Precision Medicine and Diagnostics
Modeling Colon Cancer Drug Resistance and Stemness with Tumor Spheroids
Astha Lamichhane, Pouria Rafsanjani Nejad, Jacob Heiss, Hossein Tavana
Department of Biomedical Engineering, The University of Akron, Ohio 44325, United States
Despite targeted therapies of solid tumors using molecular inhibitors, cancer cells often adapt to the treatments and develop resistance through mechanisms such as target mutation and activation of compensatory signaling pathways. Understanding mechanisms of treatment failure is critical to develop effective therapies and improve outcomes for patients. Using our aqueous two-phase system microtechnology, we robotically microprinted tumor spheroids of KRASmut and BRAFmut colon cancer cells. To mimic how patients receive chemotherapy, we cyclically treated the spheroids in a four-week regimen with MEK inhibitors (MEKi) due to the constitutive activity of MAPK/MEK pathway in the cells. Despite an initial response to the MEKi during the first treatment round, cancer cells developed resistance during subsequent cycles and gained proliferative activities. Our molecular analysis showed activation of several oncogenic pathways such as PI3K/AKT, JAK/STAT, and WNT/β-catenin in cancer cells under cyclic treatment regimen. Additionally, we investigated whether cells displayed cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) phenotypes that are associated with cancer drug resistance. We found significant upregulation of several CSC gene markers including ALDH1A3, CD166, and CD133, and enhanced clone forming capacity of the MEKi-resistant cells. Treatments with different MEKi also significantly upregulated EMT markers including ZEB1 and E47 and promoted invasion of cancer cells from spheroids into a human collagen matrix.
Next, we examined different therapeutic strategies against adaptive resistance of cancer cells to MEKi. First, we selected drug combinations based on activation of oncogenic pathways. Despite strong anti-proliferative effects of combinations of (1) MEK/ERK and PI3K/AKT inhibitors, (2) MEK/ERK and JAK/STAT inhibitors, and (3) MEK/ERK and WNT/β-catenin inhibitors, they were ineffective against CSCs and invasiveness of cancer cells. Thus, we hypothesized that direct targeting of CSCs was critical and found that a combination of MEK/ERK and CSC inhibitors to successfully downregulate the CSC and invasive phenotypes of cancer cells. Overall, our approach to use a 3D tumor model and perform high throughput screening of drug combinations that block compensatory signaling and CSC and EMT phenotypes will facilitate treatment selections for validation in animal models and progress to clinical trials.