Fig. 5: Co-administration of AMPC and Abemaciclib suppresses the metastatic colonization of dormancy-like ER+MC cells and improves prognosis.

Female BALB/c athymic mice were injected with 1 × 10⁶ FD or TD cells via the tail vein and treated with vehicle (Veh), 20 mg/kg AMPC (A), 25 mg/kg Abemaciclib (AB), or a combination of A and AB as indicated for 6 days as the 1st cycle of treatment. Administration was ceased for 6 days and subsequently started for another cycle of 6 days treatment. a Bioluminescence imaging analysis: The quantification of bioluminescence images to visualize the metastatic burden of mice that received a tail vein injection of 1 × 10⁶ FD or TD cells. Bioluminescence imaging was conducted weekly. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparisons test (n = 6). b Histology analysis: Lung specimens from mice without tail vein injection (or treatment) were devoid of metastatic nodules. MCF7-derived orthotopic xenograft specimens were used as positive controls. Metastatic nodules on lung were quantified and shown in a violin plot. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparisons test (n = 6). c qPCR: Total RNA was isolated from the whole blood, brain, lung, and liver tissue of individual mice that received a tail vein injection of FD or TD cells under the indicated treatments. qPCR was performed to measure the mRNA expression of human HYPOXANTHINE-GUANINE PHOSPHO-RIBOSYL TRANSFERASE (hHPRT) in the blood and organs of mice, with mouse Glyceraldehyde 3-phosphate dehydrogenase (mGapdh) as an internal control. The relative expression of hHPRT vs. mGapdh was calculated. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparisons test (n = 6). d Kaplan–Meier analysis: Kaplan–Meier survival curves were used to depict the indicated group median and surrogate survival rates, each comprising six animals. The log-rank test (χ2 = 18.56, p < 0.0001) was used to analyze the statistical differences between the groups (n = 6). e ELISA: Serum levels of human (h)TFF3 in mice that received a tail vein injection of FD or TD cells were determined by ELISA at 1st day post-injection and the endpoint after therapy cessation. All the measurements were normalized to the average of hTFF3 levels in the corresponding control groups as relative serum hTFF3. Statistical differences were analyzed using an unpaired two-tailed Student’s t-test (n = 6). The symbols (*) indicate statistical significance. p < 0.05(*), p < 0.01(**) and p < 0.001(***) were considered statistically significant.