Category: Assay Development and Screening
Introduction: Tumor relapse is a major clinical problem despite the early success of targeted anti-cancer drugs. Pre-existing resistant population of cells and acquired drug resistance cause chemotherapies to fail. 3D cancer spheroids are biologically relevant model of avascular tumor acquired resistance to anti-cancer drugs. We use aqueous biphasic system-mediated 3D cultures to model resistance of colon cancer cells to specific molecular inhibitors. We periodically treat tumor spheroids with molecular inhibitors followed by a recovery period to mimic how patients receive chemotherapy. We quantitatively determine emerging resistance to targeted therapies using Area Under Dose Response Curve (AUC) during periodic treatments.We co-target two mutated pathways, MAPK and PI3K in colon cancer cells to overcome resistance to single treatment of molecular inhibitors.
Methods: We suspended 15,000 cells in 12.8% Dextran (DEX), and robotically dispensed the cell suspension in microwell plate containing 5% Polyethyleneglycol (PEG) to form compact HT-29 spheroids. We checked homogenous HT-29 colon cancer spheroid formation by measuring their size after 72 hours, and monitored their growth rates daily based on volume and fluorescent intensity measurement. We treated HT-29 tumor spheroids with 1nM, 10nM, 100nM, 1uM, 10uM, and 50uM of three MEK Inhibitors: Trametinib, Selumetinib, and PD0325901. We selected multiples of IC50 concentrations of each compound (1/8*IC50, ¼*I50, ½*IC50, IC50, 2*IC50, 4*IC50 and 8*IC50). We exposed HT-29 spheroid to a pulsed dosing for two 6-day treatment cycles with a 6-day recovery phase in between. We co-targeted MAPK and PI3K/AKT by combining three MEK inhibitors and a PI3K/AKT inhibitor, dactolisib.
Discussion: Unlike a single treatment period that reduced the size/viability of tumor spheroids, we observed a significant increase in the growth/viability of spheroids during the second round of MEKi treatment and that emerging resistance overcame the initial effectiveness of the compounds. Combinations of MEKi and dactolisib synergistically and significantly inhibited the growth/viability of tumor spheroids by downregulating the inter-pathway feedback signaling. Our approach to use engineered 3D cultures and mimic clinical cyclic drug exposure and recovery offers a novel strategy to identify mechanisms of drug resistance and develop rationally-designed treatments to block resistance.
Conclusions: Our robotic 3D culture enabled exposure/recovery of tumor spheroids to specific molecular inhibitors to identify emerging drug resistance of cancer cells, and such resistance was significantly reduced by rationally-designed combination treatments of compensatory signaling pathways. The use of this model with patient derived cells will provide a major precision medicine tool to improve treatment outcomes.
Pradip Shahi Thakuri– Mr., The University of Akron, Akron, OH
The University of Akron
Pradip Shahi Thakuri is a PhD student in the Department of Biomedical Engineering at The University of Akron, Ohio. He received his Bachelor's degree in Biomedical Engineering in Nepal. His current research interests include anticancer drug screening with tumor spheroid model, developing 3D models for cancer drug resistance, and investigating cancer drug treatment regimens with the 3D cancer models.