 0.0001). A similar trend was observed in changes in gene expression, with the significant osthole efficiency at a concentration of 450 ng/µL for IL1R1 and COX-2 (p < 0.01) and 300 ng/µL for NF-κB (p < 0.001). Osthole increased Caco-2 monolayer permeability, thus if it would ever be considered as a potential drug for minimizing intestinal inflammatory symptoms, its safety should be confirmed in extended in vitro and in vivo studies.

Spectrum of Epithelial-Mesenchymal Transition Phenotypes in Circulating Tumour Cells from Early Breast Cancer Patients.

Circulating tumour cells (CTCs) can provide valuable prognostic information in a number of epithelial cancers. However, their detection is hampered due to their molecular heterogeneity, which can be induced by the epithelial-mesenchymal transition (EMT) process. Therefore, current knowledge about CTCs from clinical samples is often limited due to an inability to isolate wide spectrum of CTCs phenotypes. In the current work, we aimed at isolation and molecular characterization of CTCs with different EMT status in order to establish their clinical significance in early breast cancer patients. We have obtained CTCs-enriched blood fraction from 83 breast cancer patients in which we have tested the expression of epithelial, mesenchymal and general breast cancer CTCs markers (MGB1/HER2/CK19/CDH1/CDH2/VIM/PLS3), cancer stem cell markers (CD44, NANOG, ALDH1, OCT-4, CD133) and cluster formation gene (plakoglobin). We have shown that in the CTCs-positive patients, epithelial, epithelial-mesenchymal and mesenchymal CTCs markers were detected at a similar rate (in 28%, 24% and 24%, respectively). Mesenchymal CTCs were characterized by the most aggressive phenotype (significantly higher expression of CXCR4, uPAR, CD44, NANOG, p < 0.05 for all), presence of lymph node metastases (p = 0.043), larger tumour size (p = 0.023) and 7.33 higher risk of death in the multivariate analysis (95% CI 1.06⁻50.41, p = 0.04). Epithelial-mesenchymal subtype, believed to correspond to highly plastic and aggressive state, did not show significant impact on survival. Gene expression profile of samples with epithelial-mesenchymal CTCs group resembled pure epithelial or pure mesenchymal phenotypes, possibly underlining degree of EMT activation in particular patient's sample. Molecular profiling of CTCs EMT phenotype provides more detailed and clinically informative results, proving the role of EMT in malignant cancer progression in early breast cancer.