Figure 6.

Effect of OC-LP combination treatment on tumor growth in BT-474 BC cells xenograft model. (A) Left panel. Representative isolated mice tumors of each experimental group collected at end of the experiment. Top row is the vehicle-treated control group, then the 12.5 mg/kg LP, oral, 5X/week, the 10 mg/kg OC, ip, 3X/week, and finally the bottom row represents the combined 10 mg/kg OC, ip, 3X/week −12.5 mg/kg LP, oral, 5X/week-treated group. Right panel. The mean tumor weight at the end of the experiment vertical bars graph. (B) The mean tumor volume at the end of the experiment vertical bars graph. The tumor volume (V) was calculated as V = (L × W²)/2, where L was the length and W was the width of tumors. Bars ± SEM. *** p < 0.001 as compared with either individual OC or LP treatment or vehicle treatments. (C) Tumor volumes monitoring over the course of the experiment. Points represent the mean tumor volume of several tumors (n = 5) in each experimental group over the treatment period. Error bars indicate SEM for n = 5. (D) Body weight monitoring of mice over the experiment course. Points represent mean body weight for animals in each group (n = 5) over the experiment duration. Error bars indicate SEM for n = 5. (E) Western blot analysis of various experimental groups for the total and active levels of study target RTKs and their downstream signaling proteins. (F) H&E staining of tumor samples in different study groups. Human BC cells BT-474 cultured and suspended in serum-free RPMI medium with (30:30) µL Matrigel. Cell suspensions (5 × 10⁶ cells/60 µL) subcutaneously inoculated into the second mammary gland fat pad just beneath the nipple of each athymic nude mouse to generate orthotropic breast tumor xenografts. One day before tumor cells inoculation, a 0.72-mg 60-day release 17β-estradiol pellet (Innovative Research) surgically implanted into the interscapular region of each mouse. Once the tumor is palpable, ~35 mm³ volume around the 14th day, mice were randomly divided into four groups, n = 5 each: (i) The vehicle-treated control group; (ii) the 10.0 mg/kg OC-treated group; (iii) the 12.5 mg/kg LP-treated group; and (iv) the OC 10.0 mg/kg plus LP 12.5 mg/kg—treated group. Oral treatments, vehicle control (DMSO/saline), OC, LP, and OC-LP combination, started 14 days post-inoculation and continued until the 60th day.