Repeated exposure of anticancer agents to tumorspheres in open-surface microwell arrays for modeling chemotherapy-induced dormancy in colorectal cancer.

Abstract

Dormant cancer cells (DCCs) serve as crucial contributors to tumor drug resistance and recurrence; however, the mechanisms underlying their formation and biological characteristics remain inadequately understood. The establishment of reliable DCC models is essential for elucidating resistance mechanisms and formulating intervention strategies. Microfluidic chips represent valuable tools for conducting efficient cell-based drug testing assays; however, they encounter challenges related to cell recovery, which restricts their applicability in offline analytical contexts. This study reports the development of tumorspheres within open-surface microwell arrays, designed to establish a chemotherapy-induced colorectal cancer dormancy model. Uniform tumorspheres were generated through suspension culture in the microwells, resulting in a sphere diameter of 98.2 ± 9.8 μm and cell viability of 94.6 ± 3.0%. Following repeated exposure of an anticancer agent combination (5-fluorouracil/oxaliplatin/SN-38, FOLFIRINOX), tumorspheres were retrieved and subjected to various off-line assays. Cells within the tumorspheres demonstrated dormancy phenotypes, including diminished drug sensitivity, impaired migration, suppressed metabolism, and inhibited proliferation. Transcriptomic analysis reveals significant upregulation of drug resistance genes and cell cycle regulators, suggesting molecular mechanisms underlying dormancy. The agent verteporfin, which targets the signaling pathway associated with dormancy, exhibited improved efficacy (25.0-27.7%) in the elimination of dormant cells when administered in conjunction with FOLFIRINOX. This developed dormancy cancer model offers an efficient tool for dissecting the mechanisms of tumor dormancy and advancing the discovery of anticancer agents.