Fig. 4.

Cells expressing Rspo2 and Wnt1 individually and together are capable of tumor growth in nude mice. A: Pools of two million cells each of HC11/parental, HC11/ev, HC11/Rspo2, HC11/Wnt1, or HC11/Wnt1/Rspo2 were injected in the left and right #4 inguinal mammary gland of female nude mice. Before injection cells were placed in 12.5% Growth Factor Reduced Matrigel suspension to simulate the tumor microenvironment. Tumor growth curves represent the mean tumor size ± SD, determined weekly in two dimensions. HC11 cells did not yield tumors. B: H&E staining of representative tumors. Rspo2 cells gave rise to tumors with swirls of spindle-shaped neoplastic cells. Wnt1 tumors showed cells arranged in several types of structure within the same tumor section (Wnt1 a): Nests with microglandular features (black circle), cord-like organization (red box), spindle-shaped cells arranged in a dense fibrous pattern (green oval) as well as areas of squamous-like metaplasia (black arrows). Higher magnifications showed tumor cells arranged in glandular-like fashion compatible with an adenocarcinoma (Wnt1 b). Wnt1/Rspo2 tumor mass contained cells variable in size and shape that formed swirls containing large spindle-shaped cells. Other areas within the tumor had a more solid and undifferentiated organization characterized by cells with high-grade nuclear pleomorphism. Occasional mitotic figures can be seen (short arrows) (lower part a Wnt1/Rspo2). The original magnification was 100× (a) and 400× (b).