Generation and characterization of tumor spheroids from gastric cancer cell lines, SNU-484 and NCI-N87

In cancer, novel biomarkers are needed to predict anticancer drug efficacy and resistance. This is particularly pressing in gastric cancers, which are generally chemoresistant or acquire resistance after chemotherapy. The emerging three-dimensional (3D) in vitro models such as multicellular tumor spheroids (MCTS) which recapitulate more accurately characteristics of in vivo tumors have been researched to increase the efficacy of therapeutic candidates during the initial phases of drug development, and as a result, minimize the usage of laboratory animals. Therefore, deeper comprehension of the mechanisms of drug resistance in spheroids may assist in the producing and improving anticancer therapies that are more efficient in preclinical studies. We developed gastric MCTS models using the liquid overlay technique with histopathologically different gastric cancer cell lines; SNU-484 and NCI-N87. A compact spheroid morphology is formed three days after seeding cells in both cell lines. The phase-contrast images demonstrated that individual cells within spheroids could not be discriminated from another, only cluster of cells with tight connections were observed. We further characterize MCTS morphology and growth features by using imaging, MTT and flow cytometric analysis. The growth patterns of MCTS were different between SNU-484 and NCI-N87. SNU-484 MCTS exhibited an increase in diameter, whereas NCI-N87 MCTS demonstrated a slow decrease in their diameter during 9 days of culture. The flow cytometric quantification revealed that the spheroid viability was above 90 % for both SNU-484 and NCI-N87 spheroids at the end of a six-day culture. Immunofluorescence staining with cleaved caspase 3 (CC3) and Ki-67 also supported that spheroids on day 6 consist of viable cells. The spheroid sections were shown to have highly compact and homogenous distribution of cells within inner layers of spheroids after 6 days of generation. After establishing and characterizing of MCTS, we compared the dose-response curves of anticancer drugs in MCTS and monolayer cultures for two chemotherapeutic agents: 5-fluorouracil (5-FU) and Docetaxel. Our findings support the higher chemoresistance in MCTS with 23.8- and 3-fold increase in SNU-484 MCTS compared monolayer counterparts for 5-FU and Docetaxel respectively. NCI-N87 MCTS displayed 80% viability throughout highest applicable drug concentrations for both 5-FU and Docetaxel. We further highlighted the metabolic alterations in MCTS using targeted 1H Nuclear Magnetic Resonance Spectroscopy (NMR). This technique could be helpful for drug testing, since cancer cells demonstrate altered cellular metabolism compared to normal cells and utilize these metabolic alterations to survive, proliferate and metastasize. We have found elevated levels of lactate compared to their 2D culture models, which were previously indicated from gastric cancer studies obtained from patient tissues and biofluids. Ours is the first study demonstrating formate overflow in gastric cancer cell lines. This study advances our understanding of 3D models' capabilities for gastric cancer research and their characterization as it outlines the essential functions that make up a solid tumor. Future studies may benefit from the strategies established in this study to obtain multicellular spheroids for gastric cancer. The 3D model described in this study has the potential to be used as a high-throughput platform for anticancer drug screening that offers a more accurate prediction of the drug efficacy. NMR spectroscopy is a promising tool which further used to decipher novel metabolic biomarkers within 3D in vitro gastric cancer models in response to anticancer therapeutics.