Whereas it was not the case in monolayer. It is well known that EGF plays an important role in pancreatic cancer progression and EGF and its Roscovitine ligand over expression have been frequently observed in pancreatic cancer. A recent study reporting the effects of EGF ligands in different culture conditions of ovarian cancer cells clearly showed that in contrast to monolayer culture, spheroids facilitated growth stimulatory activity of EGF ligands. This EGF dependent proliferation of spheroids emphasized the relevance of this model by comparison with cell monolayer and with tumor context. Moreover, the EGFR systems and associated signaling pathway could be promising targets for pancreatic cancer treatment .
Consequently Capan 2 cell spheroid appears to be a relevant model to screen LY2608204 for EGF signaling targeting compounds. A proliferation gradient was observed for spheroids around 600 m diameter: proliferative cells were located in the outer layer whereas quiescent cells were located more centrally. It has been previously shown that when the central cells become deprived of oxygen and glucose, cell death and necrosis occur. According to this, we found that apoptotic cells were detected in the spheroid center after 7 days when the spheroid size reached 600 m. This proportion greatly increased until day 12. The characterization of the proliferation gradient in the spheroid of different sizes clearly showed that there was a window to test antitumoral compounds. This window started when proliferation gradient was established but before central necrosis appeared at onset of treatment.
Most in vitro studies on the response of pancreatic cancer cell to gemcitabine were based on monolayer cell culture. A study reports that gemcitabine was less potent when cancer cells were grown as multilayer compared to monolayer cultures. It is well established that for many chemotherapeutic drugs a solid tumor environment results in an increased level of drug resistance, a phenomenon called the multicellular resistance. Multicellular resistance emerges as soon as cancer cells have established contacts with their microenvironment, homologous cells, heterologous cells or extracellular matrix. This contact dependent resistance can be observed when cell are cultured as spheroid. Spheroid culture of glioblastoma cells are less sensitive to gemcitabine than monolayer cells.
Our results show that pancreatic Capan 2 cells cultured as spheroids are also less sensitive to gemcitabine than Capan 2 monolayer. This result agrees with a recent study showing that a 3 D collagen microenvironment protects pancreatic cancer cells from gemcitabine induced proliferation arrest. Spheroid permeability, presence of quiescent and hypoxic cells could explain this resistance . Our observation that gemcitabine potency was reduced in quiescent Capan 2 spheroid suggests that pancreatic cancer cell proliferation status plays a role in gemcitabine response. DNA damage induced by gemcitabine results in activation of S cell cycle checkpoint and apoptosis. In addition to assess the global cytotoxicity of anticancer agents, the spheroid model allows to image cell response in function of their position within the spheroid. H2AX phosphorylation, which ha