STAT1-/- mammary tumors respond to and depend on ovarian hormones for engraftment and tumor progression. (A) SSM1, SSM2, SSM3, or MCF7 was plated in phenol red-free media containing charcoal-treated fetal calf serum in the presence (17β-estradiol, or E2; black triangles) or absence (mock; red squares) of 10 nM E2. P values were obtained with t test comparing mock-treated and E2-treated samples. (B) SSM1, SSM2, or SSM3 (105 cells per mouse) was transplanted into the inguinal mammary glands of sham-treated (black triangles), ovariectomized (red squares), or ovariectomized wild-type or STAT1-/- mice that were supplemented with E2 pellets (blue circles). P values were obtained with t test comparing tumor growth in ovariectomized mice with that in either sham-treated or ovariectomized mice supplemented with E2 pellets . (C) Growth of tumor transplants from primary STAT1-/- mammary tumors is ovarian hormone-dependent. Fragments (1 × 1 mm2) from two primary STAT1-/- mammary tumors were transplanted into the inguinal fat pads of ovariectomized mice (red) or sham-operated mice (black). No palpable masses were detected in ovariectomized mice. In contrast, tumors grew progressively in recipients with intact ovaries. Results represent seven to eight mice in each group (P < 0.0001). (D) SSM3 tumor cells were transplanted orthotopically into the mammary fat pads of nude mice. Mice bearing established tumors around 5 mm in diameter were sham-operated (black triangles; n = 7) or ovariectomized (red squares; n = 9). SSM3 tumors failed to progress in the absence of ovarian hormones, demonstrating sensitivity to ovarian hormone deprivation therapy (P < 0.0001). (E) Fragments of primary STAT1-/- mammary tumors were transplanted into the mammary fat pads of STAT1-/- mice. When the established tumors reached 5 mm in diameter, the animals were either sham-operated (black triangles; n = 3) or ovariectomized (red squares; n = 7). Transplanted primary tumors regressed after ovarian hormone deprivation (P < 0.0001). Error bars indicate standard error of the mean. OD540, optical density at 540 nm; OVX, ovariectomy; SSM, spontaneous STAT1-/- mammary (epithelial tumor cell line).