A Proteomic Examination of Plasma Extracellular Vesicles Across Colorectal Cancer Stages Uncovers Biological Insights That Potentially Improve Prognosis

Abstract

Background: Recent advancements in understanding plasma extracellular vesicles (EVs) and their role in disease biology have provided additional unique insights into the study of Colorectal Cancer (CRC).

Methods: This study aimed to gain biological insights into disease progression from plasma-derived extracellular vesicle proteomic profiles of 80 patients (20 from each CRC stage I-IV) against 20 healthy age- and sex-matched controls using a high-resolution SWATH-MS proteomics with a reproducible centrifugation method to isolate plasma EVs.

Results: We applied the High-Stringency Human Proteome Project (HPP) guidelines for SWATH-MS analysis, which refined our initial EV protein identification from 1362 proteins (10,993 peptides) to a more reliable and confident subset of 853 proteins (6231 peptides). In early-stage CRC, we identified 11 plasma EV proteins with differential expression between patients and healthy controls (three up-regulated and eight down-regulated), many of which are involved in key cancer hallmarks. Additionally, within the same cohort, we analysed EV proteins associated with tumour recurrence to identify potential prognostic indicators for CRC. A subset of up-regulated proteins associated with extracellular vesicle formation (GDI1, NSF, and TMED9) and the down-regulation of TSG101 suggest that micro-metastasis may have occurred earlier than previously anticipated.

Discussion: By employing stringent proteomic analysis and a robust SWATH-MS approach, we identified dysregulated EV proteins that potentially indicate early-stage CRC and predict recurrence risk, including proteins involved in metabolism, cytoskeletal remodelling, and immune response. While our findings underline discrepancies with other studies due to differing isolation and stringency parameters, they provide valuable insights into the complexity of the EV proteome, emphasising the need for standardised protocols and larger, well-controlled studies to validate potential biomarkers.

Keywords: SWATH-MS; colorectal cancer; exosomes; extracellular vesicles; plasma microvesicle proteins; protein biomarkers.

Figure 1

The systematic application of high-stringency criteria to the identification of proteins for this study, resulting in a dataset of 853 proteins of high confidence. We observed a reduction of 37% in the number of proteins and 43% in that of peptides compared to the default settings. Proteolytic peptides are defined as those that are consistently identified by MS and uniquely identify each protein. A nested peptide is an identified peptide sequence that is fully subsumed within another identified peptide sequence. Supplementary Table S2 details the peptides identified for each protein across different stringency levels.

Figure 2

CRC/healthy plasma extracellular vesicle (EV) protein identification. Venn diagram comparisons between (a) EV proteins identified from our study and extracellular vesicle protein databases from ExoCarta, Vesiclepedia, and Human EV PeptideAtlas [27] and (b) EV proteins and top 100 extracellular vesicle protein markers from ExoCarta and Vesiclepedia. (c) Protein markers identified from our study represent components of EVs, including the apoptotic body, microvesicle, and exosome [24]. (d) Cellular component Gene Ontology (GO) analysis of identified EV proteins. ExoCarta and Vesiclepedia EV protein databases downloaded from http://www.exocarta.org/ (accessed on 17 December 2024) and http://microvesicles.org/ (accessed on 17 December 2024), respectively. Supplementary Table S3 provides detailed information, including the lists of proteins in each database, the detection methodologies employed, and the data accessed dates.

Figure 3

Plasma EV protein quantification in early stage I of CRC vs. healthy controls. (a) Volcano plot representations on differentially expressed proteins (FC > 1.5, p-value < 0.05) between stage I and healthy controls. Blue dots indicate up-regulated proteins and red dots indicate down-regulated proteins in stage I compared to controls. (b) Box plots illustrate the protein expression patterns between control, stage I, and stage II. *: p-value < 0.05, **: p-value < 0.01.

Figure 4

Plasma EV protein quantification comparing the non-recur group (47 CRC patients in stages I/II/III without tumour recurrence within 5 years of primary tumour resection) and recur group (13 CRC patients in stages I/II/III with tumour recurrence within 5 years). (a) Volcano plot representations of differentially expressed proteins (FC > 1.5, p-value < 0.05) between non-recurred and recurred patient groups. Blue dots indicate up-regulated proteins and red dots indicate down-regulated proteins in recurred compared to non-recurred (i.e., cured). (b) Box plots illustrate the protein expression patterns between the non-recur and recur groups. *: p-value < 0.05, **: p-value < 0.01.