Screening and validating the optimal panel of housekeeping genes for 4T1 breast carcinoma and metastasis studies in mice

Abstract The 4T1 model is extensively employed in murine studies to elucidate the mechanisms underlying the carcinogenesis of triple-negative breast cancer. Molecular biology serves as a cornerstone in these investigations. However, accurate gene expression analyses necessitate data normalization employing housekeeping genes (HKGs) to avert spurious results. Here, we initially delve into the characteristics of the tumor evolution induced by 4T1 in mice, underscoring the imperative for additional tools for tumor monitoring and assessment methods for tracking the animals, thereby facilitating prospective studies employing this methodology. Subsequently, leveraging various software platforms, we assessed ten distinct HKGs (GAPDH, 18 S, ACTB, HPRT1, B2M, GUSB, PGK1, CCSER2, SYMPK, ANKRD17) not hitherto evaluated in the 4T1 breast cancer model, across tumors and diverse tissues afflicted by metastasis. Our principal findings underscore GAPDH as the optimal HKG for gene expression analyses in tumors, while HPRT1 emerged as the most stable in the liver and CCSER2 in the lung. These genes demonstrated consistent expression and minimal variation among experimental groups. Furthermore, employing these HKGs for normalization, we assessed TNF-α and VEGF expression in tissues and discerned significant disparities among groups. We posit that this constitutes the inaugural delineation of an ideal HKG for experiments utilizing the 4T1 model, particularly in vivo settings.