Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

BACKGROUND: Glioblastoma is a malignant and aggressive type of central nevous system malignancy characterized by many distinct biological features including extensive hypoxia. Hypoxia in glioblatoma associates with complex signaling patterns including activation of several pathways such as MAPK, PI3K-AKT/mTOR and IL-6/JAK/STAT3 with the master regulator HIF-1, which in turn drive particular tumor behaviors determining, in the end, treatment outcomes and patients fate. Thus, the present study was designed to investigate the expression of selected hypoxia related factors including STAT3 in a small set of long-term surviving glioma patients. METHODS: The expression of selected hypoxia related factors including STAT3 was evaluated in a time series of formalin fixed paraffin embedded and cryopreserved glioma samples from repeatedly resected patients. In addition, comparative studies were also conducted on primary glioma cells derived from original patient samples, stabilized glioma cell lines and tumor-xenograft mice model. Obtained data were correlated with clinical findings too. RESULTS: Glioblastoma samples of the analyzed patients displayed heterogeneity in the expression of hypoxia- related and EMT markers with most interesting trend being observed in pSTAT3. This heterogeneity was subsequently confirmed in other employed models (primocultures derived from glioblastoma tissue resections, cryopreserved tumor specimens, stabilized glioblastoma cell line in vitro and in vivo) and concerned, in particular, STAT3 expression which remained stable. In addition, subsequent studies on the role of STAT3 in the context of glioblastoma hypoxia demonstrated opposing effects of its deletion on cell viability as well as the expression of hypoxia and EMT markers. CONCLUSIONS: Our results suport the importance of STAT3 expression and activity in the context of hypoxia in malignant glioblastoma long-term surviving glioma patients while emphasizing heterogeneity of biological outcomes in varying employed tumor models.

Diagnostic Utility of Immunohistochemical Detection of MEOX2, SOX11, INSM1 and EGFR in Gliomas.

Histological identification of dispersed glioma cells in small biopsies can be challenging, especially in tumours lacking the IDH1 R132H mutation or alterations in TP53. We postulated that immunohistochemical detection of proteins expressed preferentially in gliomas (EGFR, MEOX2, CD34) or during embryonal development (SOX11, INSM1) can be used to distinguish reactive gliosis from glioma. Tissue microarrays of 46 reactive glioses, 81 glioblastomas, 34 IDH1-mutant diffuse gliomas, and 23 gliomas of other types were analysed. Glial neoplasms were significantly more often (p < 0.001, χ2) positive for EGFR (34.1% vs. 0%), MEOX2 (49.3% vs. 2.3%), SOX11 (70.5% vs. 20.4%), and INSM1 (65.4% vs. 2.3%). In 94.3% (66/70) of the glioblastomas, the expression of at least two markers was observed, while no reactive gliosis showed coexpression of any of the proteins. Compared to IDH1-mutant tumours, glioblastomas showed significantly higher expression of EGFR, MEOX2, and CD34 and significantly lower positivity for SOX11. Non-diffuse gliomas were only rarely positive for any of the five markers tested. Our results indicate that immunohistochemical detection of EGFR, MEOX2, SOX11, and INSM1 can be useful for detection of glioblastoma cells in limited histological samples, especially when used in combination.

Validity of simplified self-report methods versus standard use method for pain assessment in preschool-aged children.

PURPOSE: Children experience painful invasive procedures very intensely. The aim of health professionals is to help children minimize this traumatic experience. The Simplified Faces Pain Scale (S-FPS) and Simplified Concrete Ordinal Pain Scale (S-COS) tools offer children the possibility of self-assessing their pain. This can then be the basis for tailoring pain relief to the child's individual need. The aim of this study is to present the validation procedure of the S-FPC, S-COS methods. DESIGN AND METHODS: 135 children aged 3-6 years assessed their pain using the self-reported S-FPS and S-COS methods at three consecutive times, and their results were compared with the commonly used Face, Legs, Activity, Cry, Consolability assessment scale. Intra-class correlations (ICC) were used to assess inter-rater agreement. Convergent validity was verified using Spearman's correlation coefficient. RESULTS: This study demonstrated good validity for both the S FPS and S-COS assessment tools. The ICC coefficient showed good inter-rater correlation. Spearman's correlation coefficient showed a strong correlation between the scales. PRACTICE IMPLICATIONS: It is not possible to clearly select a best method of pain assessment in preschool children. To choose the most appropriate method, it is necessary to take into account the child's cognitive development and preferences.

Small RNA Sequencing Identifies a Six-MicroRNA Signature Enabling Classification of Brain Metastases According to their Origin.

BACKGROUND/AIM: Brain metastases (BMs) are the most frequent intracranial tumors in adults and one of the greatest challenges for modern oncology. Most are derived from lung, breast, renal cell, and colorectal carcinomas and melanomas. Up to 14% of patients are diagnosed with BMs of unknown primary, which are commonly characterized by an early and aggressive metastatic spread. It is important to discover novel biomarkers for early identification of BM origin, allowing better management of patients with this disease. Our study focused on microRNAs (miRNAs), which are very stable in frozen native and FFPE tissues and have been shown to be sensitive and specific diagnostic biomarkers of cancer. We aimed to identify miRNAs with significantly different expression in the five most frequent groups of BMs and develop a diagnostic classifier capable of sensitive and specific classification of BMs. MATERIALS AND METHODS: Total RNA enriched for miRNAs was isolated using the mirVana miRNA Isolation Kit from 71 fresh-frozen histopathologically confirmed BM tissues originating in 5 cancer types. Sequencing libraries were prepared using the QIAseq miRNA Library Kit and sequenced on the NextSeq 500 platform. MiRNA expression was further validated by RT-qPCR. RESULTS: Differential analysis identified 373 miRNAs with significantly different expression between 5 BM groups (p<0.001). A classifier model was developed based on the expression of 6 miRNAs (hsa-miR-141-3p, hsa-miR-141-5p, hsa-miR-146a-5p, hsa-miR-194-5p, hsa-miR-200b-3p and hsa-miR-365b-5p) with the ability to correctly classify 91.5% of samples. Subsequent validation confirmed both significantly different expression of selected miRNAs in 5 BM groups as well as their diagnostic potential. CONCLUSION: To date, our study is the first to analyze miRNA expression in various types of BMs using small RNA sequencing to develop a diagnostic classifier and, thus, to help stratify BMs of unknown primary. The presented results confirm the importance of studying the dysregulated expression of miRNAs in BMs and the diagnostic potential of the validated 6-miRNA signature.

Cauda equina neuroendocrine tumors show biological features distinct from other paragangliomas and visceral neuroendocrine tumors.

Cauda equina neuroendocrine tumors (CENETs) are neoplasms of uncertain histogenesis with overlapping features between those of paragangliomas (PGs) and visceral neuroendocrine tumors (NETs). We have explored their biological relationship to both subsets of neuroendocrine neoplasms. The clinical and radiological features of a cohort of 23 CENETs were analyzed. A total of 21 cases were included in tissue microarrays, along with a control group of 38 PGs and 83 NETs. An extensive panel of antibodies was used to assess epithelial phenotype (cytokeratins, E-cadherin, EpCAM, Claudin-4, EMA, CD138), neuronal and neuroendocrine features (synaptophysin, chromogranin A, INSM1, neurofilaments, NeuN, internexin-α, calretinin), chromaffin differentiation (GATA3, Phox2b, tyrosine hydroxylase), and possible histogenesis (Sox2, T-brachyury, Oct3/4, Sox10). The cohort included 5 women (22%) and 18 men (78%). The average age at the time of surgery was 48.3 years (range from 21 to 80 years). The average diameter of the tumors was 39.27 mm, and invasion of surrounding structures was observed in 6/21 (29%) tumors. Follow-up was available in 16 patients (median 46.5 months). One tumor recurred after 19 months. No metastatic behavior and no endocrine activity were observed. Compared to control groups, CENETs lacked expression of epithelial adhesion molecules (EpCAM, CD138, E-cadherin, Claudin-4), and at the same time, they lacked features of chromaffin differentiation (GATA3, Phox2b, tyrosine hydroxylase). We observed no loss of SDHB. Cytokeratin expression was present in all CENETs. All the CENETs showed variable cytoplasmic expression of T-brachyury and limited nuclear expression of Sox2. These findings support the unique nature of the neoplasm with respect to NETs and PGs.

Intention to use business intelligence tools in decision making processes: applying a UTAUT 2 model.

The pressure on the speed of information processing ranks business intelligence technologies among the fastest growing decision support tools. The main goal of this article is, applying the UTAUT 2 (the unified theory of acceptance and use of technology), to verify the factors determining the implementation of business intelligence tools in business processes, especially decision-making, and their subsequent optimal use in business practice. The researched scheme was modified according to the specifics of business intelligence tools and was supplemented by user behaviour in decision-making. The verification was performed using a questionnaire survey based on UTAUT 2 theory and 152 respondents were included in the analysis. According to the results, the most important variable of influence on both the behavioural intention and the users' behaviour itself in decision-making was the factor of habit. And surprisingly, some previously recognised links were not confirmed, especially the factors influencing the intention of behaviour (effort expectancy, social influence, facilitating conditions). So, there is room after almost 10 years and experience gained during the Covid-19 pandemic to modify the latest version of a model.

Non-thermal plasma causes Pseudomonas aeruginosa biofilm release to planktonic form and inhibits production of Las-B elastase, protease and pyocyanin.

The increasing risk of antibiotic failure in the treatment of Pseudomonas aeruginosa infections is largely related to the production of a wide range of virulence factors. The use of non-thermal plasma (NTP) is a promising alternative to antimicrobial treatment. Nevertheless, there is still a lack of knowledge about the effects of NTP on the virulence factors production. We evaluated the ability of four NTP-affected P. aeruginosa strains to re-form biofilm and produce Las-B elastase, proteases, lipases, haemolysins, gelatinase or pyocyanin. Highly strains-dependent inhibitory activity of NTP against extracellular virulence factors production was observed. Las-B elastase activity was reduced up to 82% after 15-min NTP treatment, protease activity and pyocyanin production by biofilm cells was completely inhibited after 60 min, in contrast to lipases and gelatinase production, which remained unchanged. However, for all strains tested, a notable reduction in biofilm re-development ability was depicted using spinning disc confocal microscopy. In addition, NTP exposure of mature biofilms caused disruption of biofilm cells and their dispersion into the environment, as shown by transmission electron microscopy. This appears to be a key step that could help overcome the high resistance of P. aeruginosa and its eventual elimination, for example in combination with antibiotics still highly effective against planktonic cells.

Invasive Aspergillus infection of middle ear in a patient treated with secukinumab, methotrexate, and corticosteroids for psoriasis and psoriatic arthritis.

Mucocutaneous mycotic infections are common complications in patients on IL-17 inhibitor therapy. We report a case of a 33-year-old male with severe psoriasis and psoriatic arthritis on secukinumab combined with methotrexate and prednisone with swelling, otorrhea, and pain of the right ear and external auditory canal. Due to progressive hypacusis, a surgical solution was chosen. Tissue samples taken during surgery revealed the presence of Aspergillus fumigatus. Aspergillosis should be suspected in prolonged otorrhea, especially in immunocompromised patients. Without intervention, the disease could be fatal.

Cobalt Bis-Dicarbollide Enhances Antibiotics Action towards Staphylococcus epidermidis Planktonic Growth Due to Cell Envelopes Disruption.

The emergence of antibiotic resistance in opportunistic pathogens represents a huge problem, the solution for which may be a treatment with a combination of multiple antimicrobial agents. Sodium salt of cobalt bis-dicarbollide (COSAN.Na) is one of the very stable, low-toxic, amphiphilic boron-rich sandwich complex heteroboranes. This compound has a wide range of potential applications in the biological sciences due to its antitumor, anti-HIV-1, antimicrobial and antibiofilm activity. Our study confirmed the ability of COSAN.Na (in the concentration range 0.2-2.48 µg/mL) to enhance tetracycline, erythromycin, and vancomycin action towards Staphylococcus epidermidis planktonic growth with an additive or synergistic effect (e.g., the combination of 1.24 µg/mL COSAN.Na and 6.5 µg/mL TET). The effective inhibitory concentration of antibiotics was reduced up to tenfold most efficiently in the case of tetracycline (from 65 to 6.5 µg/mL). In addition, strong effect of COSAN.Na on disruption of the cell envelopes was determined using propidium iodide uptake measurement and further confirmed by transmission electron microscopy. The combination of amphiphilic COSAN.Na with antibiotics can therefore be considered a promising way to overcome antibiotic resistance in Gram-positive cocci.

Decontamination of High-Efficiency Mask Filters From Respiratory Pathogens Including SARS-CoV-2 by Non-thermal Plasma.

The current pandemic resulted in a rapidly increasing demand for personal protective equipment (PPE) initially leading to severe shortages of these items. Hence, during an unexpected and fast virus spread, the possibility of reusing highly efficient protective equipment could provide a viable solution for keeping both healthcare professionals and the general public equipped and protected. This requires an efficient decontamination technique that preserves functionality of the sensitive materials used for PPE production. Non-thermal plasma (NTP) is a decontamination technique with documented efficiency against select bacterial and fungal pathogens combined with low damage to exposed materials. We have investigated NTP for decontamination of high-efficiency P3 R filters from viral respiratory pathogens in comparison to other commonly used techniques. We show that NTP treatment completely inactivates SARS-CoV-2 and three other common human respiratory viruses including Influenza A, Rhinovirus and Adenovirus, revealing an efficiency comparable to 90°C dry heat or UVC light. Unlike some of the tested techniques (e.g., autoclaving), NTP neither influenced the filtering efficiency nor the microstructure of the filter. We demonstrate that NTP is a powerful and economic technology for efficient decontamination of protective filters and other sensitive materials from different respiratory pathogens.

Non-thermal Plasma Treatment of ESKAPE Pathogens: A Review.

The acronym ESKAPE refers to a group of bacteria consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. They are important in human medicine as pathogens that show increasing resistance to commonly used antibiotics; thus, the search for new effective bactericidal agents is still topical. One of the possible alternatives is the use of non-thermal plasma (NTP), a partially ionized gas with the energy stored particularly in the free electrons, which has antimicrobial and anti-biofilm effects. Its mechanism of action includes the formation of pores in the bacterial membranes; therefore, resistance toward it is not developed. This paper focuses on the current overview of literature describing the use of NTP as a new promising tool against ESKAPE bacteria, both in planktonic and biofilm forms. Thus, it points to the fact that NTP treatment can be used for the decontamination of different types of liquids, medical materials, and devices or even surfaces used in various industries. In summary, the use of diverse experimental setups leads to very different efficiencies in inactivation. However, Gram-positive bacteria appear less susceptible compared to Gram-negative ones, in general.

Small RNA Sequencing Identifies PIWI-Interacting RNAs Deregulated in Glioblastoma-piR-9491 and piR-12488 Reduce Tumor Cell Colonies In Vitro.

Glioblastoma (GBM) is the most frequently occurring primary malignant brain tumor of astrocytic origin. To change poor prognosis, it is necessary to deeply understand the molecular mechanisms of gliomagenesis and identify new potential biomarkers and therapeutic targets. PIWI-interacting RNAs (piRNAs) help in maintaining genome stability, and their deregulation has already been observed in many tumors. Recent studies suggest that these molecules could also play an important role in the glioma biology. To determine GBM-associated piRNAs, we performed small RNA sequencing analysis in the discovery set of 19 GBM and 11 non-tumor brain samples followed by TaqMan qRT-PCR analyses in the independent set of 77 GBM and 23 non-tumor patients. Obtained data were subsequently bioinformatically analyzed. Small RNA sequencing revealed 58 significantly deregulated piRNA molecules in GBM samples in comparison with non-tumor brain tissues. Deregulation of piR-1849, piR-9491, piR-12487, and piR-12488 was successfully confirmed in the independent groups of patients and controls (all p < 0.0001), and piR-9491 and piR-12488 reduced GBM cells' ability to form colonies in vitro. In addition, piR-23231 was significantly associated with the overall survival of the GBM patients treated with Stupp regimen (p = 0.007). Our results suggest that piRNAs could be a novel promising diagnostic and prognostic biomarker in GBM potentially playing important roles in gliomagenesis.

Low-molecular weight chitosan enhances antibacterial effect of antibiotics and permeabilizes cytoplasmic membrane of Staphylococcus epidermidis biofilm cells.

This study evaluated the effect of low-molecular weight chitosan on Staphylococcus epidermidis, a common colonizer of joint implants and other prosthetic devices. We have also attempted to elucidate its mechanism of action. Chitosan was found to be effective against both the planktonic and biofilm cells (MIC80 35-40 mg/L; MBIC80 40-150 mg/L), in contrast to the antibiotics erythromycin and tetracycline with no antibiofilm activity (MBIC80 not found). In combination, chitosan had an additive effect with antibiotics on suspension growth of S. epidermidis (FICi 0.7-1.0), and the combinatory action caused a complete inhibition of biofilm metabolic activity in some cases. In addition, chitosan caused rapid cellular damage and enhanced antihaemolytic activity of tetracycline in combination towards S. epidermidis biofilm cells. Chitosan efficiently inhibited S. epidermidis growth acting via cell membrane damage, yet the extent of antimicrobial and antibiofilm activities was quite strain-specific. It was proved to be a very efficient antimicrobial agent worth further examination as a potent candidate in pharmaceutical research. Apart from antimicrobial activity, it also acted as antivirulence enhancing agent which is a very promising strategy for alternative infectious diseases treatment.

Pathologia mutans.

The authors present results of our center retrospective study comparing autopsy findings from years 1929 (n=275) and 1989 (n=974). The male to female ratio was very similar in both cohorts (1.3:1 in 1929 and 1.4:1 in 1989). The age range in 1929 was 0-88 years with median of 50 years, whereas in 1989, the age range was 0-98 year and median was 65 years. Among lethal diseases in 1929 were namely infections and infectious complications - 61 % of all patients (out of these, 18 % were tuberculosis cases), neoplasms (12 %) and cardiovascular disorders (6.5 %). In 1989, malignant neoplasms were most frequent (31 %), followed by cardiovascular disorders (21 %) and infections (4.6 % - out of these, tuberculosis represented only 0.6 %). Our study is unique by comparing two well documented autopsy cohorts in a single center from two years being 60 years apart. The study clearly demonstrates dramatic changes in healthcare achieved during the 20th century.

Non-Thermal Plasma Sources Based on Cometary and Point-to-Ring Discharges.

A non-thermal plasma (NTP) is a promising tool against the development of bacterial, viral, and fungal diseases. The recently revealed development of microbial resistance to traditional drugs has increased interest in the use of NTPs. We have studied and compared the physical and microbicidal properties of two types of NTP sources based on a cometary discharge in the point-to-point electrode configuration and a corona discharge in the point-to-ring electrode configuration. The electrical and emission properties of both discharges are reported. The microbicidal effect of NTP sources was tested on three strains of the bacterium Staphylococcus aureus (including the methicillin-resistant strain), the bacterium Pseudomonas aeruginosa, the yeast Candida albicans, and the micromycete Trichophyton interdigitale. In general, the cometary discharge is a less stable source of NTP and mostly forms smaller but more rapidly emerging inhibition zones on agar plates. Due to the point-to-ring electrode configuration, the second type of discharge has higher stability and provides larger affected but often not completely inhibited zones. However, after 60 min of exposure, the NTP sources based on the cometary and point-to-ring discharges showed a similar microbicidal effect for bacteria and an individual effect for microscopic fungi.

Polylactic acid as a suitable material for 3D printing of protective masks in times of COVID-19 pandemic.

A critical lack of personal protective equipment has occurred during the COVID-19 pandemic. Polylactic acid (PLA), a polyester made from renewable natural resources, can be exploited for 3D printing of protective face masks using the Fused Deposition Modelling technique. Since the possible high porosity of this material raised questions regarding its suitability for protection against viruses, we have investigated its microstructure using scanning electron microscopy and aerosol generator and photometer certified as the test system according to the standards EN 143 and EN 149. Moreover, the efficiency of decontaminating PLA surfaces by conventional chemical disinfectants including 96% ethanol, 70% isopropanol, and a commercial disinfectant containing 0.85% sodium hypochlorite has been determined. We confirmed that the structure of PLA protective masks is compact and can be considered a sufficient barrier protection against particles of a size corresponding to microorganisms including viruses. Complete decontamination of PLA surfaces from externally applied Staphylococcus epidermidis, Escherichia coli, Candida albicans and SARS-CoV-2 was achieved using all disinfectants tested, and human adenovirus was completely inactivated by sodium hypochlorite-containing disinfectant. Natural contamination of PLA masks worn by test persons was decontaminated easily and efficiently by ethanol. No disinfectant caused major changes to the PLA surface properties, and the pore size did not change despite severe mechanical damage of the surface. Therefore, PLA may be regarded as a suitable material for 3D printing of protective masks during the current or future pandemic crises.

Use of non-thermal plasma pre-treatment to enhance antibiotic action against mature Pseudomonas aeruginosa biofilms.

Non-thermal plasma (NTP), generated at atmospheric pressure by DC cometary discharge with a metallic grid, and antibiotics (gentamicin-GTM, ceftazidime-CFZ and polymyxin B-PMB), either alone or in combination, were used to eradicate the mature biofilm of Pseudomonas aeruginosa formed on Ti-6Al-4V alloy. Our aim was to find the conditions for NTP pre-treatment capable of enhancing the action of the antibiotics and thus reducing their effective concentrations. The NTP treatment increased the efficacy of relatively low concentrations of antibiotics. Generally, the highest effect was achieved with GTM, which was able to suppress the metabolic activity of pre-formed P. aeruginosa biofilms in the concentration range of 4-9 mg/L by up to 99%. In addition, an apparent decrease of biofilm-covered area was confirmed after combined NTP treatment and GTM action by SYTO®13 staining using fluorescence microscopy. Scanning electron microscopy confirmed a complete eradication of P. aeruginosa ATCC 15442 mature biofilm from Ti-6Al-4V alloy when using 0.25 h NTP treatment and subsequent treatment by 8.5 mg/L GTM. Therefore, NTP may be used as a suitable antibiofilm agent in combination with antibiotics for the treatment of biofilm-associated infections caused by this pathogen.

Combination of non-thermal plasma and subsequent antibiotic treatment for biofilm re-development prevention.

The influence of non-thermal plasma (NTP) treatment on the prevention of antibiotic resistance of microbial biofilms was studied. Staphylococcus epidermidis and Escherichia coli bacteria and a yeast Candida albicans, grown on the surface of Ti-6Al-4V alloy used in the manufacture of prosthetic implants, were employed. Their biofilms were exposed to NTP produced by DC cometary discharge and subsequently treated with antibiotics commonly used for the treatment of infections caused by them: erythromycin (ERY), polymyxin B (PMB), or amphotericin B (AMB), respectively. All biofilms displayed significant reduction of their metabolic activity after NTP exposure, the most sensitive was S. epidermidis. The subsequent action of antibiotics caused significant decrease in the metabolic activity of S. epidermidis and E. coli, but not C. albicans, although the area covered by biofilm decreased in all cases. The combined effect of NTP with antibiotics was thus proved to be a promising strategy in bacterial pathogen treatment.

Combined effect of lasioglossin LL-III derivative with azoles against Candida albicans virulence factors: biofilm formation, phospholipases, proteases and hemolytic activity.

Candida albicans has several virulence factors at its disposal, including yeast-hyphal transition associated with biofilm formation, phospholipases, proteases and hemolytic activity, all of which contribute to its pathogenesis. We used synthetic derivative LL-III/43 of antimicrobial peptide lasioglossin LL-III to enhance effect of azoles on attenuation of C. albicans virulence factors. LL-III/43 was able to inhibit initial adhesion or biofilm formation of C. albicans strains at 50 µM. Azoles, however, were ineffective at this concentration. Using fluorescently labeled LL-III/43, we observed that peptide covered C. albicans cells, partially penetrated through their membranes and then accumulated inside cells. LL-III/43 (25 µM) in combination with clotrimazole prevented biofilm formation already at 3.1 µM clotrimazole. Neither LL-III/43 nor azoles were able to significantly inhibit phospholipases, proteases, or hemolytic activity of C. albicans. LL-III/43 (25 µM) and clotrimazole (50 µM) in combination decreased production of these virulence factors, and it completely attenuated its hemolytic activity. Scanning electron microscopy showed that LL-III/43 (50 µM) prevented C. albicans biofilm formation on Ti-6Al-4 V alloy used in orthopedic surgeries and combination of LL-III/43 (25 µM) with clotrimazole (3.1 µM) prevented biofilm formation on urinary catheters. Therefore, mixture of LL-III/43 and clotrimazole is suitable candidate for future pharmaceutical research.

The Evaluation of Glioblastoma Cell Dissociation and Its Influence on Its Behavior.

PURPOSE: Primary cell lines are a valuable tool for evaluation of tumor behavior or sensitivity to anticancer treatment and appropriate dissociation of cells could preserve genomic profile of the original tissue. The main aim of our study was to compare the influence of two methods of glioblastoma multiforme (GBM) cell derivation (mechanic-MD; enzymatic-ED) on basic biological properties of thus derived cells and correlate them to the ones obtained from stabilized GBM cell line A-172. METHODS: Cell proliferation and migration (xCELLigence Real-Time Cell Analysis), expression of microRNAs and protein markers (RT-PCR and Western blotting), morphology (phase contrast and fluorescent microscopy), and accumulation of temozolomide (TMZ) and its metabolite 5-aminoimidazole-4-carboxamide (AIC) inside the cells (LC-MS analysis) were carried out in five different samples of GBM (GBM1, GBM2, GBM32, GBM33, GBM34), with each of them processed by MD and ED types of isolations. The same analyses were done in the A-172 cell line too. RESULTS: Primary GBM cells obtained by ED or MD approaches significantly differ in biological behavior and properties of these cells. Unlike in primary MD GBM cells, higher proliferation, as well as migration, was observed in primary ED GBM cells, which were also associated with the acquired mesenchymal phenotype and higher sensitivity to TMZ. Finally, the same analyses of stabilized GBM cell line A-172 revealed several important differences in measured parameters. CONCLUSIONS: GBM cells obtained by MD and ED dissociation show considerable heterogeneity, but based on our results, MD approach should be the preferred method of primary GBM cell isolation.