Quantitative Proteomic Analysis of Serum Exosomes from Patients with Locally Advanced Pancreatic Cancer Undergoing Chemoradiotherapy

Abstract

Pancreatic cancer is the third leading cause of cancer-related death in the USA. Despite extensive research, minimal improvements in patient outcomes have been achieved. Early identification of treatment response and metastasis would be valuable to determine the appropriate therapeutic course for patients. In this work, we isolated exosomes from the serum of 10 patients with locally advanced pancreatic cancer at serial time points over a course of therapy, and quantitative analysis was performed using the iTRAQ method. We detected approximately 700–800 exosomal proteins per sample, several of which have been implicated in metastasis and treatment resistance. We compared the exosomal proteome of patients at different time points during treatment to healthy controls and identified eight proteins that show global treatment-specific changes. We then tested the effect of patient-derived exosomes on the migration of tumor cells and found that patient-derived exosomes, but not healthy controls, induce cell migration, supporting their role in metastasis. Our data show that exosomes can be reliably extracted from patient serum and analyzed for protein content. The differential loading of exosomes during a course of therapy suggests that exosomes may provide novel insights into the development of treatment resistance and metastasis.

Analysis of the plasma proteome using iTRAQ and TMT‐based Isobaric labeling

“…Taking a somewhat more selective approach to study the effects of chemotherapy on pancreatic cancer, An et al isolated serum exosomes from patients and healthy controls. Using serum volumes of 4 mL per sample and ultracentrifugation they were able to isolate yields of exosomal proteins in the order of 0.2–0.6 μg . After 4‐plex iTRAQ labeling and LC‐MS/MS with an Orbitrap Fusion they reported in the order of 700–800 proteins per sample (in total 1559 proteins were quantified), although it is notable that there was not any additional fractionation of the labeled mixtures.…”

Extracellular vesicles in the tumor microenvironment: Therapeutic resistance, clinical biomarkers, and targeting strategies

“…In addition to the frequently used MS‐based proteomic platforms, quantitative analysis via isobaric tags for relative and absolute quantification (iTRAQ) provides another option, as illustrated by a new study that detected approximately 700–800 EV proteins per serum sample of cancer patients, some of which are implicated in metastasis and treatment resistance . A flow field‐flow fractionation (FlFFF) approach was employed to separate urinary EVs of PCa patients, revealing the significant difference in EV sizes between these patients and healthy controls .…”

Isolation and Profiling of Circulating Tumor‐Associated Exosomes Using Extracellular Vesicular Lipid–Protein Binding Affinity Based Microfluidic Device

“…However, this method suffers from drawbacks such as lengthy processing time, low recovery rates and an inability to handle small sample volumes; these drawbacks are offset by ease of use and minimal need for technical expertise. Several groups have reported on the potential and clinical significance of these exosomes . For example, Marta et al used ultracentrifugation to isolate exosomes from ovarian cancer patients and showed that plasma from ovarian cancer patients contained higher levels of exosomal proteins compared to those from benign tumor patients or healthy controls .…”

“…For example, Marta et al used ultracentrifugation to isolate exosomes from ovarian cancer patients and showed that plasma from ovarian cancer patients contained higher levels of exosomal proteins compared to those from benign tumor patients or healthy controls . Recently, An et al isolated exosomes from pancreatic cancer patients and were able to demonstrate the role of these exosomes in inducing cell migration in ex‐vivo experiments, supporting the idea that exosomes may be involved in metastasis . However, UC‐based isolation methods are unable to distinguish exosomes from other extracellular vesicles or large protein debris having similar density.…”