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Multi-platform Affinity Proteomics Identify Proteins Linked to Metastasis and Immune Suppression in Ovarian Cancer Plasma.

ABSTRACT: A central reason behind the poor clinical outcome of patients with high-grade serous carcinoma (HGSC) of the ovary is the difficulty in reliably detecting early occurrence or recurrence of this malignancy. Biomarkers that provide reliable diagnosis of this disease are therefore urgently needed. Systematic proteomic methods that identify HGSC-associated molecules may provide such biomarkers. We applied the antibody-based proximity extension assay (PEA) platform (Olink) for the identification of proteins that are upregulated in the plasma of OC patients. Using binders targeting 368 different plasma proteins, we compared 20 plasma samples from HGSC patients (OC-plasma) with 20 plasma samples from individuals with non-malignant gynecologic disorders (N-plasma). We identified 176 proteins with significantly higher levels in OC-plasma compared to N-plasma by PEA (p < 0.05 by U-test; Benjamini-Hochberg corrected), which are mainly implicated in immune regulation and metastasis-associated processes, such as matrix remodeling, adhesion, migration and proliferation. A number of these proteins have not been reported in previous studies, such as BCAM, CDH6, DDR1, N2DL-2 (ULBP2), SPINT2, and WISP-1 (CCN4). Of these SPINT2, a protease inhibitor mainly derived from tumor cells within the HGSC microenvironment, showed the highest significance (p < 2 × 10-7) similar to the previously described IL-6 and PVRL4 (NECTIN4) proteins. Results were validated by means of the aptamer-based 1.3 k SOMAscan proteomic platform, which revealed a high inter-platform correlation with a median Spearman ? of 0.62. Likewise, ELISA confirmed the PEA data for 10 out of 12 proteins analyzed, including SPINT2. These findings suggest that in contrast to other entities SPINT2 does not act as a tumor suppressor in HGSC. This is supported by data from the PRECOG and KM-Plotter meta-analysis databases, which point to a tumor-type-specific inverse association of SPINT2 gene expression with survival. Our data also demonstrate that both the PEA and SOMAscan affinity proteomics platforms bear considerable potential for the unbiased discovery of novel disease-associated biomarkers.

SUBMITTER: Graumann J

PROVIDER: S-EPMC6839336 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Multi-platform Affinity Proteomics Identify Proteins Linked to Metastasis and Immune Suppression in Ovarian Cancer Plasma.

Graumann Johannes J Finkernagel Florian F Reinartz Silke S Stief Thomas T Brödje Dörte D Renz Harald H Jansen Julia M JM Wagner Uwe U Worzfeld Thomas T Pogge von Strandmann Elke E Müller Rolf R

Frontiers in oncology 20191101

A central reason behind the poor clinical outcome of patients with high-grade serous carcinoma (HGSC) of the ovary is the difficulty in reliably detecting early occurrence or recurrence of this malignancy. Biomarkers that provide reliable diagnosis of this disease are therefore urgently needed. Systematic proteomic methods that identify HGSC-associated molecules may provide such biomarkers. We applied the antibody-based proximity extension assay (PEA) platform (Olink) for the identification of p ...[more]

PMID: 31737572

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Project description:The peritoneal fluid (ascites), replete with abundant tumor-promoting factors and extracellular vesicles (EVs) reflecting the tumor secretome, plays an essential role in ovarian high-grade serous carcinoma (HGSC) metastasis and immune suppression. A comprehensive picture of mediators impacting HGSC progression is, however, not available. Methods: Proteins in ascites from HGSC patients were quantified by the aptamer-based SOMAscan affinity proteomic platform. SOMAscan data were analyzed by bioinformatic methods to reveal clinically relevant links and functional connections, and were validated using the antibody-based proximity extension assay (PEA) Olink platform. Mass spectrometry was used to identify proteins in extracellular microvesicles released by HGSC cells. Results: Consistent with the clinical features of HGSC, 779 proteins in ascites identified by SOMAscan clustered into groups associated either with metastasis and a short relapse-free survival (RFS), or with immune regulation and a favorable RFS. In total, 346 proteins were linked to OC recurrence in either direction. Reanalysis of 214 of these proteins by PEA revealed an excellent median Spearman inter-platform correlation of ?=0.82 for the 46 positively RFS-associated proteins in both datasets. Intriguingly, many proteins strongly associated with clinical outcome were constituents of extracellular vesicles. These include proteins either linked to a poor RFS, such as HSPA1A, BCAM and DKK1, or associated with a favorable outcome, such as the protein kinase LCK. Finally, based on these data we defined two protein signatures that clearly classify short-term and long-term relapse-free survivors. Conclusion: The ascites secretome points to metastasis-promoting events and an anti-tumor response as the major determinants of the clinical outcome of HGSC. Relevant proteins include both bone fide secreted and vesicle-encapsulated polypeptides, many of which have previously not been linked to HGSC recurrence. Besides a deeper understanding of the HGSC microenvironment our data provide novel potential tools for HGSC patient stratification. Furthermore, the first large-scale inter-platform validation of SOMAscan and PEA will be invaluable for other studies using these affinity proteomics platforms.

Project description:BackgroundIntegrated analyses of plasma proteomic and genetic markers in prospective studies can clarify the causal relevance of proteins and discover novel targets for ischemic heart disease (IHD) and other diseases.ObjectivesThe purpose of this study was to examine associations of proteomics and genetics data with IHD in population studies to discover novel preventive treatments.MethodsWe conducted a nested case-cohort study in the China Kadoorie Biobank (CKB) involving 1,971 incident IHD cases and 2,001 subcohort participants who were genotyped and free of prior cardiovascular disease. We measured 1,463 proteins in the stored baseline samples using the OLINK EXPLORE panel. Cox regression yielded adjusted HRs for IHD associated with individual proteins after accounting for multiple testing. Moreover, cis-protein quantitative loci (pQTLs) identified for proteins in genome-wide association studies of CKB and of UK Biobank were used as instrumental variables in separate 2-sample Mendelian randomization (MR) studies involving global CARDIOGRAM+C4D consortium (210,842 IHD cases and 1,378,170 controls).ResultsOverall 361 proteins were significantly associated at false discovery rate <0.05 with risk of IHD (349 positively, 12 inversely) in CKB, including N-terminal prohormone of brain natriuretic peptide and proprotein convertase subtilisin/kexin type 9. Of these 361 proteins, 212 had cis-pQTLs in CKB, and MR analyses of 198 variants in CARDIOGRAM+C4D identified 13 proteins that showed potentially causal associations with IHD. Independent MR analyses of 307 cis-pQTLs identified in Europeans replicated associations for 4 proteins (FURIN, proteinase-activated receptor-1, Asialoglycoprotein receptor-1, and matrix metalloproteinase-3). Further downstream analyses showed that FURIN, which is highly expressed in endothelial cells, is a potential novel target and matrix metalloproteinase-3 a potential repurposing target for IHD.ConclusionsIntegrated analyses of proteomic and genetic data in Chinese and European adults provided causal support for FURIN and multiple other proteins as potential novel drug targets for treatment of IHD.

Project description:The ability to quantitatively probe diverse panels of proteins and their post-translational modifications (PTMs) across multiple samples would aid a broad spectrum of biological, biochemical and pharmacological studies. We report a novel, microarray analytical technology that combines immuno-affinity capture with Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS), which is capable of supporting highly multiplexed, targeted proteomic assays. Termed "Affinity-Bead Assisted Mass Spectrometry" (Affi-BAMS), this LC-free technology enables development of highly specific and customizable assay panels for simultaneous profiling of multiple proteins and PTMs. While affinity beads have been used previously in combination with MS, the Affi-BAMS workflow uses enrichment on a single bead that contains one type of antibody, generally capturing a single analyte (protein or PTM) while having enough binding capacity to enable quantification within approximately 3 orders of magnitude. The multiplexing capability is achieved by combining Affi-BAMS beads with different protein specificities. To enable screening of bead-captured analytes by MS, we further developed a novel method of performing spatially localized elution of targets from individual beads arrayed on a microscope slide. The resulting arrays of micro spots contain highly concentrated analytes localized within 0.5 mm diameter spots that can be directly measured using MALDI MS. While both intact proteins and protein fragments can be monitored by Affi-BAMS, we initially focused on applying this technology for bottom-up proteomics to enable screening of hundreds of samples per day by combining the robust magnetic bead-based workflow with the high throughput nature of MALDI MS acquisition. To demonstrate the variety of applications and robustness of Affi-BAMS, several studies are presented that focus on the response of 4EBP1, RPS6, ERK1/ERK2, mTOR, Histone H3 and C-MET to stimuli including rapamycin, H2O2, EPO, SU11274, Staurosporine and Vorinostat.

Dataset Information

Multi-platform Affinity Proteomics Identify Proteins Linked to Metastasis and Immune Suppression in Ovarian Cancer Plasma.

Publications

Multi-platform Affinity Proteomics Identify Proteins Linked to Metastasis and Immune Suppression in Ovarian Cancer Plasma.

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