Machine learning-based analysis of cancer cell-derived vesicular proteins revealed significant tumor-specificity and predictive potential of extracellular vesicles for cell invasion and proliferation - A meta-analysis.

BACKGROUND: Although interest in the role of extracellular vesicles (EV) in oncology is growing, not all potential aspects have been investigated. In this meta-analysis, data regarding (i) the EV proteome and (ii) the invasion and proliferation capacity of the NCI-60 tumor cell lines (60 cell lines from nine different tumor types) were analyzed using machine learning methods. METHODS: On the basis of the entire proteome or the proteins shared by all EV samples, 60 cell lines were classified into the nine tumor types using multiple logistic regression. Then, utilizing the Least Absolute Shrinkage and Selection Operator, we constructed a discriminative protein panel, upon which the samples were reclassified and pathway analyses were performed. These panels were validated using clinical data (n = 4,665) from Human Protein Atlas. RESULTS: Classification models based on the entire proteome, shared proteins, and discriminative protein panel were able to distinguish the nine tumor types with 49.15%, 69.10%, and 91.68% accuracy, respectively. Invasion and proliferation capacity of the 60 cell lines were predicted with R2 = 0.68 and R2 = 0.62 (p < 0.0001). The results of the Reactome pathway analysis of the discriminative protein panel suggest that the molecular content of EVs might be indicative of tumor-specific biological processes. CONCLUSION: Integrating in vitro EV proteomic data, cell physiological characteristics, and clinical data of various tumor types illuminates the diagnostic, prognostic, and therapeutic potential of EVs. Video Abstract.

Small Extracellular Vesicles Isolated from Serum May Serve as Signal-Enhancers for the Monitoring of CNS Tumors.

Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may help to monitor brain tumors. In this proteomics-based study, we aimed to identify a characteristic protein fingerprint associated with central nervous system (CNS) tumors. Overall, 96 human serum samples were obtained from four patient groups, namely glioblastoma multiforme (GBM), non-small-cell lung cancer brain metastasis (BM), meningioma (M) and lumbar disc hernia patients (CTRL). After the isolation and characterization of small extracellular vesicles (sEVs) by nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM), liquid chromatography -mass spectrometry (LC-MS) was performed on two different sample types (whole serum and serum sEVs). Statistical analyses (ratio, Cohen's d, receiver operating characteristic; ROC) were carried out to compare patient groups. To recognize differences between the two sample types, pairwise comparisons (Welch's test) and ingenuity pathway analysis (IPA) were performed. According to our knowledge, this is the first study that compares the proteome of whole serum and serum-derived sEVs. From the 311 proteins identified, 10 whole serum proteins and 17 sEV proteins showed the highest intergroup differences. Sixty-five proteins were significantly enriched in sEV samples, while 129 proteins were significantly depleted compared to whole serum. Based on principal component analysis (PCA) analyses, sEVs are more suitable to discriminate between the patient groups. Our results support that sEVs have greater potential to monitor CNS tumors, than whole serum.

Binary mixture of Satureja hortensis and Origanum vulgare subsp. hirtum essential oils: in vivo therapeutic efficiency against Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori can cause many gastrointestinal and also extra-gastrointestinal disorders and is a major risk factor for gastric carcinoma and MALT lymphoma. Currently, numerous antibiotic-based therapies are available; however, these therapies have numerous drawbacks, mainly due to increasing prevalence of antibiotic resistant strains. Thus, there is an urgent need to develop novel therapeutic agents against H. pylori infections. MATERIALS AND METHODS: In this study, the anti-H. pylori activity of 2:1 mixture of Satureja hortensis and Origanum vulgare subsp. hirtum essential oils (2MIX) was investigated in vivo. After screening in vitro cytotoxicity of 2MIX on mammalian cell lines, the therapeutic efficiency was studied in a mouse model, where changes in H. pylori colonization were detected by PCR and histology of gastric samples. The immune reaction of mice was tested based on cytokine and chemokine production, and the in vivo toxicity of 2MIX was also investigated by measuring ALT and AST enzyme activities and Cyp3a11 and HO-1 mRNA levels in livers of mice. RESULTS: 2MIX had not shown in vitro cytotoxicity against cell lines, only the highest concentration caused significant decrease in their survival rates. In the in vivo experiments, 2MIX successfully eradicated the pathogen in 70% of the mice. We could not detect toxicity or altered cytokine and chemokine balance after in vivo treatments in mice. CONCLUSIONS: These results show that 2MIX is effective in reducing H. pylori colonization suggesting that this essential oil mixture has great potential as a new, effective, and safe therapeutic agent against H. pylori.

Stressors alter intercellular communication and exosome profile of nasopharyngeal carcinoma cells.

BACKGROUND: Head and neck cancers comprise the sixth most common cancer type worldwide. One of the most remarkable malignancies of the head and neck is the cancer of the nasopharynx, with a strong metastatic tendency already in the early stage. Besides the conventional pathways of metastasis formation, the information content of exosomes produced by the cancer cells may play a key role in metastatic transformation. The aim of this study was to investigate how stressors alter the characteristic of tumor derived exosomes. METHODS: In our experimental model, we compared the quantity and content of exosomes produced by a nasopharyngeal carcinoma cell line (5-8F) under conventional (chemotherapy) and alternative (Ag-TiO2 -catalyzed reactive oxygen species generation) cytostatic treatment. After isolation, exosomes were identified by atomic force microscopy and quantified with Nanosight NS500 device. MicroRNA content of them was analyzed using SOLiD 5500xl technology. The sequences were annotated in CLC Genomics Workbench version 5.5.1. RESULTS: Beyond the classic chemotherapeutic agent (doxorubicin), Ag-TiO2 in a photo-catalytic process also showed cytostatic activity. Tumor cell damage induced by the cytostatic treatments significantly altered the number of released exosomes and led to the predominance of tumor suppressors in the exosomal miRNA profile. CONCLUSIONS: Our results suggest that the intercellular communication between tumor cells and surrounding stroma cells can be altered by microenvironment which increased quantity of exosomes and diversity of miRNAs in this study. Imbalance of oncogenic and tumor suppressor miRNAs caused by cytostatic treatments may influence the antiproliferative and metastasis inhibitory effect of cytostatic agents.

Impact of Experimental Conditions on Extracellular Vesicles' Proteome: A Comparative Study.

Extracellular vesicle (EV) research is a rapidly developing field, mainly due to the key role of EVs in intercellular communication and pathophysiological processes. However, the heterogeneity of EVs challenges their exploration and the establishment of gold-standard methods. Here, we aimed to reveal the influence of technical changes on EV biology and the reliability of experimental data. We used B16F1 melanoma cells as a model and applied nanoparticle tracking analysis, mass spectrometry (LC-MS/MS) and pathway enrichment analysis to analyze the quantity, size distribution, proteome and function of their small EVs (sEVs) produced in sEV-depleted fetal bovine serum (FBS)-containing medium or serum-free medium. Additionally, we investigated the effects of minor technical variances on the quality of sEV preparations. We found that storage of the isolates at -80 °C has no adverse effect on LC-MS/MS analysis, and an additional washing step after differential ultracentrifugation has a minor influence on the sEV proteome. In contrast, FBS starvation affects the production and proteome of sEVs; moreover, these vesicles may have a greater impact on protein metabolism, but a smaller impact on cell adhesion and membrane raft assembly, than the control sEVs. As we demonstrated that FBS starvation has a strong influence on sEV biology, applying serum-free conditions might be considered in in vitro sEV studies.

MMP-9 as Prognostic Marker for Brain Tumours: A Comparative Study on Serum-Derived Small Extracellular Vesicles.

Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell invasion and metastasis, and its elevated level in brain tumour tissues indicates poor prognosis. High-risk tissue biopsy can be replaced by liquid biopsy; however, the blood-brain barrier (BBB) prevents tumour-associated components from entering the peripheral blood, making the development of blood-based biomarkers challenging. Therefore, we examined the MMP-9 content of small extracellular vesicles (sEVs)-which can cross the BBB and are stable in body fluids-to characterise tumours with different invasion capacity. From four patient groups (glioblastoma multiforme, brain metastases of lung cancer, meningioma, and lumbar disc herniation as controls), 222 serum-derived sEV samples were evaluated. After isolating and characterising sEVs, their MMP-9 content was measured by ELISA and assessed statistically (correlation, paired t-test, Welch's test, ANOVA, ROC). We found that the MMP-9 content of sEVs is independent of gender and age, but is affected by surgical intervention, treatment, and recurrence. We found a relation between low MMP-9 level in sEVs (<28 ppm) and improved survival (8-month advantage) of glioblastoma patients, and MMP-9 levels showed a positive correlation with aggressiveness. These findings suggest that vesicular MMP-9 level might be a useful prognostic marker for brain tumours.

Raman Spectral Signatures of Serum-Derived Extracellular Vesicle-Enriched Isolates May Support the Diagnosis of CNS Tumors.

Investigating the molecular composition of small extracellular vesicles (sEVs) for tumor diagnostic purposes is becoming increasingly popular, especially for diseases for which diagnosis is challenging, such as central nervous system (CNS) malignancies. Thorough examination of the molecular content of sEVs by Raman spectroscopy is a promising but hitherto barely explored approach for these tumor types. We attempt to reveal the potential role of serum-derived sEVs in diagnosing CNS tumors through Raman spectroscopic analyses using a relevant number of clinical samples. A total of 138 serum samples were obtained from four patient groups (glioblastoma multiforme, non-small-cell lung cancer brain metastasis, meningioma and lumbar disc herniation as control). After isolation, characterization and Raman spectroscopic assessment of sEVs, the Principal Component Analysis-Support Vector Machine (PCA-SVM) algorithm was performed on the Raman spectra for pairwise classifications. Classification accuracy (CA), sensitivity, specificity and the Area Under the Curve (AUC) value derived from Receiver Operating Characteristic (ROC) analyses were used to evaluate the performance of classification. The groups compared were distinguishable with 82.9-92.5% CA, 80-95% sensitivity and 80-90% specificity. AUC scores in the range of 0.82-0.9 suggest excellent and outstanding classification performance. Our results support that Raman spectroscopic analysis of sEV-enriched isolates from serum is a promising method that could be further developed in order to be applicable in the diagnosis of CNS tumors.

Chicken or the Egg: Microbial Alterations in Biopsy Samples of Patients with Oral Potentially Malignant Disorders.

Oral carcinogenesis often leads to the alteration of the microbiota at the site of the tumor, but data are scarce regarding the microbial communities of oral potentially malignant disorders (OPMDs). Punch biopsies were taken from healthy and non-healthy mucosa of OPMD patients to analyze the microbiome using metagenome sequencing. In healthy oral mucosa biopsies the bacterial phyla Firmicutes, Fusobacteria, Proteobacteria, Actinobacteria and Bacteroidetes were detected by Ion Torrent sequencing. The same phyla as well as the phyla Fibrobacteres and Spirochaetes were present in the OPMD biopsies. On the species level, there were 10 bacterial species unique to the healthy tissue and 35 species unique to the OPMD lesions whereas eight species were detected in both samples. We observed that the relative abundance of Streptococcus mitis decreased in the OPMD lesions compared to the uninvolved tissue. In contrast, the relative abundance of Fusobacterium nucleatum, implicated in carcinogenesis, was elevated in OPMD. We detected markedly increased bacterial diversity in the OPMD lesions compared to the healthy oral mucosa. The ratio of S. mitis and F. nucleatum are characteristically altered in the OPMD lesions compared to the healthy mucosa.

Small extracellular vesicles convey the stress-induced adaptive responses of melanoma cells.

Exosomes are small extracellular vesicles (sEVs), playing a crucial role in the intercellular communication in physiological as well as pathological processes. Here, we aimed to study whether the melanoma-derived sEV-mediated communication could adapt to microenvironmental stresses. We compared B16F1 cell-derived sEVs released under normal and stress conditions, including cytostatic, heat and oxidative stress. The miRNome and proteome showed substantial differences across the sEV groups and bioinformatics analysis of the obtained data by the Ingenuity Pathway Analysis also revealed significant functional differences. The in silico predicted functional alterations of sEVs were validated by in vitro assays. For instance, melanoma-derived sEVs elicited by oxidative stress increased Ki-67 expression of mesenchymal stem cells (MSCs); cytostatic stress-resulted sEVs facilitated melanoma cell migration; all sEV groups supported microtissue generation of MSC-B16F1 co-cultures in a 3D tumour matrix model. Based on this study, we concluded that (i) molecular patterns of tumour-derived sEVs, dictated by the microenvironmental conditions, resulted in specific response patterns in the recipient cells; (ii) in silico analyses could be useful tools to predict different stress responses; (iii) alteration of the sEV-mediated communication of tumour cells might be a therapy-induced host response, with a potential influence on treatment efficacy.

Melanoma-Derived Exosomes Induce PD-1 Overexpression and Tumor Progression via Mesenchymal Stem Cell Oncogenic Reprogramming.

Recently, it has been described that programmed cell death protein 1 (PD-1) overexpressing melanoma cells are highly aggressive. However, until now it has not been defined which factors lead to the generation of PD-1 overexpressing subpopulations. Here, we present that melanoma-derived exosomes, conveying oncogenic molecular reprogramming, induce the formation of a melanoma-like, PD-1 overexpressing cell population (mMSCPD-1+) from naïve mesenchymal stem cells (MSCs). Exosomes and mMSCPD-1+ cells induce tumor progression and expression of oncogenic factors in vivo. Finally, we revealed a characteristic, tumorigenic signaling network combining the upregulated molecules (e.g., PD-1, MET, RAF1, BCL2, MTOR) and their upstream exosomal regulating proteins and miRNAs. Our study highlights the complexity of exosomal communication during tumor progression and contributes to the detailed understanding of metastatic processes.

Gold nanoparticle-filled biodegradable photopolymer scaffolds induced muscle remodeling: in vitro and in vivo findings.

Therapeutic stem cell transplantation bears the promise of new directions in organ and tissue replacement, but a number of its difficulties and perils are also well known. Our goal was to develop a method of transplantation by which the transplanted cells remain confined to the transplantation site and induce favorable processes. With the help of mask-projection excimer laser stereolithography, 3D hybrid nanoscaffolds were fabricated from biodegradable, photocurable PPF:DEF resin with incorporated gold nanoparticles (Au NPs). The scaffolds were tested in vitro and in vivo in order to find out about their biocompatibility and fitness for our purposes. In vitro, macrophages and mouse autologous adipose stem cells (ASCs) were seeded over the hybrid scaffolds and non-hybrid (with Au NPs) scaffolds for 4days. The hybrid nanocomposite greater stem cell dispension and stem cell adhesion than PPF scaffolds without Au NPs, but such a difference was not seen in the case of macrophages. In vivo, stem cells, scaffoldings and scaffoldings covered in stem cells were transplanted under the back skin of mice. After 14days, blood samples were taken and the affected skin area was excised. Cytokine and chemokine profiling did not indicate elevated immunomediators in the sera of experimental animals. Interestingly, the autologous-stem-cell-seeded hybrid nanocomposite scaffold induced muscle tissue regeneration after experimental wound generation in vivo. We could not observe such stem cell-induced tissue regeneration when no scaffolding was used. We conclude that PPF:DEF resin nanoscaffolds with incorporated gold nanoparticles offer a safe and efficient alternative for the enhancement of local tissue remodeling. The results also support the idea that adipose derived stem cells are an optimal cell type for the purposes of regenerative musculoskeletal tissue engineering.