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 μm 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 [18
]. Spheroids from several pancreatic ductal adenocarcinoma (PDAC) cell lines were obtained on micro-patterned culture plates but no pharmacological analysis were presented with these models [19
]. 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 [20
]. 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 [3
], 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 (EGF) was necessary to maintain Capan-2 cell proliferation in a 3-D context, 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 ligand over-expression have been frequently observed in pancreatic cancer [21
]. 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 [23
]. 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 [24
]. Consequently Capan-2 cell spheroid appears to be a relevant model to screen for EGF signaling targeting compounds.
A proliferation gradient was observed for spheroids around 600 μm 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 [9
]. According to this, we found that apoptotic cells were detected in the spheroid center after 7 days when the spheroid size reached 600 μm. 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 (after 4 days) but before central necrosis appeared at onset of treatment (before 7 days).
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 [25
]. 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 [10
]. 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 [27
]. 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 [20
]. Spheroid permeability, presence of quiescent and hypoxic cells could explain this resistance [10
]. 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 [15
]. 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 [12
]. H2AX phosphorylation, which has been demonstrated as a pharmacodynamic indicator of gemcitabine-induced stalled replication forks [15
], was first used to image gemcitabine response in Capan-2 spheroid. The establishment of gemcitabine-induced S phase checkpoint was characterized by using Capan-2 cells expressing the Fucci reporters corresponding to the fluorescent protein geminin-mAG (green) that is expressed in S/G2/M phases of the cell cycle. Our results show that 16 h after gemcitabine addition only the cells located in the outer cell layer are targeted by gemcitabine. Indeed, cells of the outer cell layer are those with damaged DNA and accumulated in the S/G2/M phases of the cell cycle. This spatially confined DNA damage may result from limited drug penetration or a low sensitivity of non-proliferating cells in deeper spheroid layers. Our results do not discriminate between these two hypotheses. One limitation to gemcitabine efficacy is its poor penetration in human tumors [22
]. Using a multicellular layer method to study drug penetration it has been shown that the penetration of gemcitabine in multicellular cell layer is independent of cell concentration but decrease with the thickness of the layer [31
]. 48 h after gemcitabine addition, cells arrested in the S phase remained located in the outer cell layer whereas DNA damages and apoptosis were detected throughout the spheroid suggesting that DNA damage rather than cell cycle arrest are correlated with apoptosis. This result agrees with a previous study showing that in spheroids the persistence of DNA damage determined by γH2AX staining predicted clonogenic cell survival [32
One field of spheroid interest is the study of drug combination. Inhibition of CHK1 represents a targeted approach to selectively enhance the cytotoxicity of DNA-damaging agents in tumor cells. Whereas, p53-deficient cells have been preferentially killed by the combination of a DNA damaging agent, which arrest the cell cycle in G2, followed by CHK1 inhibitor, p53-proficient tumors could potentially be targeted by concurrent administration of an antimetabolite and a CHK1 inhibitor [33
]. CHK1 inhibitors are able to potentiate gemcitabine cytotoxicity in vitro and in vivo [3
]. In agreement with these results our data show that gemcitabine and a CHK1 inhibitor (CHIR-124) exert a synergistic cytotoxic effect on Capan-2 spheroid. This synergic cytotoxic effect was associated with an increase in the ability of gemcitabine to trigger DNA damage and apoptosis. Taken together these data indicate that the spheroid model provide new information concerning the role of cancer cell microenvironment on the gemcitabine and CHK1 inhibitor pancreatic cancer cell response.