Enhanced AkaLuc Bioluminescence Imaging for Longitudinal Intravital Monitoring of Minimal Residual Disease in a Murine Model of Triple-negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is an aggressive form of cancer with poor prognosis. Beyond the absence of targeted therapies, a major challenge is its high recurrence rate, driven by the outgrowth of residual tumor cells that survive chemotherapy and persist during minimal residual disease (MRD). To monitor the therapy response of TNBC by enhanced intravital imaging, we established a clinically relevant combination chemotherapy protocol for the treatment of mouse mammary tumors engrafted from K14cre;Brca1F/F;Trp53F/F (KB1P) organoids engineered to express an mCherry-AkaLuc dual reporter (mCA-KB1P). Reproducible MRD and relapse response patterns with significantly extended relapse-free survivals were achieved with the TAC protocol, consisting of docetaxel, doxorubicin and cyclophosphamide. AkaLuc bioluminescence imaging (AkaBLI) of mCA-KB1P organoids verified the single-cell sensitivity of the system in vitro, and showed a detection limit of approximately 1000 cells in the mammary gland of living mice. Unexpectedly, mCA-KB1P organoids elicited an immune response, which necessitated the use of immunodeficient hosts for the longitudinal intravital monitoring of MRD. AkaBLI and an adapted TAC protocol enabled, for the first time, the non-invasive intravital tracking and an estimation of the number of surviving tumor cells in the MRD state following intensive chemotherapy. Engineering KB1P organoids for Histon2B-mCherry reporter expression (HmC-KB1P) enabled the estimation of tumor cell survival also in syngeneic immunocompetent hosts. Flow cytometry and histological analysis revealed that immunocompetent hosts harbored only a few residual cells at MRD, which exhibited a transient loss of epithelial characteristics, whereas immunodeficient hosts had a greater number of surviving cells with a maintained epithelial phenotype. These findings are consistent with the immune systems role in shaping phenotypic changes influencing survival following chemotherapy Together, the results demonstrate the utility of the AkaBLI system for rare tumor cell tracking and highlight the immune systems role in triggering adaptive responses to chemotherapy.