Biology subjects

Hallal, S.

Publications and source records attributed to Hallal, S..

3 recordsLinked to original sources

bioRxiv2026

TriPort tangential flow filtration: a lab-assembled system for EV isolation across diverse cell-culture sources

Tangential flow filtration (TFF) is an effective method for scalable EV isolation. Here, we describe and validate a reference protocol for TriPort-TFF, a laboratory-assembled TFF system featuring a three- port reservoir design enabling sample circulation, retentate return, and diafiltration. Constructed from individually sourced, commercially available components with detailed assembly and operating parameters, TriPort-TFF can be built at a lower cost than commercially integrated TFF platforms. We evaluated the system using conditioned media (CM) from human MDM cultured with EV-depleted human serum, human cerebral organoids in serum-free medium, and mouse BMDM cultured with EV-depleted foetal bovine serum (FBS) unstimulated or LPS-stimulated. The system reproducibly isolated EVs from all three sources, and biological replicates of BMDM showed distinct proteomic clusters between LPS-stimulated and unstimulated conditions. The requirement for subsequent size-exclusion chromatography (SEC) after TFF depended on the sample serum-presence. In serum-containing CM, TFF alone co-concentrated serum proteins alongside EVs, subsequent SEC improved the particle-to- protein ratio, reduced inter-replicate variability and improved proteomic depth. TFF alone in serum- free organoid CM already yielded higher particle-to-protein ratios than TFF-only preparations from serum-containing CM, with SEC further improving this ratio. TriPort-TFF offers an accessible system for EV-isolation that can be combined with SEC for diverse sample types.

cell biology↗

bioRxiv2020

A comprehensive proteomic SWATH-MS workflow for profiling blood extracellular vesicles: a new avenue for glioma tumour surveillance

There is a real need for biomarkers that can indicate glioma disease burden and inform clinical management, particularly in the recurrent glioblastoma (GBM; grade IV glioma) setting where treatment-associated brain changes can confound current and expensive tumour surveillance methods. In this regard, extracellular vesicles (EVs; 30-1000 nm membranous particles) hold major promise as robust tumour biomarkers. GBM-EVs encapsulate molecules that reflect the identity and molecular state of their cell-of-origin and cross the blood-brain-barrier into the periphery where they are readily accessible. Despite the suitability of circulating-EVs for GBM biomarker discovery, sample complexity has hindered comprehensive quantitative proteomic studies. Here, sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) was used in conjunction with a targeted data extraction strategy to comprehensively profile circulating-EVs isolated from plasma. Plasma-EVs sourced from pre-operative glioma II-IV patients (n=41) and controls (n=11) were sequenced by SWATH-MS, and the identities and absolute quantities of the proteins were extracted by aligning the SWATH-MS data against a custom glioma spectral library comprised of 8662 high confidence protein species. Overall, 4054 plasma-EV proteins were quantified across the cohorts, and putative circulating-EV biomarker proteins identified (adjusted p-value<0.05) included previously reported GBM-EV proteins identified in vitro and in neurosurgical aspirates. Principle component analyses showed that plasma-EV protein profiles clustered according to glioma subtype and WHO-grade, and plasma-EV proteins reflected the extent of glioma aggression. Using SWATH-MS, we describe the most comprehensive proteomic plasma-EV profiles for glioma and highlight the promise of this approach as an accurate and sensitive tumour monitoring method. Objective blood-based measurements of glioma tumour activity will support the implementation of next-generation, patient-centred therapies and are ideal surrogate endpoints for recurrent progression that would allow clinical trial protocols to be more dynamic and adapt to the individual patient and their cancer.

pathology↗

bioRxiv2018

Deep sequencing of circulating exosomal microRNA allows non-invasive glioblastoma diagnosis

Exosomes are nano-sized extracellular vesicles released by many cells that contain molecules characteristic of their cell-of-origin, including microRNA. Exosomes released by glioblastoma cross the blood-brain-barrier into the peripheral circulation, and carry molecular cargo distinct to that of free-circulating miRNA. In this pilot study, serum exosomal-microRNAs were isolated from glioblastoma (n=12) patients and analyzed using unbiased deep sequencing. Results were compared to sera from age- and gender-matched healthy controls, and to grades II-III (n=10) glioma patients. Significant differentially expressed microRNAs were identified, and the predictive power of individual and subsets of microRNAs were tested using univariate and multivariate analyses. Additional sera from glioblastoma patients (n=4) and independent sets of healthy (n=9) and non-glioma (n=10) controls were used to further test the specificity and predictive power of this unique exosomal-microRNA signature. Twenty-six microRNAs were differentially expressed in serum exosomes from glioblastoma patients relative to healthy controls. Random forest modeling and data partitioning selected seven miRNAs (miR-182-5p, miR-328-3p, miR-339-5p, miR-340-5p, miR-485-3p, miR-486-5p and miR-543) as the most stable for classifying glioblastoma. Strikingly, within this model, six iterations of these miRNA classifiers could distinguish glioblastoma patients from controls with perfect accuracy. The seven-miRNA panel was able to correctly classify all specimens in validation cohorts (n=23). Also identified were 23 dysregulated miRNAs in IDHMUT gliomas, a partially overlapping yet distinct signature of lower grade glioma. Serum exosomal-miRNA signatures can accurately diagnose glioblastoma preoperatively. miRNA signatures identified are distinct from previously reported free-circulating miRNA studies in GBM patients, and appear to be superior.

pathology↗