Dataset Information

Dual-platform affinity proteomics identifies links between the recurrence of ovarian carcinoma and proteins released into the tumor microenvironment.

ABSTRACT: 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.

SUBMITTER: Finkernagel F

PROVIDER: S-EPMC6771240 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Dual-platform affinity proteomics identifies links between the recurrence of ovarian carcinoma and proteins released into the tumor microenvironment.

Finkernagel Florian F Reinartz Silke S Schuldner Maximiliane M Malz Alexandra A Jansen Julia M JM Wagner Uwe U Worzfeld Thomas T Graumann Johannes J von Strandmann Elke Pogge EP Müller Rolf R

Theranostics 20190822 22

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. <b>Methods:</b> Proteins in ascites from HGSC patients were quantified by the aptamer-based SOMAscan affinity proteomic platform. SOMAscan data were analyzed by ...[more]

PMID: 31588238

Similar Datasets

Project description: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.

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.

Project description:There are no blood-based molecular biomarkers of temporal lobe epilepsy (TLE) to support clinical diagnosis. MicroRNAs are short noncoding RNAs with strong biomarker potential due to their cell-specific expression, mechanistic links to brain excitability, and stable and reliable detection in biofluids. Altered expression of circulating microRNAs has been reported in human epilepsy, but most studies collected samples from one clinical site, relied on a single platform for profiling or conducted minimal validation. We collected plasma samples from video-electroencephalogram-monitored adult TLE patients at epilepsy specialist centers in two different countries, performed genome-wide PCR-based and RNA sequencing during the discovery phase and validated in a large cohort of samples (>300 samples) that included patients with psychogenic non-epileptic seizures. After profiling, validation of the discovery cohort and validation in the larger patient groups we identified miR-27a-3p, miR-328-3p and miR-654-3p with strong TLE biomarker potential. Plasma levels of these microRNAs were regulated in the same direction in plasma from epileptic mice, and furthermore were not different to healthy controls in patients with psychogenic non-epileptic seizures. The biomarker potential was extended by determining microRNA copy number in plasma and we demonstrate rapid detection of these microRNAs using an electrochemical RNA microfluidic disk as a prototype point-of-care device. Investigation of the molecular transport mechanism in plasma determined analysis of all three microRNAs within the exosome-enriched provided highest diagnostic accuracy while levels of Argonaute-bound miR-328-3p selectively increased in patient samples collected after seizures. In situ hybridization revealed the presence of miR-27a-3p and miR-328-3p within neurons in human brain and bioinformatics analysis predicted targets linked to growth factor signaling and apoptosis. Taken together, this study extends evidence for the biomarker potential of circulating microRNAs for epilepsy diagnosis and mechanistic links to underlying pathomechanisms. microRNA expression in the plasma of 16 patients with TLE, before and after seizure, and 16 controls was measured by TaqMan OpenArray Human MicroRNA Panel.

Dataset Information

Dual-platform affinity proteomics identifies links between the recurrence of ovarian carcinoma and proteins released into the tumor microenvironment.

Publications

Dual-platform affinity proteomics identifies links between the recurrence of ovarian carcinoma and proteins released into the tumor microenvironment.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets