Surface antigen profiles of leukocytes and melanoma cells in lymph node metastases are associated with survival in AJCC stage III melanoma patients

There is an urgent need to identify more accurate prognostic biomarkers in melanoma patients, particularly in those with metastatic disease. This study aimed to identify melanoma and leukocyte surface antigens predictive of survival in a prospective series of AJCC stage IIIb/c melanoma patients (n = 29). Live cell suspensions were prepared from melanoma metastases within lymph nodes (LN). The suspensions were immuno-magnetically separated into CD45+ (leukocyte) and CD45− (non-hematopoietic, enriched melanoma cell) fractions. Surface antigens on CD45− and CD45+ cell populations were profiled using DotScan™ microarrays (Medsaic Pty. Ltd.) and showed differential abundance levels for 52 and 78 antigens respectively. Associations of the surface profiles with clinicopathologic and outcome data (median follow-up 35.4 months post LN resection) were sought using univariate (log-rank test) and multivariate (Wald’s test; modelled with patient’s age, gender and AJCC staging at LN recurrence) survival models. CD9 (p = 0.036), CD39 (p = 0.004) and CD55 (p = 0.005) on CD45+ leukocytes were independently associated with distant metastasis-free survival using multivariate analysis. Leukocytes with high CD39 levels were also significantly associated with increased overall survival (OS) in multivariate analysis (p = 0.016). LNs containing leukocytes expressing CD11b (p = 0.025), CD49d (p = 0.043) and CD79b (p = 0.044) were associated with reduced OS on univariate analysis. For enriched melanoma cells (CD45− cell populations), 11 surface antigens were significantly correlated with the disease-free interval (DFI) between diagnosis of culprit primary melanoma and LN metastasis resection. Nine antigens on CD45+ leukocytes also correlated with DFI. Following validation in independent datasets, surface markers identified here should enable more accurate determination of prognosis in stage III melanoma patients and provide better risk stratification of patients entering clinical trials

Cell-derived extracellular vesicles can be used as a biomarker reservoir for glioblastoma tumor subtyping

Glioblastoma (GBM) is one of the most aggressive solid tumors for which treatment options and biomarkers are limited. Small extracellular vesicles (sEVs) produced by both GBM and stromal cells are central in the inter-cellular communication that is taking place in the tumor bulk. As tumor sEVs are accessible in biofluids, recent reports have suggested that sEVs contain valuable biomarkers for GBM patient diagnosis and follow-up. The aim of the current study was to describe the protein content of sEVs produced by different GBM cell lines and patient-derived stem cells. Our results reveal that the content of the sEVs mirrors the phenotypic signature of the respective GBM cells, leading to the description of potential informative sEV-associated biomarkers for GBM subtyping, such as CD44. Overall, these data could assist future GBM in vitro studies and provide insights for the development of new diagnostic and therapeutic methods as well as personalized treatment strategies

Proteomic insights into gliobastoma tumour invasion

Glioblastoma (GBM) is the most common, primary malignant brain tumours in adults. A better understanding of GBM biology is required to identify targets and develop new therapeutics. Tumour invasion is facilitated by cell migration and degradation of the extracellular matrix (ECM). Invadopodia are actin-rich organelles that degrade the ECM and thereby actively re-modelling the surrounding tumour microenvironment. We have characterised the invasiveness of nine established GBM cell lines using an invadopodia assay and performed quantitative MS-based proteomic analyses on enriched membrane fractions. All GBM cells produced invadopodia to some degree, with U87MG the most and LN229 the least invasive cells. Overall, 1,141 proteins were identified and the abundance levels of 49 proteins correlated with invasiveness, many of which were previously linked to invadopodia formation, epithelial-mesenchymal transition and GBM cell invasion. Invadopodia act as multi-vesicular endosome docking sites and have been shown to be a site of exosome release. Overall, 844 proteins were identified in the extracellular vesicles (EVs) protein profiles, where approximately 50% were also identified in the membrane dataset. The abundance levels of 14 proteins correlated with the invasiveness. GBM-derived EVs can cross the blood brain barrier and are detectable in peripheral blood, the profiles and significant markers presented here could be of interest to diagnostics. Bioinformatics, Western blot analysis, co-localisation immunofluorescence and a siRNA knockdown substantiated some of interesting membrane and EV proteomics findings. In silico analysis of publically available gene databanks demonstrated the clinical prognostic significant of several invasion-related targets identified in these analyses. GBM invasion is regulated by a dynamic cross-talk between tumour cells and the brain microenvironment. After confirming the uptake of fluorescently labelled GBM-EVs, astrocytes were observed to assume a more invasive phenotype, using the same invadopodia assay. Whole cell proteome analysis of astrocyte before and after exposure to GBM EVs provides insight into the intercellular communication between GBM cells and surrounding normal astrocytes. This study highlights the signalling pathways that contribute to GBM invasion and may help to understand the aggressiveness of this disease

Proteomic insights into gliobastoma tumour invasion

Glioblastoma (GBM) is the most common, primary malignant brain tumours in adults. A better understanding of GBM biology is required to identify targets and develop new therapeutics. Tumour invasion is facilitated by cell migration and degradation of the extracellular matrix (ECM). Invadopodia are actin-rich organelles that degrade the ECM and thereby actively re-modelling the surrounding tumour microenvironment. We have characterised the invasiveness of nine established GBM cell lines using an invadopodia assay and performed quantitative MS-based proteomic analyses on enriched membrane fractions. All GBM cells produced invadopodia to some degree, with U87MG the most and LN229 the least invasive cells. Overall, 1,141 proteins were identified and the abundance levels of 49 proteins correlated with invasiveness, many of which were previously linked to invadopodia formation, epithelial-mesenchymal transition and GBM cell invasion. Invadopodia act as multi-vesicular endosome docking sites and have been shown to be a site of exosome release. Overall, 844 proteins were identified in the extracellular vesicles (EVs) protein profiles, where approximately 50% were also identified in the membrane dataset. The abundance levels of 14 proteins correlated with the invasiveness. GBM-derived EVs can cross the blood brain barrier and are detectable in peripheral blood, the profiles and significant markers presented here could be of interest to diagnostics. Bioinformatics, Western blot analysis, co-localisation immunofluorescence and a siRNA knockdown substantiated some of interesting membrane and EV proteomics findings. In silico analysis of publically available gene databanks demonstrated the clinical prognostic significant of several invasion-related targets identified in these analyses. GBM invasion is regulated by a dynamic cross-talk between tumour cells and the brain microenvironment. After confirming the uptake of fluorescently labelled GBM-EVs, astrocytes were observed to assume a more invasive phenotype, using the same invadopodia assay. Whole cell proteome analysis of astrocyte before and after exposure to GBM EVs provides insight into the intercellular communication between GBM cells and surrounding normal astrocytes. This study highlights the signalling pathways that contribute to GBM invasion and may help to understand the aggressiveness of this disease

Comprehensive proteomic profiling of bevacizumab-resistant glioblastoma multiforme

Drugs that impair tumour angiogenesis, i.e. therapeutic antibody antivascular endothelial growth factor, bevacizumab (BEV), are becoming standard therapy for recurrent GBM, despite having no impact on overall survival times. Resistance to BEV is fatal, and mechanisms are largely unexplored. With access to exceedingly rare fresh-frozen serial GBM tumours, we performed comprehensive quantitative proteome analyses to identify important mechanisms of BEV escape and tumour recurrence. Tumour tissues from three patients [primary (n = 2), recurrent (n = 2) and post-BEV recurrent (n = 3)] were homogenised, clarified (1,000 x g, 4°C) and ultracentrifuged (100,000 x g, 4°C) to isolate the soluble (SOL) proteome supernatant from the microsomal (MEM) pellet. Digested SOL andMEMproteomes were analysed by two independent quantitative MS/MS approaches; Label-free quantitation performed on spectra obtained in triplicate using an Orbitrap Velos (Thermo Electron) and 4-plex iTRAQ-labelling coupled ERLIC-RP MS/MS analysis using a 5600 TripleTOFw (AB Sciex; single run for MEM; duplicate run for SOL). Spectra were processed using Mascot Distiller, Progenesis, Scaffold and ProteinPilotTM softwares. This multi-centre proteomics project has achieved a number of highly reproducible and comprehensive quantitative proteome datasets (average of 1760 MEM proteins and 2334 SOL proteins identified at 95% confidence levels) from precious serial GBM specimens. Significant differentially abundant proteins include those involved in Rho regulation of actin-based motility and cytoskeleton and endocytosis signalling. Bioinformatics analyses with captured whole exome sequencing data are underway to define novel mechanisms of evasive resistance to BEV in recurrent GBM.1 page(s