Pancreatic adenocarcinoma (PDAC) is one of the most aggressive and devastating human malignancies with a death-to-incidence ratio of 0.99. Most of the patients presents with metastatic disease at the time of diagnosis and die from metastasis within 5 years a finding that is consistent with early spread. 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 [1]. This behaviour is associated with epithelial-to-mesenchymal transition and with the establishment of circulating pancreatic cells which maintain a mesenchymal phenotype and express cancer stem cell surface markers [2]. Many, if not all, tumours and established tumour cell lines contain cancer stem cells (CSCs). Cancer stem cells are defined by the abilities to self-renew, to differentiate into progeny non-stem cancer cells (NSCCs), and to form tumours effectively upon injection into immunosuppressed mice. Increasing evidence suggests a central role for CSCs in tumourigenesis, metastasis, radioresistance, and chemoresistance as well as tumour recurrence. The aim of our study was to evaluate the differential drug sensitivity of CSCs isolated from pancreatic adenocarcinoma cell lines, relative to the parental counterpart. In order to obtain CSCs, we cultured ten cell lines in a special medium (DMEM/F-12 supplemented with B27, 1 pg/ml Fungizone, 1% penicillin/streptomycin, 5 μg/ml heparin, 20 ng/ml EGF (epidermal growth factor) and FGF (fibroblast growth factor)). The cell lines tested were: PaCa44, HPAF-II, PT45P1, CFPAC1, PSN1, PC1J, PaCa3, Panc1, MiaPaCa2 (pancreatic adenocarcinoma cell lines), and VIT-1 (normal primary pancreatic mesenchimal cells). Only five of them (PSN1, PC1J, PaCa3, Panc1, MiaPaCa2) lost their characteristic epithelial morphology and were able to form sphere after 2-3 weeks, preserving the undifferentiated state through numerous cycles of cell division. The remaining cell lines maintained their epithelial morphology or died. When cultured in RPMI medium supplemented with FBS, the pancreatic cancer spheres, formed by CSCs, were able to differentiate into adherent cells (ex-CSC) after one week of culture. The anti-proliferative activity of five anti-cancer compounds (gemcitabine, tipifarnib, sorafenib, everolimus, zoleidronic acid) was evaluated on the parental, CSC, and ex-CSC cell lines. Our data showed that CSCs, in particular those obtained from PaCa3 and Panc1 cell lines, were more resistant to the action of the drugs than parental cell lines and and ex-CSCs. Intriguingly, treatment with low doses of sorafenib drammatically induced an iper-proliferative activity of CSCs. Our results constitute a valuable prerequisite to study and to counteract pancreatic cancer resistance to standard chemotherapy.
ISOLATION AND DRUG SENSITIVITY OF CANCER STEM CELLS OBTAINED FROM PANCREATIC ADENOCARCINOMA CELL LINES
DALLA POZZA, Elisa;BRANDI, JESSICA;COSTANZO, Chiara;DANDO, Ilaria;CECCONI, Daniela;DONADELLI, Massimo;PALMIERI, Marta
2012-01-01
Abstract
Pancreatic adenocarcinoma (PDAC) is one of the most aggressive and devastating human malignancies with a death-to-incidence ratio of 0.99. Most of the patients presents with metastatic disease at the time of diagnosis and die from metastasis within 5 years a finding that is consistent with early spread. 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 [1]. This behaviour is associated with epithelial-to-mesenchymal transition and with the establishment of circulating pancreatic cells which maintain a mesenchymal phenotype and express cancer stem cell surface markers [2]. Many, if not all, tumours and established tumour cell lines contain cancer stem cells (CSCs). Cancer stem cells are defined by the abilities to self-renew, to differentiate into progeny non-stem cancer cells (NSCCs), and to form tumours effectively upon injection into immunosuppressed mice. Increasing evidence suggests a central role for CSCs in tumourigenesis, metastasis, radioresistance, and chemoresistance as well as tumour recurrence. The aim of our study was to evaluate the differential drug sensitivity of CSCs isolated from pancreatic adenocarcinoma cell lines, relative to the parental counterpart. In order to obtain CSCs, we cultured ten cell lines in a special medium (DMEM/F-12 supplemented with B27, 1 pg/ml Fungizone, 1% penicillin/streptomycin, 5 μg/ml heparin, 20 ng/ml EGF (epidermal growth factor) and FGF (fibroblast growth factor)). The cell lines tested were: PaCa44, HPAF-II, PT45P1, CFPAC1, PSN1, PC1J, PaCa3, Panc1, MiaPaCa2 (pancreatic adenocarcinoma cell lines), and VIT-1 (normal primary pancreatic mesenchimal cells). Only five of them (PSN1, PC1J, PaCa3, Panc1, MiaPaCa2) lost their characteristic epithelial morphology and were able to form sphere after 2-3 weeks, preserving the undifferentiated state through numerous cycles of cell division. The remaining cell lines maintained their epithelial morphology or died. When cultured in RPMI medium supplemented with FBS, the pancreatic cancer spheres, formed by CSCs, were able to differentiate into adherent cells (ex-CSC) after one week of culture. The anti-proliferative activity of five anti-cancer compounds (gemcitabine, tipifarnib, sorafenib, everolimus, zoleidronic acid) was evaluated on the parental, CSC, and ex-CSC cell lines. Our data showed that CSCs, in particular those obtained from PaCa3 and Panc1 cell lines, were more resistant to the action of the drugs than parental cell lines and and ex-CSCs. Intriguingly, treatment with low doses of sorafenib drammatically induced an iper-proliferative activity of CSCs. Our results constitute a valuable prerequisite to study and to counteract pancreatic cancer resistance to standard chemotherapy.
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