Proteomic profiling of cell line-derived extracellular vesicles to identify candidate circulatory markers for detection of gallbladder cancer

Abstract

Gallbladder cancer (GBC) is the sixth most common gastrointestinal tract cancer with a very low overall survival and poor prognosis. Profiling of cancer-derived extracellular vesicles (EVs) is an emerging strategy for identification of candidate biomarkers for the detection and prognosis of the disease. The aim of the study was to analyse the protein content from GBC cell line- derived EVs with emphasis on proteins which could be used as candidate biomarkers for the detection of GBC. NOZ and OCUG-1 cell lines were cultured and EVs were isolated from conditioned media. LC-MS/MS analysis of total EV proteins led to the identification of a total of 268 proteins in both the cell lines. Of these, 110 proteins were identified with ≥2 unique peptides with ≥2 PSMs in at least two experimental and technical replicate runs. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database was used to perform bioinformatics analysis of 110 proteins which showed ‘cell adhesion molecule binding’, ‘integrin binding’, ‘cadherin binding’ among the top molecular functions and ‘focal adhesion’ to be among the top pathways associated with the EV proteins. A total of 42 proteins including haptoglobin (HP), pyruvate kinase (PKM), annexin A2 (ANXA2), thrombospondin 1 (THBS1), were reported to be differentially abundant in GBC tissue. Of these, 16 proteins were reported to be differentially abundant in plasma and plasma-derived EVs. We infer these proteins to be highly important to be considered as potential circulatory biomarkers for the detection of GBC. To check the validity of this hypothesis, one of the proteins, haptoglobin (HP) as a representative case, was analysed in plasma by quantitative Enzyme- linked immunosorbent assay (ELISA) and we observed its increased levels in GBC in comparison to controls (p value= 0.0063). Receiver operating characteristic (ROC) curve analysis for GBC vs controls showed an Area under the ROC Curve (AUC) of 0.8264 for HP with 22% sensitivity against 100% specificity. We propose that HP along with other candidate proteins may be further explored for their clinical application.

Proteomic analysis of human serum Extracellular Vesicles reveals early diagnostic markers for Amyotrophic Lateral Sclerosis

“…The detection of increased EV haptoglobin in an in vitro setting suggests haptoglobin is not a contaminant and its packaging into EVs is being disrupted in disease settings (Zheng et al, 2020;Priya et al, 2022). The detection of elevated levels of hemoglobin subunits and the identification of the hemoglobin complex in the GO's cellular component analysis of the EVs, suggests an increased extracellular iron concentration consistent with the literature (Yu et al, 2018).…”

“…Increased iron has been detected in ALS patients' motor cortex, cerebral spinal fluid, and serum (Ignjatović et al, 2013;Veyrat-Durebex et al, 2014;Zheng et al, 2017;Sanghai and Tranmer, 2021). Additionally, haptoglobin has not only been detected in EVs but, has been found to be elevated in EVs isolated from the plasma of cancer patients and supernatant of cell culture cancer models (Zheng et al, 2020;Priya et al, 2022).…”

Gallbladder cancer: Progress in the Indian subcontinent

“…Analysis of cancer-derived extracellular vesicles (EVs) is an emerging method employed to identify potential biomarkers for disease detection and prognosis. Priya et al [ 108 ] studies GBC cell line- derived EVs and found 16 proteins including haptoglobin which can be served as circulating biomarkers for early detection of GBC. Akhtar et al [ 109 ] studied specific proteins (MPO, MMP9, and DEFA1) associated with neutrophil degranulation possess signal sequences.…”