Platelets constitute a major reservoir of platelet-derived growth factor B (PDGFB) and are continuously activated in the tumor microenvironment, exposing tumors to the plethora of growth factors contained in platelet granules. To address the specific role of platelet-derived PDGFB in the tumor microenvironment, we have created a mouse model with conditional knockout of PDGFB in platelets (pl-PDGFB KO). Lack of PDGFB in platelets resulted in 10-fold lower PDGFB concentration in the tumor microenvironment, fewer cancer-associated fibroblasts and reduced deposition of the extracellular matrix (ECM) molecules fibronectin and collagen I in the orthotopic RIP1-Tag2 model for pancreatic neuroendocrine cancer. Myosin light chain phosphorylation, promoting cell contraction and, consequently, the mechano-induced release of active transforming growth factor (TGF) β from extracellular compartments, was reduced in tumors from pl-PDGFB KO mice. In agreement, TGFβ signaling, measured as phosphorylated Smad2, was significantly hampered in tumors from mice lacking PDGFB in their platelets, providing a plausible explanation for the reduced deposition of extracellular matrix. These findings indicate a major contribution of platelet-derived PDGFB to a malignant transformation of the tumor microenvironment and address for the first time the role of PDGFB released specifically from platelets in the remodeling of the ECM in tumors.

Extracellular vesicles (EVs) are mediators of intercellular communication and a promising class of biomarkers. Surface proteins of EVs play decisive roles in establishing a connection with recipient cells, and they are putative targets for diagnostic assays. Analysis of the surface proteins can thus both illuminate the biological functions of EVs and help identify potential biomarkers. We developed a strategy combining high-resolution mass spectrometry (HRMS) and proximity ligation assays (PLA) to first identify and then validate surface proteins discovered on EVs. We applied our workflow to investigate surface proteins of small EVs found in seminal fluid (SF-sEV). We identified 1,014 surface proteins and verified the presence of a subset of these on the surface of SF-sEVs. Our work demonstrates a general strategy for deep analysis of EVs’ surface proteins across patients and pathological conditions, proceeding from unbiased screening by HRMS to ultra-sensitive targeted analyses via PLA.

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1⁺ group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1⁺ NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.

Influenza A virus belongs to the Orthomyxoviridae family and, to date, is one of the most important pathogens causing acute respiratory infections, such as the recent pandemic of 2009. Hemagglutinin (HA) is one of the surface proteins of the virus that allow it to interact with cellular molecules. Due to the fact that it is the most abundant protein in the virus capsule, it is the best target in the detection of the Influenza A H1N1 virus through biosensing devices. Our aim is to develop an electrochemical biosensor to detect H1 by modifying carbon screen-printed electrodes (CSPE) with gold nanoparticles and to add further functionalization with monoclonal antibodies that are specific to this protein. The electrodes were characterized by the means of cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy. Our preliminary results suggest that the selected monoclonal antibodies have acceptable affinity and bind effectively to the H1 protein and that the electrodes have a wide potential window in the presence of [Fe(CN)₆]^3−/4−. In the future, we will continue to develop this biosensor in hope that it will be commercialized and be common in medical procedures during flu seasons and future influenza pandemics.

Host proteins incorporated into virus particles have been reported to contribute to infectivity and tissue-tropism. This incorporation of host proteins is expected to be variable among viral particles, however, protein analysis at single-virus levels has been challenging. We have developed a method to detect host proteins incorporated on the surface of virions using the in situ proximity ligation assay (isPLA) with rolling circle amplification (RCA), employing oligonucleotide-conjugated antibody pairs. The technique allows highly selective and sensitive antibody-based detection of viral and host proteins on the surface of individual virions. We detected recombinant noninfectious sub-viral particles (SVPs) of tick-borne encephalitis virus (TBEV) immobilized in microtiter wells as fluorescent particles detected by regular fluorescence microscopy. Counting the particles in the images enabled us to estimate individual TBEVSVP counts in different samples. Using isPLA we detected individual calnexin-, CD9-, CD81-, CD29-and CD59-positive SVPs among the viral particles. Our data suggests that a diversity of host proteins may be incorporated into TEBV, illustrating that isPLA with digital counting enables single-virus analysis of host protein incorporation. (C) 2020 The Authors. Published by Elsevier Inc.