Chemicals
Gefinitib, cediranib, bevacizumab and paclitaxel were purchased from Selleck Chemicals, LLC (Houston, Texas, USA) and suspended in DMSO.
Patient-derived organoid models of endometrial cancer
All studies using human tissues have been approved by the University of Iowa Institutional Review Board (IRB), protocol #201809807. Patient tumor specimens were obtained within 30 min of surgical resection. Organoids were created per our protocol for ascites fluid [16], with modifications to digest tissue and isolate single cells. Specifically, freshly resected tissue was washed with 10 ml PBS and cut into small pieces. The minced fragments were collected in a 50 ml tube and digested in 5ml AdDF+++ media (Advanced DMEM-F12 with1X Glutamax,10mM HEPES and Pen strep) supplemented with 2U/ml Dispase ǁ, 1mg/ml collagenase P and 50µg/ml Dnase I, then incubated at 37°C for 0.5-1 h. Dissociated cells were filtered through a 40 μm cell strainer, centrifuged at 300 x g for 10 min, washed twice with PBS and pelleted. Erythrocytes were removed by incubating the dissociated cells with 2 ml red blood cell lysis buffer for 5 min at room temperature followed by an additional wash with 10 ml AdDF+++ and centrifugation at 300 x g for 5 minutes. Finally, the cells were counted and embedded in Matrigel on ice and seeded on pre-warmed 24-well cell culture plates; 500 µl AdDF+++ media was added on the top of the Matrigel to each well.
Cell culture
Hec50 cells were kindly provided by Dr. Erlio Gurpide (New York University) [17] and grown in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Gibco Corporation, USA) supplemented with 10% fetal bovine serum. KLE cells were purchased from ATCC and grown in RPMI-1640 medium supplemented with 10% fetal bovine serum. Human umbilical vascular endothelial cells (HUVEC) were purchased from ATCC and grown in EGMTM -2 MV Microvascular Endothelial Cell Growth Medium (2 BulletKit, Lonza, GA, USA). To ensure rigor and reproducibility, the identity of all cell lines was confirmed using the CODIS genotyping test (Cat. No. CL1003, Bio-Synthesis).
Western blot
Cells were collected and lysed with NP-40 lysis buffer with protease inhibitors. Lysates were analyzed for protein expression/phosphorylation as described previously [18, 19]. The following antibodies were used at the indicated dilutions; all antibodies were purchased from Cell Signaling: anti-p-AKT-S473 (1:1000, #4060), anti-AKT (1:1000, #4685), anti-p-p44/42 MAPK (Erk1/2) -Thr202/Tyr204 (1:1000, #4370), anti- p44/42 MAPK (Erk1/2) (1:1000, #4695), anti-p-p38 MAPK-Thr180/Tyr182 (1:1000, #9211), anti-p38 MAPK (1:1000, #8690), anti-p-cdc2-Tyr15 (1:1000, #4539), anti-cdc2 (1:1000, #9116), anti-p-CDC25C-Ser216 (1:1000, #4901), anti-CDC25C(1:1000, #4688), anti-p-Histone H3-ser10 (1:1000, #53348).
Cell viability assay
Organoids: Viability of the tumor organoids following drug treatment was performed as previously described [16]. Briefly, patient-derived organoids were collected with organoid harvesting solution (Cultrex, USA), and then digested to single cells with TrypLE Express supplemented with 4 µl 10 mg/ml DNAse I stock and 4 µl 10 mM Y-27632 stock. Single cells were suspended in AdDE+++ medium with 10% Matrigel and seeded at a density of 10,000 cells/well (50 μl/well) in an ultra-low attachment 96-well U-bottom white plate. After 24 hrs, cells were exposed to paclitaxel (10 nM), bevacizumab (1 µM), or cediranib (1 µM) for 72h at 37°C. At the end of incubation, equal volume of CellTiter-Glo 3D reagent (Promega) was added to each well and incubated for 25 min at room temperature. The luminescence was measured using the Gen5 Microplate Reader (BioTek, Vermont, USA). All the tests were conducted in triplicate and data normalized to untreated control (set at 100% viability).
Cell lines: Cell viability was determined by WST-1 assay as previously described [20]. Briefly, Hec50 and KLE cells were seeded into 96-well plates (10,000 cells per well) for 24 hrs and then cultured with increased concentrations of the drugs for an additional 72 hrs. Cell viability was evaluated using the cell proliferation reagent WST-1 (Roche, Germany) according to the manufacturer’s protocol. The absorbance was measured with a micro-plate reader (Biorad). All the tests were conducted in triplicate and data normalized to untreated control (set at 100% viability).
Cell cycle analysis
Cell cycle analysis was performed by flow cytometry as described previously [18]. Equal number of cells were plated in 10 cm plates and treated with different drugs for 24 hrs as for cell viability assays. Cell pellets were harvested and suspended separately in Krishan’s solution (3.8 mM sodium citrate, 0.014 mM propidium iodide, 1% NP-40 and 2.0 mg/ml RNase A). Cell suspensions were analyzed using a FacScan Flow Cytometer (Becton, Dickinson and Company, San Jose, CA) and data were analyzed by CellQuest software version 3.3.
Shedding assay
HUVEC, Hec50, and KLE cells were grown to 70-80% confluence and serum-starved in Opti-MEM for one hour prior to transfection. Transfection of 1 µg alkaline phosphatase (AP)-tagged transforming growth factor (TGF)-α were performed according to manufacturer’s protocols using Lipofectamine2000 (Invitrogen, Carlsbad, CA). Cells were starved for at least four hours before the stimulation. Recombinant human vascular endothelial growth factor (VEGF-A) was obtained from R&D Systems (Minneapolis, MN) and used at a concentration of 100 ng/ml. The concentration of bevacizumab was 1µg/ml. PMA (phorbol-12-myristate-13-acetate) and the AP-tagged TGF-α plasmid have been previously published [21]. Evaluation of AP activity was determined by colorimetric assay as described previously [22]. Briefly, the ratio between the total AP activity in the supernatant and the total AP activity in the cell lysate plus supernatant was computed for normalization. The presented ratios reflect the relative proteolytic activity of a given sheddase toward the AP-tagged ligand TGF-α. No AP activity was detected in the conditioned media of untransfected cells.
Phosphoproteomic microarray
Hec50 cells were plated in 15 cm plates and treated with DMSO, 1µM bevacizumab or 1µM cediranib for 24 hrs. Cell pellets were collected and lysed with Kinexus Lysis Buffer with chemical cleavage. The microarray assay and data analysis were performed with Kinex™ KAM-1325 Antibody Microarray Kit by Kinexus Bioinformatics Corporation. To qualify as a lead, the percent change from control (%CFC) value should be at least 45% higher or lower with fluorescent signals that were at least 1,000 counts [23].
Statistical analysis
Data were analyzed using GraphPad Prism software. Statistical significance of differences was determined using one-way ANOVA with Sidak’s post-hoc test or two-way ANOVA with Tukey’s post-hoc test, as specified. All values are expressed as mean ± standard deviation (SD) of at least three independent experiments unless otherwise indicated.