MSLN-mediated activation of EGFR-ERK1/2 signaling drives liver metastasis in breast cancer

To facilitate the study of organ metastasis of triple-negative breast cancer (TNBC), liver metastatic models of TNBC were developed using 4T1 cell lines via repeated fat pad injections and the selection and growth of metastatic clones in vivo . The obtained cell lines were designated based on their source organs and injection cycles. Through ex vivo bioluminescence imaging (BLI) and metastatic nodule quantification, liver metastasis potential was validated in HM3 (third-passage liver metastatic cells). These cells exhibited preferential metastasis to the liver. Metastatic burden progressively increased across serial transplantations, as evidenced by escalating nodule counts in target organs. For the liver-specific metastasis mouse model, 100 μl of PBS containing 1×10⁶ 4T1 (BALB/c mice) cells was injected in situ into mammary fat pad of mice. Cells from primary tumors and liver metastases were separately isolated and cultured (cells isolated from mouse primary tumors were named 4T1-Pri, the metastatic tumor cells isolated from liver organs were named HM). After stable clones were established, liver organ metastatic cells were reinoculated into new mice to generate metastatic tumors. After at least three rounds of screening, highly liver organotropic metastasis cells were successfully established, designated as 4T1/HM3 (liver-preferential metastases). The firefly luciferase reporter gene (for bioluminescence tracking) retrovirus was infected with 4T1-derived cells and puromycin (1 μg/mL, Sigma Aldrich, USA) was selected for 14 days.