DETERMINATION cancer burden to world health population is a

DETERMINATION OF STATIN ACTIVITY AGAINST PANCREATIC CANCER CELL LINES IN 2D AND 3D CULTURES1.0 INTRODUCTIONPancreatic cancer (PC) continues to have one of the poorest prognoses among all cancers. PC accounts for about 3% of all cancers in the US and about 7% of all cancer deaths (American Cancer Society, 2017). Cancer death rates trend has continued to go down for other types of cancers such as breast and prostate cancers, this is due to the availability of early detection biomarkers, improved treatments, better knowledge of risk factors and good funding and awareness programs (Stewart et al., 2016). However the cause of pancreatic cancer still remain unknown, Cancer status is usually determined when the disease is well beyond established and intervention at this period only through surgery, and remission works only in few cases. The reason behind being luck of screening or diagnostics tests (Herreros-Villanueva & Bujanda, 2016). 1.1 SIGNIFICANCE OF THIS RESEARCH STUDYPancreatic cancer burden to world health population is a major concern. Pancreatic cancer contributes to loss to an individual utility to the quality of life through its debilitating nature and compromising economic objectives of a person. Currently, there is no effective mode of treatment for pancreatic cancer, and long-term survival of cancer patients remains poor. A lot of research is focused on finding better treatments for pancreatic cancer. Many clinical trials are testing new combinations of chemotherapy drugs for pancreatic cancer. This study will validate the effectiveness of statins against pancreatic cancer cell lines. 2.0 RESEARCH BACKGROUNDPancreatic ductal adenocarcinoma (PDAC) comprise 80% of pancreatic cancer cases (Ying et al., 2016). A number of predisposing factors have been attributed to etiology of PDAC and some of the factors include genetic predisposition, environmental exposure and chronic inflammation (Knudsen et al., 2017). PDAC of particular concern has shown to be resistant to all currently available treatment regimens such as chemotherapy, radiotherapy, and immunotherapy.  PDAC common feature is the growth of dense fibrous or connective tissue called desmoplasia. Desmoplasia is a reaction secondary to an insult where overactive tumor-associated fibroblasts deposit excessive extracellular matrix (ECM), stellate cells as well as immune cells, which play a significant role in tumor progression and resistance to therapy (Nielsen, Mortensen, & Detlefsen, 2016).  Pancreatic cancer poor prognosis was believed to be due to quick progression to metastatic disease. However, recent sequencing data from excised tumor and metastatic tissue shows that there is a period of over ten years between initiating mutation and formation of the tumor mass. Metastasis occurs five years later with death following after two years (Roe et al., 2017). Therefore, proving this theory in the general population, and if biomarkers for early diagnosis can be achieved, there is ample time to treat pancreatic cancer before the metastatic disease. 2.1 EVALUATION OF DRUG IN 3D CULTURE MODEL3D in vitro tumor model has been adopted as a preclinical tool for evaluating anticancer therapeutic candidates (Fang & Eglen, 2017). Research shows that 3D culture accurately reflects in vivo drug responses and mechanisms of chemotherapy than 2D monolayer based cultures (Adcock, Trivedi, Edmondson, & Yang, 2015). It has been reported cells grown in 3D cultures show high resistance to anticancer drugs than cells grown in monolayer culture. This corresponds to massive resistance to anticancer agents in vivo shown in clinical settings (Breslin & O’Driscoll, 2016). The increased resistance to chemotherapeutic agents has been attributed to intrinsic mechanism in tumor microenvironment which includes factors such as increased cell to cell contact, pro-cell survival signaling, general 3D cellular architecture, lowered proliferation rate, and oxygen and drug diffusion gradient of site-specific non cancer cells (Senthebane et al., 2017).2.2 USE OF STATINS AS ANTICANCER AGENTSStatins work by targeting rate-limiting enzyme of the mevalonate pathway, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Mullen, Yu, Longo, Archer, & Penn, 2016). The products of mevalonate pathway: Sterols, such as cholesterol are involved in membrane integrity and steroid production. Ubiquinone (coenzyme Q) is involved in electron transport and cell respiration. Farnesyl and geranylgeranyl isoprenoids involved in covalent binding of proteins such as the Ras family to membranes. Dolichol is required for glycoprotein synthesis. Isopentenyl adenine is essential for certain tRNA function and protein synthesis (Tricarico, Crovella, & Celsi, 2015). Importantly statins inhibition of HMG-CoA is applied in clinical settings to treat hypercholesterolemia and as prophylaxis for cardiovascular disease (Yang et al., 2017). As an anticancer agent, statins inhibit HMG-CoA reductase, which results in a reduction of isoprenoids synthesis, geranylgeranyl, and farnesyl (Likus et al., 2016). These substances bind to Ras protein, which is involved in signaling pathways essential for cell growth, proliferation, differentiation, and cancer development.  Lack of isoprenoids leads to Ras protein inactivation, blockade of cell signaling, and tumor regression (Jonckheere, Vasseur, & Van Seuningen, 2017). Based on recent studies, we will analyze the activity of four lipophilic statins from different classes (I class: lovastatin (LOVA), mevastatin (MEVA), and simvastatin (SIMVA); and II class: pitavastatin (PITA)) in 2D and 3D human pancreatic cancer cell (BxPC-3, MIA PaCa-2, and PANC-1) cultures3.0 HYPOTHESIS Inhibition of mevalonate will result in depletion of downstream products needed for cell proliferation, which will result in the arrest of cell growth and eventual cell death.  HMG-CoA reductase is the major rate-limiting enzyme of the mevalonate pathway. Inhibition of HMG-CoA reductase prevents the conversion of HMG-CoA to mevalonate, and thereby reduce levels of mevalonate and its downstream products. Many products of the mevalonate pathway are necessary for critical cellular functions such as membrane integrity, cell signaling, protein synthesis, and cell cycle progression. Disruptions of these processes result in cell death. 4.0 STUDY AIMS• To determine the activity of statin against pancreatic cancer cell lines in 2D and 3D cultures. • To compare drug interaction in traditional 2D cell monolayer and 3D culture model.5.0 MATERIALS AND METHODS5. 1 STUDY DESIGNReproducibility of results study will be conducted.  All data will be obtained by performing experiments. All experiments will be done in professor’s Chi-Tsai laboratory at Kaohsiung Medical University, Kaohsiung, Taiwan.5.2 STUDY METHODSMETHOD AIMCell culture To grow and maintain pancreatic cancer cell lines in appropriate media until further analysisCell viability assay (MTT) To determine effectiveness of a therapeutic agent to inhibit or kill pancreatic cancer cellsspheroid 3d culture To determine drug interaction against pancreatic cancer cell lines in 3D tissue-mimicking environmentCell colony formation To determine proliferative ability of cells and long-term survival after treatment  with cytotoxic drugCell death assay To evaluate type of cell death whether due to apoptosis or necrosis after statins treatment to cellsStatistical analysis To determine the significance of data obtained through experiments.5.3 MaterialsAll materials including culture media, reagents, consumables and equipment will be provided by professor Chi-Tsai laboratory.6.0 BIOMEDICAL TECHNIQUES IN THIS RESEARCHIn this project will use the following biomedical techniques: cell culture, cell viability test, spheroid growth test, cell colony formation, cell death and statistical analysis.6.1 CELL CULTURESAseptic technique and good cell culture practice should be practiced all the time to ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. Human pancreatic cancer cell lines: BxPC-3, MIA PaCa-2, and PANC-1 will be obtained from the American Type Culture Collection (ATCC, Taiwan R.O.C). Human foreskin fibroblast cells CRL-4001 labeled with green fluorescent protein (HF-GFP) will be obtained from ATCC, Taiwan R.O.C. BxPC-3 cells will be grown in Roswell Park Memorial Institute 1640 GlutaMAX medium, MIA PaCa-2 and PANC-1 cell lines will be cultured in Dulbecco’s Modified Eagle’s Medium GlutaMAX medium. Both media will be supplemented with 10,000 U/mL penicillin, 10 mg/mL streptomycin, and 10% fetal bovine serum. HF-GFP cells will be grown in Medium 106 with Low Serum Growth Supplement. Media and supplements will be purchased from Gibco (Carlsbad, CA, USA). Cells will be maintained in a humidified atmosphere containing 5% CO2 at 37°C. The cells will be quickly thawed in warm media to stop DSMO activity.  The cells will then be cultured in respective media as mentioned above until 80 – 85% confluence of growth is reached. Subculture or passage of cells by removing all the old media, and wash with pre-warmed PBS to remove FBS. Trypsinization to break adhesion and free up cells. Check under inverted microscope and tap the dish against the base of microscope to ensure all the cells are released. Stop the action of trypsin by adding medium containing 10% FBS. Pipette the cells up and down to ensure all the cells are resuspended. Perform cell count and subculture as required.6.2 CELL VIABILITY TEST (MTT ASSY)To determine the effect of drug on cell viability, 3-(4, 5-dimethylthiazol-2-yr)-2, 5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Co., St Louis, MO, USA) assay will be performed. BxPC-3, MIA PaCa-2, and PANC-1 cells will be seeded in 96-well plates in a volume of 100 ?L (5,000 cells/well). After 24 h pre-incubation, the cells will be treated with 100 ?L of different concentrations of statins. Only medium without cells will be used as a positive control, and the medium with 0.5% DMSO (Sigma-Aldrich Co.) will serve as a negative control. After 24, 48, and 72 hours the cells will be incubated for 3 h with the MTT solution (Sigma-Aldrich Co.). The absorbance will be measured at a wavelength of 570 and 630 nm.6.3 SPHEROID GROWTHSpheroids growth will be performed on BxPC-3, MIA PaCa-2, and PANC-1 cells by 3D Bioprinting method (Tseng et al., 2015).  The cancer cells will be mixed with human fibroblasts (1:1), to better represent the tumor microenvironment. The cells will be incubated with nanoparticles NanoShuttle (Nano3D Biosciences Inc., Houston, TX, USA) for 8–10 h. Then cells will be resuspended and seeded into ultra-low attachment 96-well plates in a volume of 100 ?L (2,000 pancreatic cancer cells and 2,000 human fibroblasts per well). The plate will be placed on a magnetic drive, and will be incubated in humidified atmosphere containing 5% CO2 at 37°C until spheroids formation. Photos of spheroids will be taken after 2 days of incubation. The medium will be replaced by new medium containing 5, 10, and 20 ?M of statins. Photos will be taken every 48 h, and medium will be replaced every 96 hours of incubation. The effect of statins in 3D pancreatic cancer cell cultures will be examined by measuring the size change of spheroids using ImageJ software (National Institutes of Health).6.4 CELL COLONY FORMATIONBxPC-3, MIA PaCa-2, and PANC-1 cells will be seeded in 12-well plates in a volume of 1 mL (100 cells/well) and will be treated with 100 ?L of 10 and 90% half maximal effective concentration (EC50) of statin solutions. Cells will be incubated in a humidified atmosphere containing 5% CO2 at 37°C. The cells will be incubated for 12 days, after which they will be rinsed with phosphate-buffered saline (PBS, Gibco) and then fixed with 4% paraformaldehyde (Thermo Scientific, Waltham, MA, USA) solution in PBS for 15 min. Then the cells will be rinsed with PBS, incubated with 0.1% aqueous crystal violet solution for 15 minutes. Finally, the cells will be washed with sterile deionized water. Pictures will be taken using G:BOX gel documentation system (Syngene International Ltd, Bengaluru, India) and Genesys software (Syngene International Ltd). The number and percentage area of colonies will be calculated.6.5 TYPE OF CELL DEATHPancreatic cancer cells will be seeded in 24-well plates in a volume of 0.5 mL (1,500 cells/well), and incubated in a humidified atmosphere containing 5% CO2 at 37°C for 24 hours. After incubation, cells will be treated with 10, 50, and 90% EC50 of statins. After 72 hours, cells will be rinsed with PBS and the medium will be replaced with fresh one. 3 ?L of 10 mg/mL Hoechst 33342 aqueous solution and 1 ?L of 1 mg/mL propidium iodide will be added into each well and cells will be incubated for 10 min. The type of cell death will be examined (apoptotic and necrotic cells) by fluorescence microscopy, and the number and percentage of cells will be calculated.6.6 STATISTICAL ANALYSISStatistical analysis will be performed using Microsoft Office Excel 2016 software (Microsoft Corporation, Redmond, WA, USA). All the experiments will be done in triplicate independent measurements and the obtained values were reported as mean ± standard deviation. Student’s t-test will be used and p-values will be calculated. A value of p<0.05 will be considered as statistically significant.7.0 EXPECTED RESULTSAs with other studies, in this study theoretically statins are expected to inhibit growth of pancreatic cancer cell lines through apoptosis or necrosis. In 3D culture statins are expected to inhibit growth and reduce the size of the spheroids