Fig. 2. KISS1R expression regulates primary breast tumor growth and metastasis in xenograft models.

a–e Depletion of endogenous KISS1R in human metastatic triple-negative MDA-MB-231 breast cancer cells inhibits primary tumor growth and lung metastases in a spontaneous metastasis xenograft model. a Representative western blot showing KISS1R expression in lysates from MDA-MB-231 cells stably expressing KISS1R shRNA and scrambled control used in xenograft experiments; β-actin, loading control. Densitometric analysis of blots shown in Supplementary Fig. 1b. b Primary orthotopic tumor volumes: mouse mammary fat pad, 10⁶ cells/mouse, n = 5 mice/group. Points represent each mouse mean tumor volume and bars represent mean volume ± SEM. c Representative images of lung metastasis (outlined in green, left panels) subjected to either hematoxylin and eosin, antihuman Ki67 (middle panels), or human anti-cytochrome C oxidase. Scale bar, 100 μm. The extent of lung metastasis measured by (d) number of lung metastases (normalized to 100 mm²) lung and (e) tumor burden in the lung (percentage of lung occupied by tumor); four sections/tissue/mouse, n = 5 mice per group). Bars represent lung area and number of metastases ± SEM, respectively. One-way ANOVA followed by Dunnett’s multiple comparison test: *p < 0.05. f KISS1R overexpression promotes lung colonization in a experimental metastasis xenograft model. Lung metastases (n = 5 mice/group, outlined in green) formed by human ERα-negative SKBR3 breast cancer cells stably expressing FLAG-KISS1R or pFLAG vector control. Bars represent mean ± SEM. Metastases quantified blindly in hematoxylin and eosin and human anti-cytochrome C oxidase stained slides. Student’s unpaired t test: *p < 0.05. Scale bars, 200 μm.