Angiostatin-mediated suppression of cancer metastases by primary neoplasms engineered to produce granulocyte/macrophage colony-stimulating factor

Abstract

We determined whether tumor cells consistently generating granulocyte/macrophage colony- stimulating factor (GM-CSF) can recruit and activate macrophages to generate angiostatin and, hence, inhibit the growth of distant metastasis. Two murine melanoma lines, B16-F10 (syngeneic to C57BL/6 mice) and K-1735 (syngeneic to C3H/HeN mice), were engineered to produce GM-CSF. High GM-CSF (>1 ng/10(6) cells)- and low GM-CSF (<10 pg/10(6) cells)-producing clones were identified. Parental, low, and high GM-CSF-producing cells were injected subcutaneously into syngeneic and into nude mice. Parental and low-producing cells produced rapidly growing tumors, whereas the high-producing cells produced slow-growing tumors. Macrophage density inversely correlated with tumorigenicity and directly correlated with steady state levels of macrophage metalloelastase (MME) mRNA. B16 and K-1735 subcutaneous (s.c.) tumors producing high levels of GM-CSF significantly suppressed lung metastasis of 3LL, UV-2237 fibrosarcoma, K-1735 M2, and B16-F10 cells, but parental or low-producing tumors did not. The level of angiostatin in the serum directly correlated with the production of GM-CSF by the s.c. tumors. Macrophages incubated with medium conditioned by GM-CSF- producing B16 or K-1735 cells had higher MME activity and generated fourfold more angiostatin than control counterparts. These data provide direct evidence that GM-CSF released from a primary tumor can upregulate angiostatin production and suppress growth of metastases.

Figure 1

Northern blot analysis. Polyadenylated mRNA (2 μg/lane) from cultured cells or tumors of B16-F10 (top) or K-1735 M2 (bottom) was separated, blotted, and hybridized with cDNA fragments corresponding to murine GM-CSF, MME, or rat GAPDH. P, Parental B16-F10 or K-1735 M2; L, B16-L or K-1735-L; H, B16-H or K-1735-H.

Figure 2

Macrophage infiltration into s.c. tumors. B16-F10 (top) or K-1735 M2 (bottom) tumors (8–10 mm in diameter) were resected, fixed in formalin for hematoxylin and eosin staining (H&E) or in liquid nitrogen for immunohistochemical staining using antimacrophage-specific scavenger receptor antibody (reference 42).

Figure 3

Suppression of the growth of K-1735 M2 cells implanted subcutaneously by distant s.c. B16-H tumors. Nude mice (n = 10) were injected subcutaneously with 4 × 10⁵ B16-L (open squares) or 5 × 10⁴ B16-H (filled circles) cells. When the tumors reached 8–10 mm in diameter, K-1735 M2 cells (10⁵/mouse) were injected subcutaneously into the contralateral flank of tumor-bearing mice or normal mice (open circles) serving as additional controls. Tumor size was measured twice weekly using a caliper, and tumor volume was calculated by the formula V = (A × B ²)/2.

Figure 4

Identification of angiostatin in the serum. Angiostatin in the serum of control or tumor-bearing mice was purified by a lysine–Sepharose 4B column, separated on 10% SDS-PAGE, blotted, and identified using an mAb against plasminogen LBS-1. N, Normal mice; L, mice bearing B16-L tumors; H, mice bearing B16-H tumors.

Figure 5

In vitro generation of MME and angiostatin. Mouse PEM were treated for 24 h in medium conditioned by B16-F10 or K-1735 M2 cells. The cultures were briefly rinsed and incubated for 72 h in serum-free DMEM/F12 medium in the absence (a) or presence (b) of 100 μg/ml of human plasminogen. MME activity (a) and angiostatin activity (b) in the culture supernatants were determined as described above. Plasminogen fragments in the supernatants were identified by Western blot analysis using an antibody against plasminogen LBS-1 (c). The angiostatin assay was conducted in the presence of anti–LBS-1 (1, 5, or 10 μg/ ml) or control IgG (10 μg/ml) (d). M, Medium; P, parental B16-F10 or K-1735 M2; L, B16-L or K-1735-L; H, B16-H or K-1735-H.