Fig. 7

UNC5A knockdown cells form tumors independent of E2 supplementation. a The number of animals with tumors after injection of sh-Control, sh2- and sh5-UNC5A TMCF7 cells in the absence of implanted estradiol (E2) pellet. Number of sh2-UNC5A (p < 0.05) and sh5-UNC5A (p < 0.001) mammary fat pad tumor-positive mice at the time of euthanasia (11 weeks postinjection). b Tumor volume in animals injected with sh-Control, sh2-UNC5A, or sh5-UNC5A TMCF7 cells with or without E2 pellets (mean ± SEM). c Lung metastasis pattern in animals injected with sh-Control or sh-UNC5A cells into the mammary fat pad in the presence of implanted E2 pellets. The number of animals with and without lung metastasis is shown on the left (p < 0.05), whereas representative lung sections stained with hematoxylin and eosin (H&E) from sh-Control, sh2-, and sh5-UNC5A TMCF7 cell inoculated mice with an E2 pellet is shown on the right. Arrowheads point to metastatic tumor cells (al, alveolus; scale bar = 200 μm). Data were analyzed using the Fisher’s exact test (two-tailed). Lungs from only three sh-Control cell injected animals were examined because we had previously shown a lack of lung metastasis in animals injected with TMCF7 cells [16]. d sh-UNC5A TMCF7 cell-derived tumors show significant levels of angiogenesis. PECAM1 staining was performed to measure endothelial cells in tumors. The number of PECAM1⁺ cells in at least 12 fields per tumor is shown on the left, whereas representative PECAM1 staining pattern is shown on the right (scale bar = 200 μm; *p < 0.05 and ***p < 0.001). e UNC5A knockdown cells show enhanced metastases to ovaries and adrenal glands. The number of animals with metastases to ovaries and adrenal glands is shown on the left, whereas the gross appearance of ovaries and adrenal glands of sh-UNC5A TMCF7 cells injected via the intracardiac route and the H&E staining pattern of ovary and adrenal gland with metastasis are shown on the right. Scale bars = 3 mm and 200 μm. f Model depicting crosstalk between UNC5A, epidermal growth factor receptor (EGFR), and E2 signaling. Unliganded UNC5A likely inhibits E2 signaling, which may be reversed upon binding of NTN1. Question marks indicates unknown mechanisms of regulation. ER, estrogen receptor