Platelet and osteoclast beta3 integrins are critical for bone metastasis

Abstract

Mice with a targeted deletion of beta3 integrin were used to examine the process by which tumor cells metastasize and destroy bone. Injection of B16 melanoma cells into the left cardiac ventricle resulted in osteolytic bone metastasis in 74% of beta3+/+ mice by 14 days. In contrast, only 4% of beta3-/- mice developed bone lesions. Direct intratibial inoculation of tumor resulted in marrow replacement by tumor in beta3-/- mice, but no associated trabecular bone resorption as seen inbeta3+/+ mice. Bone marrow transplantation studies showed that susceptibility to bone metastasis was conferred by a bone marrow-derived cell. To dissect the roles of osteoclast and platelet beta3 integrins in this model of bone metastasis, osteoclast-defective src-/- mice were used. Src-null mice were protected from tumor-associated bone destruction but were not protected from tumor cell metastasis to bone. In contrast, a highly specific platelet aggregation inhibitor of activated alphaIIbbeta3 prevented B16 metastases. These data demonstrate a critical role for platelet alphaIIbbeta3 in tumor entry into bone and suggest a mechanism by which antiplatelet therapy may be beneficial in preventing the metastasis of solid tumors.

Fig. 1.

β₃^–/– mice are protected from osteolytic bone metastases. (A) Visible pigmented B16 melanoma cell bone metastases were seen in β₃^+/+ but not in β₃^–/– mice, 14 days after B16 tumor cell LV injection. Pathologic fracture (F) developed in β₃^+/+ femur. (B) TRAP-stained femur cross section of a B16 LV-injected β₃^+/+ mouse (×4 and ×40 objectives). Pigmented B16 cells (T) are growing throughout the bone marrow (M). Tumor-associated osteolysis induced fracture (F) of bone cortex. Arrows mark TRAP-positive OCs recruited to B16 tumor cells within the bone matrix in β₃^+/+ mice and to trabecular bone/marrow interface in β₃^–/– mice. No B16 cells were evident in β₃^–/– femurs in 23/24 mice. (C) Percent of mice with bone metastases in femur and tibia 14 days after LV injection of B16 cells for β₃^+/+ compared with β₃^–/– mice (P < 0.0001, Fisher's exact t test). (D) Percent of mice with pigmented visceral metastases (P = 0.2 by Fisher's exact t test).

Fig. 2.

β3^–/– mice are protected from osteolytic bone invasion after direct inoculation of tumor cells into the bone marrow cavity. (A) TRAP/hematoxalin staining of β₃^+/+ tibia (Left) and β₃^–/– tibia (Right) 14 days after B16 intratibial injection. (B) Histomorphometric analysis of trabecular bone area for saline (S) and B16 (T) intratibial-injected tibia (each data point is a compilation of 12 equivalent tibial cross-section measurements taken from four mice with standard error bars). B16 cells induced significant trabecular bone destruction in the β₃^+/+ tibia compared with saline injection (P < 0.01 measured by paired t test), whereas there was no significant difference seen in β₃^–/– tibia. β₃^–/– (T) tibia were protected from tumor-associated trabecular bone loss compared with β₃^+/+ (T) tibia (P < 0.01, two-sample t test).

Fig. 3.

BMT of β₃^–/– marrow confers protection from osteolytic metastases. (A) (Left) TRAP staining of femur 10 days after 950-rad γ-irradiation in untransplanted control mouse demonstrating fatty marrow devoid of red marrow cells and loss of TRAP+ OCs at growth plate. Recovery of TRAP+ OCs is seen at growth plates of femurs 10 days after BMT of β₃^+/+ marrow into β₃^+/+-positive control (Center) or into β3^–/– mouse (Right). (B) In vitro TRAP staining of cultured OCs results in multinucleated β₃^+/+ OCs with well formed actin rings, compared with β₃^–/– OCs at day 5. Three weeks after BMT with β₃^+/+ marrow into β₃^–/– mice restores OC with β₃^+/+ phenotype (Right). (C) Bleeding times returned to normal range in β₃^–/– mice 3 weeks after transplantation with β₃^+/+ marrow. (D) Percentage of transplanted mice with visible bone metastases (B) and visceral metastases (V) 14 days after LV injection of B16 cells. β₃^+/+>β₃^+/+ is positive control β₃^+/+ marrow transplanted into β₃^+/+ mouse (n = 10). β^+/+>β₃^–/– is β₃^+/+ marrow transplanted into β₃^–/– animals (n = 13), demonstrating that β^+/+ marrow can restore ability of B16 to induce bone metastases in β₃^–/– mice. β₃^–/–>β₃^+/+ is β₃^–/– marrow transplanted into β₃^+/+ mice (n = 7), demonstrating that β₃^–/– bone marrow can protect WT mice from the bone metastases susceptibility as compared with β₃^+/+ mice (P = 0.0004 using the Fisher exact t test).

Fig. 4.

OC-defective src^–/– mutant mice develop bone lesions without tumor-associated bone destruction. (A) Visible pigmented B16 melanoma cells bone lesions were seen in src^+/+ and src^–/– mice 14 days after B16 tumor cell LV injection. (B) Histology of TRAP-stained tibias from saline LV-injected (S) src^+/+ and src^–/– mice compared with B16 LV-injected (T) mice. B16 cells proliferate to fill available marrow space in both src^–/– and src^+/+ tibia. (C) Histomorphometry results show tumor-induced trabecular bone loss in src^+/+ mice compared with saline-injected mice (P < 0.01 by two-sample t test) and no tumor-induced bone loss in B16-injected src^–/– mice.

Fig. 5.

α_IIbβ₃ inhibitor of platelet aggregation reduces metastases in β₃^+/+ mice. ML464 is an oral α_IIbβ₃ antagonist. ML728 is the active metabolite of ML464. (A) B16 cells spreading on fibrinogen-coated surface (♦) were not inhibited by 37.5 μM ML728 (▪) but were completely inhibited by RGD peptide (▴). (B) ML464 (inhibitor) was administered to WT (β₃^+/+ mice) 30 min before B16 LV injection and then every 12 h for 2.5 days. Placebo in DMSO carrier was also administered by oral gavage. Mice were evaluated 14 days after B16 injection for bone and visceral metastases. Percentage of mice with bone (B) or visceral (V) metastases or placebo-treated mice (n = 17) and ML464 inhibitor-treated mice (n = 26) is shown. *, Metastases were decreased in inhibitor-treated mice compared with placebo-treated mice (P = 0.0013 for bone and P = 0.0125 for visceral using Fisher's exact t test). (C) B16 cells adhered to spread platelets in the presence (+Fib) or absence (–Fib) of fibrinogen, which was not inhibited by ML728 α_IIbβ₃ inhibitor (Inh). Platelets alone (PLT no B16) and B16 cells on BSA-coated surface (B16 BSA) served as controls. (D) B16 cells added to unactivated platelets induce platelet aggregation as measured in an aggregometer. The arrow represents the addition of B16 cells to platelets. The blue line represents microaggregates, and the black line is total aggregates (micro and large). Addition of 5 μM ML728, an α_IIbβ₃ antagonist, to stirred platelets before addition of B16 cells completely inhibited platelet aggregation. (E) Calcein-labeled fluorescent mouse platelets adhere to unlabeled B16 tumor cells (arrows) and form aggregates of platelets and tumor cells (Left). Addition of 5 μM ML728 inhibited tumor cell and platelet aggregation/clumping but not platelet–tumor cell adhesion (Right).