Innovative methodology for the identification of soluble biomarkers in fresh tissues

Abstract

The identification of diagnostic and prognostic biomarkers from early lesions, measurable in liquid biopsies remains a major challenge, particularly in oncology. Fresh human material of high quality is required for biomarker discovery but is often not available when it is totally required for clinical pathology investigation. Hence, all OMICs studies are done on residual and less clinically relevant biological samples. Here after, we present an innovative, simple, and non-destructive, procedure named EXPEL that uses rapid, pressure-assisted, interstitial fluid extrusion, preserving the specimen for full routine clinical pathology investigation. In the meantime, the technique allows a comprehensive OMICs analysis (proteins, metabolites, miRNAs and DNA). As proof of concept, we have applied EXPEL on freshly collected human colorectal cancer and liver metastases tissues. We demonstrate that the procedure efficiently allows the extraction, within a few minutes, of a wide variety of biomolecules holding diagnostic and prognostic potential while keeping both tissue morphology and antigenicity unaltered. Our method enables, for the first time, both clinicians and scientists to explore identical clinical material regardless of its origin and size, which has a major positive impact on translation to the clinic.

Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

“…Consistent with the long-time observations that most tumors cells display an altered redox status, 52 the malignant cells used in this study have been shown to represent a major source of ROS ( figure 6A, B ). Using a novel innovative methodology (called EXPEL), 43 soluble proteins contained in tumor-extruded fluids were retrieved and directly alkylated in order to maintain their original redox state. As shown in figure 6C, D , all HMGB1 forms are encountered within tumor microenvironment with an approximate 2:1 reduced-disulfide/oxidized ratio.…”

“…When the volume reached 1000 mm 3 , tumors were retrieved and soluble proteins/interstitial fluids were collected using a pressure-assisted innovative methodology (EXPEL) recently described. 43 In order to irreversibly freeze the redox state of HMGB1 molecules contained in tumor-extruded fluids, an alkylation step (50 mM iodoacetamide for 30 min at 25°C) was then directly conducted. Proteins were quantified using the Pierce BCA protein assay (ThermoFisher Scientific) and 15 µg were separated by electrophoresis on 10% polyacrylamide gels and transferred onto polyvinylidene difluoride membranes.…”

Normalization Method Utilizing Endogenous Proteins for Quantitative Proteomics

“…Using a normalization method based on selection and validation of EPNS, we identified five protein markers (NUP205, CNN2, GNL3, GTPBP4, S100A11) that may be useful to distinguish colorectal cancer from normal tissues. Compared with other cancer biomarker screening methods, − we rely on machine learning for marker selection to improve the accuracy, rather than finding the markers that are differentially expressed. From the literature, all biomarkers selected by our approach have been reported to be associated with the prognosis of colorectal cancer.…”

New Proteins Contributing to Immune Cell Infiltration and Pannus Formation of Synovial Membrane from Arthritis Diseases

“…Each sample was then spiked with a standard MassPREP digestion mixture (MPDS Mix) (Waters Corp., Milford, MA, USA) at a quantity of 150 fmoles of ADH digest per injection. All samples were injected on a 2D-nanoAquity UPLC (Waters, Corp., Milford, MA, USA) coupled online with an ESI-Q-Orbitrap (Q Exactive, Thermo Fisher Scientific, Waltham, MA, USA) in positive ion mode, as previously described [ 72 ]. Briefly, for the 2-dimensional method (2D-LC), three steps were applied on a high pH column with an increasing percentage of acetonitrile.…”