As showncrease in gemcitabine induced DNA damage. As shown in Figure 6, a low gemcitabine concentration had minimal effect on the induction of DNA damage, apoptosis and accumulation of cells in S G2 M phases in the outer cell layer. CHIR 124 at a low dose showed a very weak effect on cell accumulation in the S G2 M phase, apoptosis and DNA damage. When CHIR 124 was combined with PLX-4720 gemcitabine at these same doses, the effect on DNA damage and apoptosis was significantly improved mainly in the outer layer of the MCTS. All these measurements were performed in several independent experiments as shown for apoptosis image analysis. From these images, induced apoptosis was quantified by counting PARP C positive cells per spheroid section and showed a clear synergic effect.
It has been reported that CHK inhibitors abrogate the gemcitabine induced S phase checkpoint. However, in our combination experiments, the gemcitabineinduced accumulation of cells in S G2 M phases is too low to observe a clear reversion of this effect in presence of CHIR 124. These results indicated that the potentiation of spheroid proliferation inhibition LY2603618 of gemcitabine by CHIR 124 was associated with a cell cycle checkpoint abrogation leading to the induction of DNA damage and apoptosis. Discussion Standard chemotherapeutic drugs have limited effect in large scale clinical trials for pancreatic cancer. Because of the very poor prognosis of this type of cancer, novel approaches are therefore urgently needed. Most in vitro screening approaches are based on monolayer culture of pancreatic cancer cells but it is well established that tumor microenvironment plays an important role in response to chemotherapy.
It is therefore of major importance that more predictive pharmacological models be developed for the assessment of new therapeutic strategies. Multicellular Tumor Spheroids are of particular interest as they offer a level of intermediate complexity that recapitulate the three dimensional organization of a tumor and integrate the notion of microenvironment. The production of 500 600 m large spheroids from various epithelial cancer cell lines has already been shown for colon, breast, prostate and kidney but not pancreas with the liquid overlay technology. Spheroids from several pancreatic ductal adenocarcinoma cell lines were obtained on micro patterned culture plates but no pharmacological analysis were presented with these models.
Recently, PDAC cell lines grown in 3D collagen microenvironment were shown to proliferate in the presence of gemcitabine whereas they stopped growing when cultivated on tissue culture plastic indicating that 3D cell organisations could have an impact on pancreatic cancer cell drug sensitivity. Then, the development of new MCTS models represents an interesting way to improve the discovery of new treatment. By using the in vivo validated gemcitabine and CHIR124 molecules , we show here that our Capan 2 MCTS model for pancreatic cancer could detect effective drug combinations. In this study we developed an automation friendly spheroid model of Capan 2 pancreatic cancer cell spheroids in 96 well plates. We chose ATP quantification to measure the effect of chemical compounds on cell viability and proliferation. We showed that epidermal growth factor was necessary