Evaluating KRAS-Associated Responses to Sulfasalazine and 5-Fluorouracil in Colorectal Cancer Using Integrated 2D and PEGDA Microwell-Based 3D Tumor Models

Colorectal cancer (CRC) is a major cause of cancer-related mortality among adults younger than 50 years of age, and many tumors show incomplete response or develop resistance to 5-fluorouracil (5-FU)-based chemotherapy. Therefore, new therapeutic approaches that improve CRC sensitivity to existing chemotherapeutic agents are needed. KRAS-associated signaling contributes to CRC growth, metabolic adaptation and treatment resistance. In this study, we investigated whether sulfasalazine (SSZ), a U.S. Food and Drug Administration (FDA)-approved anti-inflammatory drug, could enhance the response of CRC cells to 5-FU and modulate KRAS/mitogen-activated protein kinase (MAPK)-associated signaling. Public dataset analysis using cBioPortal, Kaplan–Meier Plotter and DepMap showed that KRAS is commonly altered in CRC. The analysis also showed that higher KRAS expression was associated with shorter overall survival in 1061 CRC patients, while CRC cell-line models demonstrated KRAS dependency. In 2D cultures, both KRAS-mutant HCT116 and KRAS-wild-type RKO cells showed lower cell viability, reduced colony formation and decreased KRAS expression after SSZ treatment. In 3D cultures, exposure to SSZ reduced early spheroid formation, both as a single treatment and when combined with 5-FU. In established spheroids, SSZ-containing treatments affected cell viability, spheroid growth and morphology, with the most noticeable suppressive effect observed in RKO aggregates. SynergyFinder+ dose-matrix analysis identified dose ranges where SSZ and 5-FU showed additive-to-synergistic effects, leading us to select 600 μM SSZ with 25 μM 5-FU for further validation. Western blot results from PEGDA microwell-derived 3D spheroids showed that SSZ + 5-FU treatment reduced KRAS expression and affected KRAS/MAPK-related signaling. This effect was more pronounced in RKO cells, where downstream pathway suppression was stronger. The combination treatment also increased apoptosis-associated PARP cleavage. At the same time, it reduced Cyclin D1 and GPX4 protein levels and changed the expression of stemness-related markers, including ALDH1A3, CD44, and CD133. Together, these results support SSZ as a candidate repurposed adjuvant that may improve the response to 5-FU in CRC spheroid models and support the use of PEGDA microwell-based 3D platforms for testing combination therapy approaches.