Secretome protein signature of human pancreatic cancer stem-like cells
Graphical abstract
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and devastating human malignancies with a death-to-incidence ratio of 0.99. Most patients have metastatic disease at the time of diagnosis. More than 75% of patients who undergo surgical resection of small pancreatic tumours with clear surgical margins and no evidence of metastasis nonetheless die from metastasis within 5 years [1], a finding that is consistent with early spread. In addition to late diagnosis, high resistance to chemotherapy and radiation seems to be responsible for the dismal outcome of PDAC. Recent studies have demonstrated that in a mouse model of PDAC cellular dissemination leading to metastasis occurs prior to the formation of an identifiable primary tumour [2]. This behaviour is associated with epithelial-to-mesenchymal transition (EMT) and with the establishment of circulating pancreatic cells which maintain a mesenchymal phenotype and express typical markers of cancer stem cells (CSCs) [3]. Evidence for the existence of CSCs has also been provided in primary human pancreatic adenocarcinomas grown in immunocompromised mice [4]. At present, PDAC CSCs may be considered a subpopulation of cells in the bulk of the tumour characterised by the exclusive ability to drive tumourigenesis and metastasis and to play a fundamental role in disease relapse. Hence, to substantially impact long-term survival of PDAC patients, the study of the biological features and of the secretome of PDAC CSCs is critical, and will inform the development of more efficient therapies and the identification of early biomarkers.
The high heterogeneity of CSCs, which originates from genotypic and phenotypic plasticity, and their low presence in cancer sample tissues make their isolation and correct identification extremely difficult, strongly limiting the realization of biochemical studies. The current approach to isolate CSCs from tissue samples is mainly based on the difference in cell size or on the expression of specific antigens. However, these methods do not permit the recovery of sufficient cells to perform proteome or secretome studies. In order to obtain valid and reproducible results, the biochemical approach to CSC pathophysiology can take advantage of the observation that CSCs can be isolated and enriched from several human cancer cell lines [5]. Recently, our group has been able to isolate cancer stem-like cells from five out of nine PDAC cell lines [6]. In particular, we have demonstrated that Panc1 cancer stem-like cells (Panc1 CSCs) isolated from parental (P) cell line by using the CSC selective medium, represent a model of great importance to deepen the understanding of the biology of pancreatic adenocarcinoma. Panc1 CSCs showed the highest tumorsphere-forming ability, were more resistant to the action of the anticancer drugs, had typical surface stem cell markers, and when subcutaneously injected into nude female mice were more tumorigenic than parental cells. Thus far, proteomic approaches have been applied to pancreatic CSCs isolated from xenografted tumours in mice [7], early stage tumours [8], or established cell lines [9]. However, secretome analysis of pancreatic CSCs has not yet been reported, although the secreted proteins may serve as a valuable tool to obtain insight into interaction of the tumour with its microenvironment as well as intracellular processes, taking into account the observation that many tumour cells shed intracellular and even nuclear proteins into the extracellular space. Furthermore, these studies may allow the identification of potential PDAC biomarkers. Contrarily to PDAC, secretome approaches have been used to investigate CSCs of colon [10] and prostate cancer [11]. In general, secretome studies on cell lines grown in culture medium are limited by contamination from intracellular proteins originating from spontaneous cell autolysis. For this reason, a filtering criterion [12] must be established to select bona fide secreted proteins while avoiding the contaminants for downstream validation works. In this study, we have adopted a shotgun proteomics approach using iTRAQ 8-plex coupled with 2D-LC–MS/MS to compare the secretome of Panc1 pancreatic adenocarcinoma cell line with that of their derived stem-like cells (Panc1 CSCs). In order to identify only secreted proteins, we have compared the protein expression levels of the conditioned medium (CM) with those of the whole cell lysate, taking into account that the relative abundance of secreted proteins should be higher in CM than in cell lysate. Following this approach, we have identified 43 proteins secreted at higher level by Panc1 CSCs relative to the parental cells. In silico functional pathway analysis has demonstrated a predominant association of these proteins to glycolysis, gluconeogenesis, IGF-1 signalling, atherosclerosis signalling, pyruvate fermentation to lactate, and pentose phosphate pathway. Among the identified proteins, ceruloplasmin was the most abundant detected in CM from Panc1 CSCs and showed promise as predictors for PDAC, particularly for patients negative for CA19-9.
To our knowledge, this is the first proteomic study of pancreatic CSCs secretome. Our findings advance the understanding of the pathways implicated in tumourigenesis, metastasis and chemoresistance of pancreatic cancer, and also identify a pool of proteins from which novel candidate diagnostic and therapeutic biomarkers could be discovered.
Section snippets
Cell culture
The human PDAC cell line Panc1 was grown in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, and 50 μg/ml gentamicin sulfate (Gibco, Life Technologies). Adherent cells were maintained in standard conditions for a few passages at 37 °C with 5% CO2. Panc1 CSCs were obtained as previously described [6]. Briefly, adherent cells were cultured in CSC medium (i.e. DMEM/F-12 supplemented with glucose, B27, fungizone, penicillin/streptomycin, heparin, epidermal growth factor and fibroblast growth
Protein identification and quantification in Panc1 cell and Panc1 CSC conditioned media using iTRAQ
The iTRAQ-labeled CM protein samples of Panc1 cells and Panc1 CSCs were analyzed together with their respective whole cell lysates as shown in Fig. 1. A total of 2045 proteins with at least 93.8% confidence and an Unused ProteinPilot scores > 1.09 (equating to a global FDR of 1%) were identified, among these a total of 1157 proteins were quantified with a peptide confidence cut-off of 95% (Supplemental Table 3). Of these, 608 were identified via a single peptide with a confidence of 99% (
Discussion
The ability to identify and isolate CSCs in various tumour models has led to the possibility to study the mechanisms by which CSCs can contribute to tumour initiation as well as continued tumour progression. Up to now, a deep comprehension of CSC biology, and in particular that of pancreatic CSCs, is lacking. In the present study, a comparative secretome approach was taken to dissect the differences in protein level between pancreatic cancer and pancreatic cancer stem cells. Secreted proteins
Conflict of interest
The authors declare no conflicts of interest.
Competing financial interests
The authors declare no competing financial interests.
Acknowledgements
We thank Dr. Salvagno Gianluca for the technical assistance with the CA19-9 ELISA assays. This work was supported by AIRC-Fondazione CariPaRo, Padova, Italy; AIRC 5 per mille grant n. 12182, Italy and the NIHR Liverpool Pancreas Biomedical Research Unit. JB received a travel Fellowship from the European Pancreatic Club.
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