Profiling of metalloprotease activities in cerebrospinal fluids of patients with neoplastic meningitis

Abstract

BackgroundNeoplastic invasion into leptomeninges and subarachnoid space, resulting in neoplastic meningitis (NM) is a fatal complication of advanced solid and hematological neoplasms. Identification of malignant involvement of the cerebrospinal fluid (CSF) early in the disease course has crucial prognostic and therapeutic implications, but remains challenging. As indicators of extracellular matrix (ECM) degradation and breakdown of the blood–brain-barrier, Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloproteases (ADAMs) are potential analytes for cerebral pathophysiology and metastatic dissemination of tumor cells into the CSF.MethodsWe compared protease activities in CSF samples from patients with NM and control individuals using FRET-based metalloprotease substrates with distinct enzyme selectivity profiles in a real-time, multiplex approach termed “proteolytic activity matrix assay” (PrAMA). Protease activity dynamics can be tracked by fluorescence changes over time. By simultaneously monitoring a panel of 5 FRET-substrate cleavages, a proteolytic signature can be identified and analyzed to infer the activities of multiple specific proteases. Distinct patterns of substrate cleavage comparing disease vs. control samples allow rapid, reproducible and sensitive discrimination even in small volumes of CSF.ResultsIndividual substrate cleavage rates were linked to distinct proteases, and PrAMA computational inference implied increased activities of MMP-9, ADAM8 and ADAM17 (4–5-fold on average) in CSF samples from NM patients that were inhibitable by the metalloprotease inhibitor batimastat (BB-94). The activities of these proteases correlated with blood–brain barrier impairment. Notably, CSF cell counts were not found to directly reflect the protease activities observed in CSF samples from NM patients; this may explain the frequent clinical observation of negative cytology in NM patients.ConclusionPrAMA analysis of CSF samples is a potential diagnostic method for sensitive detection of NM and may be suitable for the clinical routine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12987-017-0070-5) contains supplementary material, which is available to authorized users.

Concepts of extracellular matrix remodelling in tumour progression and metastasis

“…Cleavage patterns of FRET-based polypeptides thereby reflect specific enzyme activities when compared to purified proteases as standard. This method allows the detection of enhanced protease activity in patient samples such as cerebrospinal fluid, which is indicative of neoplastic meningitis and metastasis to the brain 117 .…”

Leptomeningeal metastases from non-small cell lung cancer: state of the art and recent advances

“…In particular, C3 promotes the disruption of blood-CSF barrier, leading to the passage of some mitogens, such as amphiregulin, that promotes tumor cell growth within leptomeninges [39] . Similarly, Matrix Metalloproteases (MMPs) type 9, A Disintegrin and Metalloproteases (ADAMs) type 8 and 17 interfere with the integrity of the blood-CSF barrier, facilitating the passage of tumor cells into the subarachnoid space [40] . Some driver mutations select clonal tumor cells making them more prone to metastasize to the CNS.…”

New Frontiers in Diagnosis and Therapy of Circulating Tumor Markers in Cerebrospinal Fluid In Vitro and In Vivo

“…In the past few years, the clinical potential of some other technological platforms including microfluidic technology, immunomagnetic platform, high performance liquid chromatography-mass spectrometry, next generation sequencing (NGS) and proteolytic activity matrix assay (PrAMA) has also been demonstrated [25,50,55,66,67,68,69]. Despite interest and promises, the singularity of these reports does not allow yet making conclusions on suitability of these methods to improve prognosis in patients.Overall, despite CSF liquid biopsy is expected to yield clinically significant biomarkers and assays, the main drawback to all aforementioned approaches in vitro is that their sensitivity is substantially limited by the volume of the sample [70,71].…”