5-Fluorouracil (5-FU) is a cornerstone in colorectal cancer therapy, but resistance has compromised its efficacy, necessitating detailed research into resistance mechanisms. Traditional methods for developing 5-FU-resistant cell lines are lengthy, unstable, and often unrepresentative of clinical scenarios.

We devised a rapid approach to create 5-FU-resistant colorectal cancer cells using an integrated in vivo/in vitro methodology. HCT116 cells were pretreated with 5-FU, then implanted into nude mice. Tumor growth was monitored, and cells from the tumors were cultured to establish the HCT116-Tumor cell line. Cells from 5-FU-exposed tumors received increasing 5-FU doses to induce resistance, creating the tumor-derived resistant (TR) cell line. Cells cultured without 5-FU were termed tumor-derived parental (TP) cells. An in vitro 5-FU resistance model, CR, served as a benchmark. Resistance metrics were evaluated using CCK-8 assays, Western Blotting, flow cytometry, and in vivo studies. Proteomics identified resistance-related differentially expressed proteins (DEPs).

Low-dose 5-FU pretreatment accelerated tumor growth. Combining in vivo and in vitro methods, we developed 5-FU-resistant TR cells within two and a half months, faster than the ten-month conventional protocol. TR cells showed stronger and more durable 5-FU resistance than CR cells, with inhibited apoptosis, autophagy, and ferroptosis, and activation of MDR1. Proteomic analysis indicated more DEPs in TR cells, suggesting unique resistance mechanisms. Animal studies confirmed enhanced drug resistance in TR cells.

Our integrated approach rapidly develops colorectal cancer cells with robust 5-FU resistance, offering a potent model for exploring multiple resistance pathways and counter-resistance strategies.