A robust strategy for proteomic identification of biomarkers of invasive phenotype complexed with extracellular heat shock proteins

Abstract

As an extension of their orchestration of intracellular pathways, secretion of extracellular heat shock proteins (HSPs) is an emerging paradigm of homeostasis imperative to multicellular organization. Extracellular HSP is axiomatic to the survival of cells during tumorigenesis; proportional representation of specific HSP family members is indicative of invasive potential and prognosis. Further significance has been added by the knowledge that all cancer-derived exosomes have surface-exposed HSPs that reflect the membrane topology of cells that secrete them. Extracellular HSPs are also characteristic of chronic inflammation and sepsis. Accordingly, interrogation of extracellular HSPs secreted from cell culture models may represent a facile means of identifying translational biomarker signatures for targeting in situ. In the current study, we evaluated a simple peptide-based multivalent HSP affinity approach using the Vn96 peptide for low speed pelleting of HSP complexes from bioreactor cultures of cell lines with varying invasive phenotype in xenotransplant models: U87 (glioblastoma multiforme; invasive); HELA (choriocarcinoma; minimally invasive); HEK293T (virally transformed immortalized; embryonic). Proteomic profiling by bottom-up mass spectrometry revealed a comprehensive range of candidate biomarkers including primary HSP ligands. HSP complexes were associated with additional chaperones of prognostic significance such as protein disulfide isomerases, as well as pleiotropic metabolic enzymes, established as proportionally reflective of invasive phenotype. Biomarkers of inflammatory and mechanotransductive phenotype were restricted to the most invasive cell model U87, including chitinase CHI3L1, lamin C, amyloid derivatives, and histone isoforms.

Proteomic Characterization of Two Extracellular Vesicle Subtypes Isolated from Human Glioblastoma Stem Cell Secretome by Sequential Centrifugal Ultrafiltration

“…Moreover, we detected FKBP4, the upregulated expression of which was also found in GBM [ 56 ]. Additionally, the presence of Lamin A/C isoforms together with HSP was found in GBM cell lines U87, which may represent an index of disease progression [ 74 , 75 ].…”

Enriching extracellular vesicles for mass spectrometry

“…In addition to antibody reagents, vesicles have been selected using peptides (Ghosh et al, 2014; Knol et al, 2016; Taylor et al, 2020) or heparin (Balaj et al, 2015; Reiter et al, 2019; Wang & Turko, 2018). The most widely reported use of a peptide affinity reagent is Vn96, a peptide designed to have high affinity for heat shock proteins (Griffiths et al, 2017, 2019). Incubation of samples with µM concentrations of the Vn96 peptide causes aggregation of vesicles so that they can be pelleted by low‐speed centrifugation (Ghosh et al, 2014; Griffiths et al, 2017, 2019; Knol et al, 2016).…”

“…The most widely reported use of a peptide affinity reagent is Vn96, a peptide designed to have high affinity for heat shock proteins (Griffiths et al, 2017, 2019). Incubation of samples with µM concentrations of the Vn96 peptide causes aggregation of vesicles so that they can be pelleted by low‐speed centrifugation (Ghosh et al, 2014; Griffiths et al, 2017, 2019; Knol et al, 2016). The limitation of the Vn96 affinity enrichment strategy is that the presence of heat shock proteins on exosome vesicles has not been established to be a universal feature.…”

Characterization of miRNAs in Extracellular Vesicles Released From Atlantic Salmon Monocyte-Like and Macrophage-Like Cells

“…EVs were isolated using the Vn96 peptide (New England Peptide, Gardner, MA, USA) following the manufacturer’s instructions. Vn96 binds to at least 5 unique HSPs secreted by a variety of different cell types ( 55 , 56 ). In addition, Vn96 isolates EVs with reduced contamination from protein aggregates or lipoproteins, compared to other methods of EV isolation (i.e.…”