Development of 4T1 breast cancer mouse model system for preclinical carbonic anhydrase IX studies.

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer, for which targeted treatment is currently lacking. Carbonic anhydrase IX (CAIX) is a known cancer target due to its selective overexpression in hypoxia, a hallmark of many solid cancers including TNBC. This study aimed to develop a robust murine TNBC cell line 4T1-based model system that could be used in the comprehensive preclinical evaluation of targeting CAIX. The model is based on the original 4T1 breast cancer cell line and two genetically edited versions of it-one with biallelic CRISPR/Cas9-mediated Car9 inactivation and another with constitutively expressed Car9, thus ensuring negative and positive controls for CAIX production in the model system, respectively. The generated cell lines were validated for CAIX production and characterised functionally in vitro and in vivo after orthotopic implantation in syngeneic BALB/c mice. Results demonstrated significantly reduced primary tumour growth and metastatic progression rates in animals with CAIX-deficient tumours, while the CAIX-expressing tumours had vascularised phenotypes with prominent central areas of coagulative necrosis. The differential CAIX expression levels in the model were preserved during tumour growth in syngeneic mice, as verified by in vivo imaging using a novel high-affinity CAIX-specific near-infrared (NIR) fluorescent imaging probe, GZ22-4. Constitutive overexpression of autologous CAIX did not elicit specific autoantibody responses in vivo, demonstrating the suitability of this model for evaluating the efficacy of anti-CAIX vaccination as a therapeutic strategy. The in vivo study was repeated as an independent experiment and demonstrated good robustness of the developed model.