Fig. 4. Orthotopic growth of HER2-positive trastuzumab-resistant breast cancer xenografts is suppressed by the Entrectinib-Pacritinib combination therapy.

(A) Scheme for the mammary fat pad (MFP) tumor treatment model. Trastuzumab-refractory BT474 (BT474-TtzmR) cells stably expressing luciferase were inoculated into the right inguinal MFP of female nude mice and assessed bi-weekly for tumor volume via caliper measurement. Once MFP tumors reached an average tumor volume of ~100 mm³, mice were randomized and began receiving vehicle, Entrectinib (25 mg/kg), Pacritinib (50 mg/kg), or combination orally b.i.d. 5 times per week (N = 10–11/group). (B) MFP tumor growth curve for each treatment condition. V: vehicle; E: Entrectinib; P: Pacritinib; C: combination. Tx: treatment. (C) Average ex vivo MFP tumor mass at study endpoint. (D-J) Percent nuclear positivity or H-score quantitation of MFP tumors after IHC staining. For all analyses, at least 3 fields were quantified per tumor section (N = 3–5 tumors/group). (K) Representative IHC images of MFP tumors for each treatment group. All images were captured at 40x magnification. Scale bar, 50 μm. (L) Average animal weights for each treatment group throughout the course of the study. (M) Liver toxicity following systemic therapy administration was measured using ALT activity assay. Data of at least three experimental repeats are presented as mean ± SEM. One-way ANOVA with Tukey’s multiple comparison post hoc test was used to compare p values.