A phase I clinical trial of 17AAG and irinotecan has been conducted. Pancreatic ductal adenocarcinoma is one of the most lethal cancers with less than 5 of overall patient survival after 5 years. Local and distant invasion, resistance to chemotherapy and BMS-387032 radiotherapy and lack of early detection are responsible for this poor prognosis. Gemcitabine chemotherapy, is the standard treatment of the patients. The combination of gemcitabine with other chemo or biotherapies has resulted in a very limited prognostic improvement. Recently, a high throughput RNAi screen identified the checkpoint kinase 1 as a gene conferring resistance to gemcitabine in pancreatic cancer cells. CHK1 is a key component of the cell cycle checkpoints that are activated by genomic and replicative stress.
This checkpoint activation Limonin is known to facilitate DNA repair. Consequently, CHK1 may play an important role in the resistance of tumor cells to genotoxic therapy, raising the possibility that inhibitors of checkpoint kinases may be useful adjuvant agents in chemotherapy of cancer. In the case of pancreatic cancer, in vitro and in vivo studies have shown that CHK inhibitors enhance the antitumor activity of gemcitabine. The MultiCellular Tumor Spheroid model is generally considered as a better model than two dimensional culture to predict the in vivo response to drug treatments and it is now widely accepted that MCTS reproduce more accurately the tumor microenvironment than monolayer cell cultures.
While growing, spheroids display a gradient of proliferating cells from the outer cell layers with quiescent cells located more centrally. When deprived of oxygen and glucose, central cells die and a necrotic zone is formed. This cell heterogeneity is similar to that found in avascular microregions of tumors. It is well established that solid tumor environment induces the level of drug resistance to many chemotherapeutic agents. This phenomenon, called multicellular resistance, emerges as soon as cancer cells have established contacts with surrounding cells or extracellular matrix, i.e. its microenvironment. In MCTS, cancer cells can acquire this multicellular resistance by interacting efficiently in 3 dimensions with their environment.
In order to contribute to the discovery of new anti pancreatic cancer agents or new potent combinations with gemcitabine, we describe here the development and the validation of a new spheroid model mimicking the structure and chemo resistance of pancreatic solid tumors compared to conventional 2D cell culture models. We also present the spatio temporal parameters of the biological response of gemcitabine alone or combined with a CHK1 inhibitor, CHIR 124. Materials and methods Reagents Gemcitabine was purchased from Sigma. CHIR 124 was a generous gift of Dr Alain Pierr?. Cell culture Capan 2 pancreatic cancer cells were cultured in DMEM F12 containing 10 FCS with 2 mmol l glutamine and penicillin streptomycin in a humidified atmosphere of 5 CO2 at 37. Capan 2 cells were transduced with a lentiviral vectors coding for fused green emitting fluorescent proteins to Geminin . Spheroid generation Spheroids were prepared according to. A Capan 2 cell suspension containing 104 cells ml of DMEM F12 supplem