Tumor growth and metastatic ability of breast cancer cells is impaired in Sema4D KO mice. (A) TSA breast cancer cells were subcutaneously injected in WT and Sema4D KO mice. Tumor volume was evaluated at the indicated days. As shown, tumor burden was dramatically less in Sema4D KO versus WT mice. Bars indicate mean tumor volume ± SEM. *, P < 0.0001 (n = 10 mice per group). (B and C) Macro- and micrometastases present in the lungs of mice bearing tumors of comparable size were evaluated as indicated in Materials and methods and scored. Error bars indicate mean ± SD. *, P < 0.0001 (n = 10 lungs). (D) Representative pictures of lungs and lung slices for WT and Sema4D KO mice. As shown, the number of metastases was significantly lower in Sema4D KO mice. Statistical differences between groups were determined by using the two-tailed Student's t test. Bars: (whole mount) 0.5 cm; (micrograph) 0.2 mm.

Bone marrow–derived cells produce Sema4D that contributes to tumor growth and vessel organization. (A) CD45⁺ cells express Sema4D. To evaluate whether CD45⁺ cells express Sema4D, tumor slices were costained with CD45 and Sema4D antibodies. Double-positive cells were then counted on 10 slices per group (one slice per tumor). (right) As shown, ∼85% of the leukocytes express Sema4D. Error bars indicate mean ± SD. *, P < 0.0001. Bar, 50 μm. (B) WT-derived bone marrow cells rescue tumor burden. (top) The mean volume ± SEM of grafted tumors in WT mice, Sema4D KO mice, and Sema4D KO mice transplanted with WT-derived bone marrow (BMT; n = 6 tumors per group). As shown, tumors grown in transplanted KO mice were bigger than those grown in nontransplanted KO mice. (bottom) PCR amplification of the ZFY1 gene on the Y chromosome from DNA derived from peripheral leukocytes. As shown, transplanted mice were chimeric, displaying ∼70% of leukocytes derived from the donors (for experimental details, see Materials and methods). W1 and W2 indicate representative transplanted mice, and female and male DNA are shown. (C) Sema4D KO bone marrow transplantation leads to decreased tumor burden in WT females. Lethally irradiated (900 cGy) WT females were transplanted with bone marrow cells derived from KO or WT male mice (n = 8 tumors per group). Bars indicate mean volume ± SEM. As in B, engraftment was confirmed by PCR analysis (bottom). K, Sema4D KO mice. (D and E) Tumor vessels resemble the phenotype of the original bone marrow donor when stained by CD31 antibody (compare with Fig. 2). D shows vessels, stained with CD31 antibodies, in a tumor grown in Sema4D KO mice transplanted with WT bone marrow. E shows slices derived from tumors grown in WT animals transplanted with WT (top) or KO (bottom) bone marrow. Statistical differences between groups were determined by using the two-tailed Student's t test. Bars, 100 μm.

Sema4D produced by TAMs promotes angiogenesis and increases the tumorigenic ability of cancer cells. (A) Matrigel plugs containing either WT or KO TAMs were subcutaneously implanted in KO mice. After 14 d, mice received dextran-FITC and were killed, and plugs were microscopically analyzed. As shown, plugs containing WT TAMs were well vascularized and showed well-formed and branched vessels, whereas plugs containing KO TAMs displayed small and poorly organized vessels. Addition of blocking plexin B1 antibodies, as well as of the c-Met inhibitor PHA-665752, resulted in the lack of an organized vessel tree. Plugs containing PBS were used as a negative control, whereas plugs containing known angiogenic factors (HGF and b-FGF) were used as positive controls. (n = 3 plugs per group). Bar, 2 mm. (B) TSA tumor cells were injected in KO mice alone or together with TAMs obtained either from WT or KO animals. TSA cells alone were also injected in WT mice as a control. Coinjection of TSA cells and WT TAMs resulted in the formation of significantly larger tumor masses in comparison to those originated by TSA plus KO TAMs. Bars represent the mean ± SEM.

Tumor vascularization is altered in Sema4D KO mice. (A and B) Evaluation of tumor vascularization. Blood vessel staining of tumor histological sections was performed with an anti-CD31 antibody. The number and area of vessels were evaluated by fluorescence microscopy. As shown, although the number of vessels is not significantly different, vessel area and size are significantly reduced in Sema4D KO mice. Error bars indicate mean ± SD. *, P < 0.0001 (n = 8 mice per group, with 10 fields per animal). Bars, 100 μm. (C) To evaluate the presence of vessel-associated pericytes, tumor sections were costained with the pericyte-specific antibodies NG2 (red) and CD31 (green). Analysis was performed on one slice per mouse (n = 8 mice per group). As shown, vessels of tumors that originated in KO mice very rarely showed a normal interaction between ECs and pericytes, and in most cases they stained negative for the presence of pericytes (right), which were instead normally present in tumors originated in control mice (left). Statistical differences between groups were determined by using the two-tailed Student's t test. v.f., visual field. Bars, 50 μm.

TAMs produce Sema4D that acts on ECs. (A and B) Immunofluorescence analysis of tumors grown in WT or Sema4D KO animals. In A, staining with CD45 and F4/80 (specific for macrophages) reveals that most of the leukocytes in the tumor stroma are macrophages (∼90%; not depicted). In B, staining with F4/80 and Sema4D antibody reveals that TAMs express Sema4D. Bars, 50 μm. (C) Western blot analysis of total cell lysates of TAMs derived from WT or Sema4D KO mice. The blot was probed with anti-Sema4D antibodies and, as a loading control, with anti–β-actin. (D) PCR analysis performed on RNAs obtained from peritoneal macrophages of control mice (left) or of mice injected i.p. with CFA. As shown, only activated macrophage express Sema4D. (E) Transwell motility assays. The ability of supernatants of cultures of TAMs, purified from tumors grown either in WT or KO mice, to promote EC (HUVEC) migration was evaluated. Supernatants of WT TAMs efficiently promoted EC migration, whereas supernatants of KO TAMs were significantly less effective. Moreover, the chemoattractive activity of the WT supernatant was significantly decreased in the presence of Sema4D or plexin B1 blocking antibodies, and of the c-Met inhibitor PHA-665752. Chemoattractive positive controls included Sema4D (4D) and HGF, whereas anti-VSV was used as a control antibody. Bars represent the mean ± SD (experiments were performed twice in triplicate). Statistical differences between groups were determined by using the two-tailed Student's t test. *, P = 0.0003; **, P = 0.0002; ***, P = 0.0013; and #, P = 0.0001.