Integrin-associated transcriptional characteristics of circulating tumor cells in breast cancer patients.

Background: Integrins enable cell communication with the basal membrane and extracellular matrix, activating signaling pathways and facilitating intracellular changes. Integrins in circulating tumor cells (CTCs) play a significant role in apoptosis evasion and anchor-independent survival. However, the link between CTCs expressing different integrin subunits, their transcriptional profile and, therefore, their functional activity with respect to metastatic potential remains unclear. Methods: Single-cell RNA sequencing of CD45-negative cell fraction of breast cancer patients was performed. All CTCs were divided into nine groups according to their integrin profile. Results: СTCs without the gene expression of integrins or with the expression of non-complementary α and ß subunits that cannot form heterodimers prevailed. Only about 15% of CTCs expressed integrin subunits which can form heterodimers. The transcriptional profile of CTCs appeared to be associated with the spectrum of expressed integrins. The lowest potential activity was observed in CTCs without integrin expression, while the highest frequency of expression of tumor progression-related genes, namely genes of stemness, epithelial-mesenchymal transition (EMT), invasion, proinflammatory chemokines and cytokines as well as laminin subunits, were observed in CTCs co-expressing ITGA6 and ITGB4. Validation on the protein level revealed that the median of integrin ß4+ CTCs was higher in patients with more aggressive molecular subtypes as well as in metastatic breast cancer patients. One can expect that CTCs with ITGA6 and ITGB4 expression will have pronounced metastatic potencies manifesting in expression of EMT and stemness-related genes, as well as potential ability to produce chemokine/proinflammatory cytokines and laminins.

Genetic Discrimination of Grade 3 and Grade 4 Gliomas by Artificial Neural Network.

Gliomas, including anaplastic gliomas (AG; grade 3) and glioblastomas (GBM; grade 4), are malignant brain tumors associated with poor prognosis and low survival rates. Current classification systems based on histopathology have limitations due to intratumoral heterogeneity. The treatment and prognosis are distinctly different between grade 3 and grade 4 gliomas patients. Therefore, there is a need for molecular markers to differentiate these tumors accurately. In this study, we aimed to identify a gene expression signature using an artificial neural network (ANN) in application to microarray and serial analysis of gene expression (SAGE) data for grade 3 (AG) and grade 4 (GBM) gliomas discrimination. We acquired gene expression data from publicly available datasets on glial tumors of grades 3 and 4-a total of 93 grade 3 gliomas and 224 grade 4 gliomas. To select genes for classification, we implemented an artificial neural network-based method using a combination of self-organized maps (SOM) and perceptron. In general, we implemented a multi-stage procedure that involved multiple runs of a genetic algorithm to identify genes that provided optimal clusterization on the SOM. We performed this procedure multiple times, resulting in different sets of genes each time. Eventually, we selected several genes that appeared most frequently in the reduced sets and performed classification using them. Our analysis identified a set of seven genes (BCAS4, GLUD2, KCNJ10, KCND2, AKR7A2, FOLR1, and KIAA0319). The classification accuracy using this gene set was 87.5%. These findings suggest the potential of this gene set as a molecular marker for distinguishing grade 3 (AG) from grade 4 (GBM) gliomas.

The Association of Integrins ß3, ß4, and αVß5 on Exosomes, CTCs and Tumor Cells with Localization of Distant Metastasis in Breast Cancer Patients.

Integrins are cell adhesion receptors, which play a role in breast cancer invasion, angiogenesis, and metastasis. Moreover, it has been shown that exosomal integrins provide organotropic metastasis in a mouse model. In our study, we aimed to investigate the expression of integrins ß3, ß4, and αVß5 on exosomes and tumor cells (circulating tumor cells and primary tumor) and their association with the localization of distant metastasis. We confirmed the association of exosomal integrin ß4 with lung metastasis in breast cancer patients. However, we were unable to evaluate the role of integrin ß3 in brain metastasis due to the rarity of this localization. We established no association of exosomal integrin αVß5 with liver metastasis in our cohort of breast cancer patients. The further evaluation of ß3, ß4, and αVß5 integrin expression on CTCs revealed an association of integrin ß4 and αVß5 with liver, but not the lung metastases. Integrin ß4 in the primary tumor was associated with liver metastasis. Furthermore, an in-depth analysis of phenotypic characteristics of ß4+ tumor cells revealed a significantly increased proportion of E-cadherin+ and CD44+CD24- cells in patients with liver metastases compared to patients with lung or no distant metastases.

Heterogeneity of Circulating Epithelial Cells in Breast Cancer at Single-Cell Resolution: Identifying Tumor and Hybrid Cells.

Circulating tumor cells and hybrid cells formed by the fusion of tumor cells with normal cells are leading players in metastasis and have prognostic relevance. This study applies single-cell RNA sequencing to profile CD45-negative and CD45-positive circulating epithelial cells (CECs) in nonmetastatic breast cancer patients. CECs are represented by transcriptionally-distinct populations that include both aneuploid and diploid cells. CD45- CECs are predominantly aneuploid, but one population contained more diploid than aneuploid cells. CD45+ CECs mostly diploid: only two populations have aneuploid cells. Diploid CD45+ CECs annotated as different immune cells, surprisingly harbored many copy number aberrations, and positively correlated to tumor grade. It is noteworthy that cancer-associated signaling pathways areabundant only in one aneuploid CD45- CEC population, which may represent an aggressive subset of circulating tumor cells. Thus, CD45- and CD45+ CECs are highly heterogeneous in breast cancer patients and include aneuploid cells, which are most likely circulating tumor and hybrid cells, respectively, and diploid cells. DNA ploidy analysis can be an effective instrument for identifying tumor and hybrid cells among CECs. Further follow-up study is needed to determine which subsets of circulating tumor and hybrid cells contribute to breast cancer metastasis.

The Novel Association of Early Apoptotic Circulating Tumor Cells with Treatment Outcomes in Breast Cancer Patients.

Stemness and epithelial-mesenchymal plasticity are widely studied in the circulating tumor cells of breast cancer patients because the roles of both processes in tumor progression are well established. An important property that should be taken into account is the ability of CTCs to disseminate, particularly the viability and apoptotic states of circulating tumor cells (CTCs). Recent data demonstrate that apoptosis reversal promotes the formation of stem-like tumor cells with pronounced potential for dissemination. Our study focused on the association between different apoptotic states of CTCs with short- and long-term treatment outcomes. We evaluated the association of viable CTCs, CTCs with early features of apoptosis, and end-stage apoptosis/necrosis CTCs with clinicopathological parameters of breast cancer patients. We found that the proportion of circulating tumor cells with features of early apoptosis is a perspective prognosticator of metastasis-free survival, which also correlates with the neoadjuvant chemotherapy response in breast cancer patients. Moreover, we establish that apoptotic CTCs are associated with the poor response to neoadjuvant chemotherapy, and metastasis-free survival expressed at least two stemness markers, CD44 and CD133.

Heterogeneous Manifestations of Epithelial-Mesenchymal Plasticity of Circulating Tumor Cells in Breast Cancer Patients.

To date, there is indisputable evidence of significant CTC heterogeneity in carcinomas, in particular breast cancer. The heterogeneity of CTCs is manifested in the key characteristics of tumor cells related to metastatic progression - stemness and epithelial-mesenchymal (EMT) plasticity. It is still not clear what markers can characterize the phenomenon of EMT plasticity in the range from epithelial to mesenchymal phenotypes. In this article we examine the manifestations of EMT plasticity in the CTCs in breast cancer. The prospective study included 39 patients with invasive carcinoma of no special type. CTC phenotypes were determined by flow cytometry before any type of treatment. EMT features of CTC were assessed using antibodies against CD45, CD326 (EpCam), CD325 (N-cadherin), CK7, Snail, and Vimentin. Circulating tumor cells in breast cancer are characterized by pronounced heterogeneity of EMT manifestations. The results of the study indicate that the majority of heterogeneous CTC phenotypes (22 out of 24 detectable) exhibit epithelial-mesenchymal plasticity. The variability of EMT manifestations does not prevent intravasation. Co-expression of EpCAM and CK7, regardless of the variant of co-expression of Snail, N-cadherin, and Vimentin, are associated with a low number of CTCs. Intrapersonal heterogeneity is manifested by the detection of several CTC phenotypes in each patient. Interpersonal heterogeneity is manifested by various combinations of CTC phenotypes in patients (from 1 to 17 phenotypes).

Heterogeneity of Stemlike Circulating Tumor Cells in Invasive Breast Cancer.

The presence of stem and epithelial-mesenchymal-transition (EMT) features in circulating tumor cells (CTCs) determines their invasiveness, adaptability to the microenvironment, and resistance to proapoptotic signals and chemotherapy. It also allows them to fulfil the role of metastatic "seeds". We evaluated the heterogeneity of stem CTCs by their CD44, ALDH1, and CD133 expression depending on N-cadherin expression in breast-cancer patients. A total of 38 female patients were selected for this study. CTC phenotypes were determined by flow cytometry before any type of treatment. Multiplex immunofluorescence was used for the evaluation of tumor-cell heterogeneity in primary lesions. In patients who had CD44-CD24- CTCs, a subset of cells with the expression of other stem-cell markers (CD133 and ALDH1) were detected. Expression of CD133 and/or ALDH1 may be associated with expression of N-cadherin: all populations of N-cadherin+ CTCs demonstrate stem features; in the absence of N-cadherin expression, true nonstem (CD44-CD24-CD133-ALDH1-) cells are found. The heterogeneity of stem marker expression in CTCs was observed regardless of N-cadherin expression. In our study, stromal cell-derived factor-1 (SDF-1) receptor expression in CTCs did not depend on stemlike traits, but was instead associated with N-cadherin expression. Subpopulations of tumor cells, detected both in tumors and blood, were identified. Breast cancer was characterized by pronounced interpersonal and intrapersonal heterogeneity of CTCs by the presence and combination of various stem features and N-cadherin expression. To complete the characterization of stemlike features of CTCs, we suggest the simultaneous use of the three stem markers.

Influenza a H1N1 associated acute glomerulonephritis in an adolescent.

Influenza virus primarily affects the respiratory system. It rarely causes extrapulmonary complications, with otitis media and febrile seizures being the most common in children. Acute glomerulonephritis as a complication of H1N1 influenza virus infection has been described only sporadically. Herein we present a case of acute glomerulonephritis in a previously healthy adolescent, in the context of infection with influenza A H1N1 virus. A 15-year old adolescent was admitted to our pediatric department due to fever, pharyngitis, cough, vomit, dizziness and fatigue. Based on his symptoms and the seasonal epidemiology, empiric treatment with oseltamivir was initiated while waiting for RT-PCR for influenza virus in pharyngeal swab, which was positive for A H1N1 influenza virus. In the first 24 h of admission, the patient presented macroscopic haematuria, which completely subsided in the following days, along with fever recession. The urine microscopic analysis showed findings compatible with acute glomerulonephritis. The patient remained normotasic while his biochemical profile including renal function, as well as further investigation of hematuria (immunoglobulins, C3, C4, ANA, anti-DNA, U/S) were all normal. ASTO levels, which were initially above normal (562 IU/ml), did not increase significantly in the following days, and given the fact that C3 levels were constantly within normal limits and pharyngeal culture was negative for pyogenic streptococcus, they were not considered sufficient for poststreptococcal glomerulonephritis diagnosis. Physicians should be suspicious and include influenza in the differential diagnosis when children present with uncommon symptoms such as hematuria along with even mild respiratory symptoms, during seasonal influenza period.

Single Tumor Cells With Epithelial-Like Morphology Are Associated With Breast Cancer Metastasis.

Introduction: The identification of tumor cells that can be potential metastatic seeds would reach two key aims-prognosis of metastasis risk and appointment of the optimal adjuvant therapy to prevent metastatic disease. Single tumor cells (STCs) located out of multicellular structures can most likely demonstrate features that are needed to initiate metastasis. Methods: One-hundred-and-thirty-five patients with invasive breast carcinoma of no special type have been enrolled. Molecular subtypes of breast cancer were categorized according to St. Gallen recommendations. Hematoxylin and eosin staining was used to identify STCs with epithelial-like morphology (eSTCs) in breast tumors. Immunofluorescence staining was applied to evaluate stemness and epithelial-mesenchymal transition (EMT) in STCs. The correlation between STCs and recurrence and metastasis-free survival (MFS) was performed using the Kaplan-Meier method and the log-rank test. Results: Distant metastasis was more frequent in eSTC-positive than eSTC-negative patients (28.0% vs. 9.4%, p = 0.007). When tumor types were analyzed separately, distant metastasis tended to be more frequent in eSTC-positive than eSTC-negative patients for HER2-positive cancer [75.0% (3/4) vs. 12.5% (1/8), p = 0.066]. In luminal A [22.7% (5/22) vs. 10.0% (3/30), p = 0.259], luminal B [21.1% (4/19) vs. 6.7% (2/30), p = 0.189], and triple-negative [40.0% (2/5) vs. 11.8% (2/17), p = 0.209] cancers, distance metastasis was not associated with eSTCs. Median MFS was not reached in eSTC-positive and eSTC-negative patients. eSTC-positive patients had a higher risk of breast cancer metastasis [hazard ratio (HR) 3.57, 95% confidence interval (CI): 1.46-8.71; p = 0.001]. When tumor types were analyzed separately, a higher risk of breast cancer metastasis occurred only in HER2-positive patients (HR 8.49, 95% CI: 1.29-55.59; p = 0.016). Immunofluorescence analysis revealed mesenchymal-like STCs (mSTCs) and inter- and intra-tumor heterogeneity in STCs. There were breast tumors with either eSTCs or mSTCs and tumors with both types of STCs. Both eSTCs and mSTCs were represented by cells with different stem and/or EMT phenotypes. Conclusions: STCs with epithelial-like morphology contribute to breast cancer metastasis and represent an attractive model for studying mechanisms of metastatic seeding. The assessment of STCs in histological sections of breast tumors can be a simple and effective method for the prediction of metastasis risk.

Mechanisms behind prometastatic changes induced by neoadjuvant chemotherapy in the breast cancer microenvironment.

Chemotherapy, along with surgery and radiotherapy, is a key treatment option for malignant tumors. Neoadjuvant chemotherapy (NACT) reduces the tumor size and enables total tumor resection. In addition, NACT is believed to be more effective in destroying micrometastases than the same chemotherapy performed after surgery. To date, various NACT regimens have been tested and implemented, which provide a favorable outcome in primary tumors and reduce the risk of progression. However, there is increasing evidence of the NACT ability to increase the risk of cancer progression. This review discusses potential mechanisms by which NACT promotes distant metastasis of breast cancer through changes in the microenvironment of tumor cells. We describe prometastatic NACT-mediated changes in angiogenesis, immuno-inflammatory reactions in the stroma, intravasation, and amount of circulating tumor cells. The role of NACT-related cellular stress in cancer metastasis is also discussed.

Heterogeneity of Circulating Tumor Cells in Neoadjuvant Chemotherapy of Breast Cancer.

The biological properties of circulating tumor cells (CTCs), and their dynamics during neoadjuvant chemotherapy are important, both for disease progression prediction and therapeutic target determination, with the aim of preventing disease progression. The aim of our study was to estimate of different CTC subsets in breast cancer during the NACT (neoadjuvant chemotherapy). The prospective study includes 27 patients with invasive breast cancer, T2-4N0-3M0, aged 32 to 60 years. Venous heparinized blood samples, taken before and after biopsy, after each courses of chemotherapy (on days 3-7), and before surgical intervention, served as the material for this study. Different subsets of circulating tumor cells were determined on the basis of the expression of EpCAM, CD45, CD44, CD24, and N-Cadherin using flow cytometry. As the result of this study, it has been observed that significant changes in the quantity of the different subsets of circulating tumor cells in patients' blood were observed after carrying out the 3rd course of NACT. NACT causes significant changes in the quantity of six CTC subsets, with various combinations of stemness and epithelial-mesenchymal transition (EMT) properties.

Tissue necrosis following extravasation of acyclovir in an adolescent: A case report.

OBJECTIVE: Extravasation of intravenously infused vesicant solutions is a common problem in medical practice, which can lead to severe and progressive tissue dysfunction, ranging from persistent tissue oedema and fibrosis to delayed tissue necrosis. Acyclovir is a known vesicant medication administrated in paediatric patients, which appears to irritate venous and soft tissue if extravasated. CASE REPORT: We present the first case involving the extravasation of intravenously infused acyclovir in a female adolescent patient, which caused tissue necrosis and left behind a residual scar lesion. Nursing and medical staff should be aware of the potential dermatological side effects of intravenously infused acyclovir and other medications, even a long time after infusion, and the possible lack of initial local symptoms and signs. CONCLUSION: Early recognition of extravasation and prompt management are critical in preventing further morbidity, and optimizing outcomes.

Monoclinic honeycomb-layered compound Li3Ni2SbO6: preparation, crystal structure and magnetic properties.

Two synthetic routes-ion-exchange preparation from layered Na(3)Ni(2)SbO(6) at 300 °C and direct solid-state synthesis at 1150 °C resulted in layered Li(3)Ni(2)SbO(6), a cation-ordered derivative from the rocksalt type. The Fddd form reported earlier could not be reproduced. According to the XRD Rietveld analysis, Li(3)Ni(2)SbO(6) is a pseudohexagonal monoclinic structure, C2/m, with a = 5.1828(2) Å, b = 8.9677(3) Å, c = 5.1577(2) Å, ß = 109.696(2)°. No Li/Ni mixed occupancy was detected. At high temperatures, the magnetic susceptibility follows the Curie-Weiss law with a positive value of Weiss temperature, ∼8 K, indicating a predominance of ferromagnetic interactions. However, Li(3)Ni(2)SbO(6) orders antiferromagnetically at T(N)∼ 15 K. The effective magnetic moment is 4.3 µ(B)/f.u. which satisfactorily agrees with theoretical estimations assuming high-spin configuration of Ni(2+) (S = 1). Electron spin resonance (ESR) spectra show single Lorentzian shape line attributed to Ni(2+) ion in octahedral coordination. The absorption is characterized by isotropic temperature independent effective g-factor g = 2.150 ± 0.005. In accordance with the layered honeycomb crystal structure determined for Li(3)Ni(2)SbO(6), the superexchange interaction between Ni(2+) ions through Ni-O-Ni pathways within Ni(2)SbO(6) layers are assumed to be ferromagnetic, while the dominant interaction between layers is antiferromagnetic.

Utilization of aminoaromatic acids by a methanogenic enrichment culture and by a novel Citrobacter freundii strain.

Following incubation of mesophilic methanogenic floccular sludge from a lab-scale upflow anaerobic sludge bed reactor used to treat cattle manure wastewater, a stable 5-aminosalicylate-degrading enrichment culture was obtained. Subsequently, a Citrobacter freundii strain, WA1, was isolated from the 5-aminosalicylate-degrading methanogenic consortium. The methanogenic enrichment culture degraded 5-aminosalicylate completely to CH4, CO2 and NH4+, while C. freundii strain WA1 reduced 5-aminosalicylate with simultaneous deamination to 2-hydroxybenzyl alcohol during anaerobic growth with electron donors such as pyruvate, glucose or serine. When grown on pyruvate, C. freundii WA1 converted 3-aminobenzoate to benzyl alcohol and also reduced benzaldehyde to benzyl alcohol. Pyruvate was fermented to acetate, CO2, H2 and small amounts of lactate, succinate and formate. Less lactate (30%) was produced from pyruvate when C. freundii WA1 grew with 5-aminosalicylate as co-substrate.