Cyclical Treatment of Colorectal Tumor Spheroids Induces Resistance to MEK Inhibitors

Adaptive drug resistance is a major obstacle to successful treatment of colorectal cancers. Physiologic tumor models of drug resistance are crucial to understand mechanisms of treatment failure and improve therapy by developing new therapeutics and treatment strategies. Using our aqueous two-phase system microtechnology, we developed colorectal tumor spheroids and periodically treated them with sub-lethal concentrations of three Mitogen Activated Kinase inhibitors (MEKi) used in clinical trials. We used long-term, periodic treatment and recovery of spheroids to mimic cycles of clinical chemotherapy and implemented a growth rate metric to quantitatively assess efficacy of the MEKi during treatment. Our results showed that efficacy of the MEKi significantly reduced with increased treatment cycles. Using a comprehensive molecular analysis, we established that resistance of colorectal tumor spheroids to the MEKi developed through activation of the PI3K/AKT/mTOR pathway. We also showed that other potential feedback mechanisms, such as STAT3 activation or amplified B-RAF, did not account for resistance to the MEKi. We combined each of the three MEKi with a PI3K/mTOR inhibitor and showed that the combination treatments synergistically blocked resistance to the MEKi. Importantly, and unlike the individual inhibitors, we demonstrated that synergistic concentrations of combinations of MEK and PI3K/mTOR inhibitors effectively inhibited growth of colorectal tumor spheroids in long-term treatments. This proof-of-concept study to model treatment-induced drug resistance of cancer cells using 3D cultures offers a unique approach to identify underlying molecular mechanisms and develop effective treatments.