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Background

Gastric cancer (GC), the world’s fifth most common malignancy, remains a major cause of death, motivating minimally invasive protein measurements. Small extracellular vesicles (sEVs) mirror cellular state but are difficult to profile from limited material. Proximity extension assay (PEA) enables multiplex, sensitive detection. We developed a PEA-centred workflow to profile GC cell-line sEVs, benchmarked against cells and an EV-rich control, and nominate hypothesis-generating candidates.

Methodss

EVs from four GC cell lines (Kato III, AGS, MKN45, MKN7) were isolated by differential centrifugation/filtration and ultracentrifugation; prostasomes (seminal-fluid EVs; ethics-approved) were purified by ultracentrifugation, Superdex 200 size-exclusion, and sucrose gradient. EVs were characterized by TEM, NTA, and Western blot (CD9/CD63/CD81; Calnexin). Protein profiling used Olink PEA across five Target 96 panels, two commercial (Oncology, Inflammation) and three experimental (Cancer, Cellular Pathways, Neurology) with availability-based selection; single replicate per condition. NPX values were background/LOD-corrected in R, with PCA. STRING PPI used medium confidence (0.4) with FDR-adjusted enrichment. Selected proteins were validated by SP-PLA (qPCR), EV-PLA (flow cytometry), and WB.

Resultss

EVs and prostasomes were purified and characterized per MISEV2023 (TEM/NTA; CD9/CD63/CD81+, calnexin–). Using five Olink PEA panels, 460 proteins were profiled; principal component analysis separated sEVs from parental cells. We identified 71 proteins enriched in GC sEVs, including EphA2, cMet, IL-6, SCF, AXIN1, NOTCH1, and CAV1; 17 were unique to Kato III sEVs. STRING showed a highly connected network, with top pathways: Pathways in cancer, Cytokine–cytokine receptor interaction, and PI3K–Akt. Orthogonal assays (SP-PLA, EV-PLA, WB densitometry) confirmed the presence of EphA2 and cMet on GC sEVs.

Discussions

PEA-based profiling of GC cell-line sEVs nominates 71 enriched proteins (e.g., EphA2, cMet, NOTCH1, CAV1) as hypothesis-generating candidates that may reflect cell-type–specific cargo and tumor–microenvironment signaling. Key limitations include the in-vitro design, single replicates, preselected PEA panels, and the use of prostasomes rather than healthy plasma sEVs, which may affect generalizability and specificity. Translational relevance requires validation in patient biofluids with orthogonal assays and cohort-level evaluation.

Extracellular vesicles (EVs), whose main subtypes are exosomes, microparticles, and apoptotic bodies, are secreted by all cells and harbor biomolecules such as DNA, RNA, and proteins. They function as intercellular messengers and, depending on their cargo, may have multiple roles in cancer development. Thymidine kinase 1 (TK1) is a cell cycle-dependent enzyme used as a biomarker for cell proliferation. TK1 is usually elevated in cancer patients' serum, making the enzyme a valuable tumor proliferation biomarker that strongly correlates with cancer stage and metastatic capabilities. Here, we investigated the presence of TK1 in EVs derived from three prostate cancer cell lines with various p53 mutation statuses (LNCaP, PC3, and DU145), EVs from the normal prostate epithelial cell line RWPE-1 and EVs isolated from human seminal fluid (prostasomes). We measured the TK1 activity by a real-time assay for these EVs. We demonstrated that the TK1 enzyme activity is higher in EVs derived from the malignant cell lines, with the highest activity from cells deriving from the most aggressive cancer, compared to the prostasomes and RWPE-1 EVs. The measurement of TK1 activity in EVs may be essential in future prostate cancer studies.

Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from & oslash;1.2 mm discs cut from dried blood and saliva spots (DBS and DSS) without the need for elution. For total Ig detection, antigen probes were prepared by conjugating recombinant spike protein subunit 1 (S1-RBD) to a pair of oligonucleotides. To detect isotype-specific antibody reactivity, one antigen probe was replaced with oligonucleotide-conjugated antibodies specific for antibody isotypes. Binding of pairs of oligonucleotide-conjugated probes to antibodies in patient samples brings oligonucleotides in proximity. An added DNA polymerase uses a transient hybridization between the oligonucleotides to prime synthesis of a DNA strand, which serves as a DNA amplicon that is quantified by real-time PCR. The S1-RBD-specific IgG, IgM, and IgA antibodies in DBS samples collected over the course of a first and second vaccination exhibited kinetics consistent with previous reports. Both DBS and DSS collected from 42 individuals in the autumn of 2023 showed significant level of total S1-RBD antibodies with a correlation of R = 0.70. However, levels in DSS were generally 10 to 100-fold lower than in DBS. Anti-S1-RBD IgG and IgA in DSS demonstrated a correlation of R = 0.6.

The glycoprotein CD47 regulates antiphagocytic activity via signal regulatory protein alpha (SIRPa). This study investigated CD47 expression on Hodgkin and Reed–Sternberg (HRS) cells in the classical Hodgkin lymphoma (cHL) tumour microenvironment and its correlation with prognosis, programmed-death (PD) immune markers, and SIRPa⁺ leukocytes. We conducted immunohistochemistry with CD47 and SIRPa antibodies on diagnostic biopsies (tissue microarrays) from cHL patients from two cohorts (n = 178). In cohort I (n = 136) patients with high expression of CD47 on HRS cells (n = 48) had a significantly inferior event-free survival [hazard ratio (HR) = 5.57; 95% confidence interval (CI), 2.78–11.20; p < 0.001] and overall survival (OS) (HR = 8.54; 95% CI, 3.19–22.90; p < 0.001) compared with patients with low expression (n = 88). The survival results remained statistically significant in multivariable Cox regression adjusted for known prognostic factors. In cohort II (n = 42) high HRS cell CD47 expression also carried shorter event-free survival (EFS) (HR = 5.96; 95% CI, 1.20–29.59; p = 0.029) and OS (HR = 5.61; 95% CI, 0.58–54.15; p = 0.136), although it did not retain statistical significance in the multivariable analysis. Further, high CD47 expression did not correlate with SIRPa⁺ leukocytes or PD-1, PD-L1 and PD-L2 expression. This study provides a deeper understanding of the role of CD47 in cHL during an era of emerging CD47 therapies.

A homogeneous PCR-based assay for sensitive and specific detection of antibodies in serum or dried blood spots (DBS) is presented and the method is used to monitor individuals infected with or vaccinated against SARS-CoV-2. Detection probes were prepared by conjugating the recombinant spike protein subunit 1 (S1), containing the receptor binding domain (RBD) of SARS-CoV-2, to each of a pair of specific oligonucleotides. The same was done for the nucleocapsid protein (NP). Upon incubation with serum or DBS samples, the bi- or multivalency of the antibodies (IgG, IgA or IgM) brings pairs of viral proteins with their conjugated oligonucleotides in proximity, allowing the antibodies to be detected by a modified proximity extension assay (PEA). Anti-S1 and anti-NP antibodies could be detected simultaneously from one incubation reaction. This Antibody PEA (AbPEA) test uses only 1 µl of neat or up to 100,000-fold diluted serum or one ø1.2 mm disc cut from a DBS. All 100 investigated sera and 21 DBS collected prior to the COVID-19 outbreak were negative, demonstrating a 100% specificity. The area under the curve, as evaluated by Receiver Operating Characteristic (ROC) analysis reached 0.998 (95%CI: 0.993–1) for samples taken from 11 days after symptoms onset. The kinetics of antibody responses were monitored after a first and second vaccination using serially collected DBS from 14 individuals. AbPEA offers highly specific and sensitive solution-phase antibody detection without requirement for secondary antibodies, no elution step when using DBS sample in a simple procedure that lends itself to multiplex survey of antibody responses.

In classical Hodgkin Lymphoma (cHL), immunoediting via protein signaling is key to evading tumor surveillance. We aimed to identify immune-related proteins that distinguish diagnostic cHL tissues (=diagnostic tumor lysates, n = 27) from control tissues (reactive lymph node lysates, n = 30). Further, we correlated our findings with the proteome plasma profile between cHL patients (n = 26) and healthy controls (n = 27). We used the proximity extension assay (PEA) with the OlinkTM multiplex Immuno-Oncology panel, consisting of 92 proteins. Univariate, multivariate-adjusted analysis and Benjamini–Hochberg’s false discovery testing (=Padj) were performed to detect significant discrepancies. Proteins distinguishing cHL cases from controls were more numerous in plasma (30 proteins) than tissue (17 proteins), all Padj < 0.05. Eight of the identified proteins in cHL tissue (PD-L1, IL-6, CCL17, CCL3, IL-13, MMP12, TNFRS4, and LAG3) were elevated in both cHL tissues and cHL plasma compared with control samples. Six proteins distinguishing cHL tissues from controls tissues were significantly correlated to PD-L1 expression in cHL tissue (IL-6, MCP-2, CCL3, CCL4, GZMB, and IFN-gamma, all p ≤0.05). In conclusion, this study introduces a distinguishing proteomic profile in cHL tissue and potential immune-related markers of pathophysiological relevance

Adenine nucleotide translocases (ANTs) and uncoupling proteins (UCPs) are known to facilitate proton leak across the inner mitochondrial membrane. However, it remains to be unravelled whether UCP2/3 contribute to significant amount of proton leak in vivo. Reports are indicative of UCP2 dependent proton-coupled efflux of C4 metabolites from the mitochondrial matrix. Previous studies have suggested that UCP2/3 knockdown (KD) contributes to increased ANT-dependent proton leak. Here we investigated the hypothesis that interaction exists between the UCP2 and ANT2 proteins, and that such interaction is regulated by the cellular metabolic demand. Protein-protein interaction was evaluated using reciprocal co-immunoprecipitation and in situ proximity ligation assay. KD of ANT2 and UCP2 was performed by siRNA in human embryonic kidney cells 293A (HEK293A) cells. Mitochondrial and cellular respiration was measured by high-resolution respirometry. ANT2-UCP2 interaction was demonstrated, and this was dependent on cellular metabolism. Inhibition of ATP synthase promoted ANT2-UCP2 interaction whereas high cellular respiration, induced by adding the mitochondrial uncoupler FCCP, prevented interaction. UCP2 KD contributed to increased carboxyatractyloside (CATR) sensitive proton leak, whereas ANT2 and UCP2 double KD reduced CATR sensitive proton leak, compared to UCP2 KD. Furthermore, proton leak was reduced in double KD compared to UCP2 KD. In conclusion, our results show that there is an interaction between ANT2-UCP2, which appears to be dynamically regulated by mitochondrial respiratory activity. This may have implications in the regulation of mitochondrial efficiency or cellular substrate utilization as increased activity of UCP2 may promote a switch from glucose to fatty acid metabolism.

Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug–target interactions at spatial resolution in protein arrays, cells and in tissues.

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.

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.