Identification of novel candidate circulating biomarkers for malignant soft tissue sarcomas: Correlation with metastatic progression

Abstract

Soft tissue sarcomas (STS) are a heterogeneous group of rare tumors for which identification and validation of biological markers may improve clinical management. The fraction of low-molecular-weight (LMW) circulating proteins and fragments of proteins is a rich source of new potential biomarkers. To identify circulating biomarkers useful for STS early diagnosis and prognosis, we analyzed 53 high-grade STS sera using hydrogel core-shell nanoparticles that selectively entrap LMW proteins by size exclusion and affinity chromatography, protect them from degradation and amplify their concentration for mass spectrometry detection. Twenty-two analytes mostly involved in inflammatory and immunological response, showed a progressive increase from benign to malignant STS with a relative difference in abundance, more than 50% when compared to healthy control. 16 of these were higher in metastatic compared to non-metastatic tumors. Cox's regression analysis revealed a statistical significant association between the abundance of lactotransferrin (LTF) and complement factor H-related 5 (CFHR5) and risk of metastasis. In particular, CFHR5 was associated with the risk of metastasis. The role of circulating proteins involved in metastatic progression will be crucial for a better understanding of STS biology and patient management.

Circulating Candidate Biomarkers in Giant Cell Tumors of Bone

“…Particle synthesis and bait coupling were performed to eliminate high abundant proteins and concentrate LMW proteins and/or peptides present in low‐abundance in serum, as described previously …”

“…To test protein binding and sieving properties of the dye‐loaded particles, they were incubated in a protein solution consisting of a combination of molecular weight markers . Image analysis of each gel was performed with a PC scanner (HP ScanJet 5400c, Hewlett‐Packard, Palo Alto, CA).…”

“…In order to evaluate the nanoparticle capture workflow process performance and the reproducibility of the data, 500 μL of serum were centrifuged at 4 °C, and diluted 1:2 with 50 mM Tris–HCl, pH 7.0. Insulin‐like growth factor 1 (IGF1) (Antigenix America; MW = 7.6kDa) and chemokine CXCL12 (SDF‐1β) (Antigenix America; MW = 11kDa) were added to the sera as internal standards . Tumor serum was spiked with CXCL12/SDF‐1 at 0.01 μg μL –1 concentration, while 0.1 μg μL –1 SDF‐1β were added to control serum.…”

“…Subsequently, nanoparticles were incubated with each diluted serum sample for 30 min at room temperature under slow rotation, and centrifugation for 20 min at 25 °C. Then, the pellets were processed as previously described, dried and stored at –20 °C until use. Eluted proteins from the nanoparticles were reconstituted, digested, and purified as described …”

“…As serum constantly perfuses tissues, circulating serum biomarkers derived from proteolytic activity of tumor cells may be useful for early diagnosis and prognosis as they are indicators of pathway activity . In particular, low molecular weight (LMW) serum proteins and protein fragments are associated with pathological conditions, such as cancer, diabetes, and infectious diseases, probably reflecting the state of cells or tissues .…”