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Supplementary Figure 1 from CD47 Regulates Bone Mass and Tumor Metastasis to Bone

Supplementary Figure Legends 1-2 from CD47 Regulates Bone Mass and Tumor Metastasis to Bone

CD47, also called integrin-associated protein, plays a critical role in the innate immune response and is an atypical member of the immunoglobulin superfamily that interacts with and activates β3 integrins. β3 integrin−/− mice have defective platelet and osteoclast function and are protected from bone metastasis. The role of CD47 in skeletal homeostasis and bone metastasis has not been described. CD47−/− mice had increased bone mass and defective osteoclast function in vivo. Although the number of functional osteoclasts formed by differentiating CD47−/− bone marrow macrophages was decreased, high doses of RANKL rescued differentiation and function of CD47−/− osteoclasts ex vivo and rescued the osteoclast defect in CD47−/− mice. Inhibition of nitric oxide (NO) synthase, which is expressed at higher levels in CD47−/− osteoclasts, also rescued the osteoclast defect in CD47−/− cells. We then examined the consequences of this osteoclast defect in bone metastasis. In a model of tumor metastasis to bone, bone tumor burden was decreased in the CD47−/− mice compared with wild-type (WT) controls, with no decrease in s.c. tumor growth in CD47−/− mice. There was decreased tumor-associated bone destruction in the CD47−/− mice compared with WT controls, consistent with a defect in osteoclast function that was not rescued by the presence of tumor. Our data show that CD47 regulates osteoclastogenesis, in part, via regulation of NO production, and its disruption leads to a decrease in tumor bone metastasis. CD47 is a novel therapeutic target to strengthen bone mass and diminish metastatic tumor growth in bone. [Cancer Res 2009;69(7):3196–204]

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Supplementary Figure 2 from CD47 Regulates Bone Mass and Tumor Metastasis to Bone

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The as-cast Mg–6Li–4Zn-xMn alloys were prepared and extruded at 280 ​°C with an extrusion ratio of 25:1. The effects of Mn content on the microstructure and mechanical properties of Mg–6Li–4Zn-xMn alloys were investigated in this study. The XRD results show that Mg–6Li–4Zn–xMn alloys consisted of α-Mg (hcp) ​+ ​β-Li (bcc) duplex structured matrix, MgLi2Zn and Mn phases. The grains of the extruded Mg–6Li–4Zn–xMn alloys were refined by dynamic recrystallization during the extrusion process. The EBSD results show that the extruded alloys had basal textures. The grain size of the extruded alloys decreased while the basal texture was strengthened with the increasing Mn addition. The TEM results show that a large amount of nanoscale Mn precipitates existed in the extruded Mg–6Li–4Zn–1.2Mn alloy, which can effectively inhibit the dynamic recrystallized (DRXed) grains growth during the hot extrusion and is beneficial to the improvement of mechanical properties. Mg–6Li–4Zn–1.2Mn alloy in this research possesses the best mechanical properties with the ultimate tensile strength and yield strength of 321 ​MPa, 250 ​MPa, respectively.

Effects of annealing temperature on the microstructure and mechanical properties of cold-rolled LZ91 alloy are investigated. The results indicate that LZ91 alloy is composed of α-Mg, β-Li, MgLiZn a...

The effect of rolling reduction and annealing process on the microstructure and corrosion behavior of Mg−9Li−1Zn (LZ91) alloy was investigated. The test alloy sheets were cold rolled with the reduction of 50% and 75%, respectively, and then were annealed at 200 °C for 1 h. The microstructure of test alloys was observed by OM and SEM while the phase composition was determined by XRD. The corrosion property was evaluated by electrochemical measurements and immersion tests. The results show that LZ91 alloy sheet consists of α-Mg, β-Li and precipitated Mg−Li−Zn compounds (MgLi2Zn and MgLiZn phases). Dynamic recrystallization grains appear in β-Li phase during annealing process, leading to grain refinement. The results indicate that the increasing rolling reduction and performing the annealing process can enhance the corrosion resistance of LZ91 alloy. The 75% cold-rolled and annealed LZ91 alloy shows the best corrosion resistance.

Mutations in the macroautophagy/autophagy gene

Mutations in the macroautophagy/autophagy gene EPG5 are responsible for Vici syndrome, a human genetic disease characterized by combined immunodeficiency. Previously, we found that epg5−/- mice exhibit hyperinflammation in the lungs mediated by IL1B/IL-1β and TNF/TNFα, resulting in resistance to influenza. Here, we find that disruption of Epg5 results in protection against multiple enteric viruses including norovirus and rotavirus. Gene expression analysis reveals IFNL/IFN-λ responsive genes as a key alteration. Further, mice lacking Epg5 exhibit substantial alterations of the intestinal microbiota. Surprisingly, germ-free mouse studies indicate Epg5-associated inflammation of both the intestine and lung is microbiota-independent. Genetic studies support IFNL signaling as the primary mediator of resistance to enteric viruses, but not of microbial dysbiosis, in epg5−/- mice. This study unveils an important role, unexpectedly independent of the microbiota, for autophagy gene Epg5 in host organism protection by modulating intestinal IFNL responses. Abbreviations: CTNNB1: catenin (cadherin associated protein), beta 1; DAPI: 4′,6-diamidino-2-phenylindole; EPG5: ectopic P-granules autophagy protein 5 homolog (C. elegans); FT: fecal transplant; IFI44: interferon-induced protein 44; IFIT1: interferon-induced protein with tetratricopeptide repeats 1; IFNG/IFN-γ: interferon gamma; IFNL/IFN-λ: interferon lambda; IFNLR1: interferon lambda receptor 1; IL1B/IL-1β: interleukin 1 beta; ISG: interferon stimulated gene; GF: germ-free; LEfSe: linear discriminant analysis effect size; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MNoV: murine norovirus; MX2: MX dynamin-like GTPase 2; OAS1A: 2’-5’ oligoadenylate synthetase 1A; RV: rotavirus; SPF: specific-pathogen free; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK-binding kinase 1; TNF/TNFα: tumor necrosis factor

The effect of Ca addition on the microstructure, mechanical properties, and corrosion behaviors of the extruded Mg&ndash, 7Li&ndash, 3Al alloys was investigated. The results showed that the extruded Mg&ndash, 3Al&ndash, xCa alloys consisted of &alpha, Mg (hcp) + &beta, Li (bcc) matrix phases and Al2Ca. With increasing Ca content, the amount and morphology of the Al2Ca phase changed significantly. The grains of the extruded Mg&ndash, xCa alloys were refined by dynamic recrystallization during the extrusion process. The tensile tests results indicated that the extruded Mg&ndash, 0.4Ca alloy exhibited favorable comprehensive mechanical properties, its ultimate tensile strength was 286 MPa, the yield strength was 249 MPa, and the elongation was 18.7%. The corrosion results showed that this alloy with 0.4 wt.% Ca addition exhibited superior corrosion resistance, with a corrosion potential Ecorr of &minus, 1.48742 VVSE, attributed to the formation of protective Al2Ca phases.

The as-cast Mg-6.5Li-xY-yZn alloys (Mg-6.5Li-0.8Y, Mg-6.5Li-0.8Y-0.3Zn and Mg-6.5Li-1.0Y-1.0Zn) were extruded at 553 K with an extrusion ratio of 25:1 and a constant extrusion speed of 1.6 mm/s. The microstructure and corrosion behavior of as-extruded Mg-6.5Li-xY-yZn alloys were studied by microstructure observation, weight loss test, hydrogen evolution test and electrochemical test in present work. The microstructure shows that the Mg-6.5Li-0.8Y alloy contains α-Mg, β-Li and Mg2Y phase, the Mg-6.5Li-0.8Y-0.3Zn alloy is composed of α-Mg, β-Li and X phase (Mg12ZnY, LPSO phase) and the Mg-6.5Li-1.0Y-1.0Zn alloy consists of α-Mg, β-Li and W phase (Mg3Zn3Y2). The corrosion results indicate that the combining addition of Y and Zn can improve the corrosion resistance of Mg-6.5Li alloys and Mg-6.5Li-0.8Y-0.3Zn alloy with LPSO phase possesses the best corrosion resistance. The surface microstructure of test alloys after immersed in 3.5 wt% NaCl solution for 48 h, showing that the Mg-6.5Li-0.8Y alloy has the most corrosion pits while the Mg-6.5Li-0.8Y-0.3Zn alloy has the least corrosion pits due to the minimal micro-galvanic formed on its surface.

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12–/– OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12–/–, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent αIIbβ3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β3 gene knockout (Itgb3–/–) OCs, but its effects were substantially blunted in P2ry12–/– OCs. In vivo, P2ry12–/– mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss.

Bisphosphonates (BPs), bone targeted drugs that disrupt osteoclast function, are routinely used to treat complications of bone metastasis. Studies in preclinical models of cancer have shown that BPs reduce skeletal tumor burden and increase survival. Similarly, we observed in the present study that administration of the Nitrogen-containing BP (N-BP), zoledronic acid (ZA) to osteolytic tumor-bearing Tax+ mice beginning at 6 months of age led to resolution of radiographic skeletal lesions. N-BPs inhibit farnesyl diphosphate (FPP) synthase, thereby inhibiting protein prenylation and causing cellular toxicity. We found that ZA decreased Tax+ tumor and B16 melanoma viability and caused the accumulation of unprenylated Rap1a proteins in vitro. However, it is presently unclear whether N-BPs exert anti-tumor effects in bone independent of inhibition of osteoclast (OC) function in vivo. Therefore, we evaluated the impact of treatment with ZA on B16 melanoma bone tumor burden in irradiated mice transplanted with splenic cells from src(-/-) mice, which have non-functioning OCs. OC-defective mice treated with ZA demonstrated a significant 88% decrease in tumor growth in bone compared to vehicle-treated OC-defective mice. These data support an osteoclast-independent role for N-BP therapy in bone metastasis.

Interferon-gamma (IFN-gamma) has been shown to enhance anti-tumor immunity and inhibit the formation of bone-resorbing osteoclasts. We evaluated the role of IFN-gamma in bone metastases, tumor-associated bone destruction, and hypercalcemia in human T cell lymphotrophic virus type 1-Tax transgenic mice. Compared with Tax(+)IFN-gamma(+/+) mice, Tax(+)IFN-gamma(-/-) mice developed increased osteolytic bone lesions and soft tissue tumors, as well as increased osteoclast formation and activity. In vivo administration of IFN-gamma to tumor-bearing Tax(+)IFN-gamma(-/-) mice prevented new tumor development and resulted in decreased bromodeoxyuridine uptake by established tumors. In vitro, IFN-gamma directly decreased the viability of Tax(+) tumor cells through inhibition of proliferation, suppression of ERK phosphorylation, and induction of apoptosis and caspase 3 cleavage. IFN-gamma also inhibited macrophage colonystimulating factor-mediated proliferation and survival of osteoclast progenitors in vitro. Administration of IFN-gamma to C57BL/6 mice decreased Tax(+) tumor growth and prevented tumor-associated bone loss and hypercalcemia. In contrast, IFN-gamma treatment failed to protect IFN-gammaR1(-/-) mice from Tax(+) tumor-induced skeletal complications, despite decreasing tumor growth. These data demonstrate that IFN-gamma suppressed tumor-induced bone loss and hypercalcemia in Tax(+) mice by inhibiting both Tax(+) tumor cell growth and host-induced osteolysis. These data suggest a protective role for IFN-gamma in patients with bone metastases and hypercalcemia of malignancy.

Background: To investigate whether estrogen may attenuate neointima formation in hyperhomocysteinemic rat carotid endarterectomy. Methods: Rats were divided into 6 groups: ovariectomized estradiol-treated homocysteine or chow; ovariectomized placebo-treated homocysteine or chow; intact placebo-treated homocysteine or chow. Chow served as controls while homocysteine served as exaggerated intimal hyperplasia. Prior to endarterectomy, rats were implanted with estradiol mini-pump or placebo, diets given 2 weeks before and after surgery. Homocysteine, estrogen, and neointimal hyperplasia were determined. Results: Homocysteine was elevated in homocysteine groups versus controls except in estradiol-treated group. Intimal hyperplasia increased in placebo-treated ovariectomized homocysteine versus intact group. Exaggerated intimal hyperplasia in placebo-treated ovariectomized homocysteine was reduced by estrogen and so was homocysteine. Estrogen replacement in ovariectomized homocysteine group reduced intimal hyperplasia to that of intact or ovariectomized controls. Conclusion: Estradiol treatment in this ovariectomized hyperhomocysteinemia carotid endarterectomy and resultant attenuation of homocysteine and neointima may have relevance to the beneficial effects of estrogen on hyperplastic response.

Hedgehog (Hh) signaling is implicated in bone development and cellular transformation. Here we show that inhibition of Hh pathway activity inhibits tumor growth through effects on the microenvironment. Pharmacologic inhibition of the Hh effector Smoothened (Smo) increased trabecular bone in vivo and inhibited osteoclastogenesis in vitro. In addition, enhanced Hh signaling due to heterozygosity of the Hh inhibitory receptor Patched (Ptch1+/−) increased bone resorption, suggesting direct regulation of osteoclast (OC) activity by the Hh pathway. Ptch1+/− mice had increased bone metastatic and subcutaneous tumor growth, suggesting that increased Hh activation in host cells promoted tumor growth. Subcutaneous growth of Hh-resistant tumor cells was inhibited by LDE225, a novel orally bioavailable SMO antagonist, consistent with effects on tumor microenvironment. Knockdown of the Hh ligand Sonic Hh (SHH) in these cells decreased subcutaneous tumor growth and decreased stromal cell production of interleukin-6, indicating that tumor-derived Hh ligands stimulated tumor growth in a paracrine fashion. Together our findings show that inhibition of the Hh pathway can reduce tumor burden, regardless of tumor Hh responsiveness, through effects on tumor cells, OCs, and stromal cells within the tumor microenvironment. Hh may be a promising therapeutic target for solid cancers and bone metastases. Cancer Res; 72(4); 897–907. ©2011 AACR.

This article describes the evaluation of the radiopharmaceutical (64)Cu-CB-TE2A-c(RGDyK) ((64)Cu-RGD) as an imaging agent for osteolytic bone metastases and their associated inflammation by targeting of the alpha(v)beta(3) integrin on osteoclasts and the proinflammatory cells involved at the bone metastatic site.The (64)Cu-RGD radiotracer was evaluated in the transgenic mouse expressing Tax (Tax(+)), which spontaneously develops osteolytic tumors throughout the vertebrae and hind limbs, using biodistribution studies and small-animal PET/CT. Histologic analysis was also performed on Tax(+) mouse tails, using hematoxylin and eosin and tartrate-resistant acid phosphatase to confirm the presence of osteolytic bone lesions and the presence of osteoclasts, respectively. Additionally, a proof-of-principle study was conducted with a small group of Tax(+) animals presenting with osteolytic lesions. These animals were treated with the bisphosphonate zoledronic acid and imaged with (64)Cu-RGD to determine whether this radiopharmaceutical was sensitive enough to detect a response to the bisphosphonate therapy.Biodistribution studies using (64)Cu-RGD demonstrated that Tax(+) mice between the ages of 6 and 12 mo had a greater accumulation of activity in their tail vertebrae than did the wild-type (WT) cohort (P = 0.013). Additionally, Tax(+) mice between the ages of 6 and 12 mo had significantly more tracer activity associated with their tail vertebrae than did Tax(+) mice older than 12 mo (P = 0.003), suggesting that earlier bone metastases cause an increased recruitment of alpha(v)beta(3)-expressing cells. Small-animal PET/CT with (64)Cu-RGD was conducted on Tax(+) and WT mice. On the basis of standardized uptake value analysis, Tax(+) mice had approximately 2-fold more tail-associated activity than did WT animals (P = 0.0157). Additionally, decreases in uptake were observed in the tails of Tax(+) mice after treatment with the osteoclast inhibitor zoledronic acid, and histologic analysis of Tax(+) mouse-tail vertebrae revealed the presence of Tax(+) tumor cells, osteoclasts, and proinflammatory cells within the bone microenvironment.Together, these data suggest that (64)Cu-RGD has the potential to effectively image osteolytic bone metastases and monitor the physiologic changes in the bone metastatic microenvironment after osteoclast-inhibiting bisphosphonate therapy.

CD47, also called integrin-associated protein, plays a critical role in the innate immune response and is an atypical member of the immunoglobulin superfamily that interacts with and activates β3 integrins. β3 integrin−/− mice have defective platelet and osteoclast function and are protected from bone metastasis. The role of CD47 in skeletal homeostasis and bone metastasis has not been described. CD47−/− mice had increased bone mass and defective osteoclast function in vivo. Although the number of functional osteoclasts formed by differentiating CD47−/− bone marrow macrophages was decreased, high doses of RANKL rescued differentiation and function of CD47−/− osteoclasts ex vivo and rescued the osteoclast defect in CD47−/− mice. Inhibition of nitric oxide (NO) synthase, which is expressed at higher levels in CD47−/− osteoclasts, also rescued the osteoclast defect in CD47−/− cells. We then examined the consequences of this osteoclast defect in bone metastasis. In a model of tumor metastasis to bone, bone tumor burden was decreased in the CD47−/− mice compared with wild-type (WT) controls, with no decrease in s.c. tumor growth in CD47−/− mice. There was decreased tumor-associated bone destruction in the CD47−/− mice compared with WT controls, consistent with a defect in osteoclast function that was not rescued by the presence of tumor. Our data show that CD47 regulates osteoclastogenesis, in part, via regulation of NO production, and its disruption leads to a decrease in tumor bone metastasis. CD47 is a novel therapeutic target to strengthen bone mass and diminish metastatic tumor growth in bone. [Cancer Res 2009;69(7):3196–204]

One in 20 carriers of human T-cell leukemia virus type 1 (HTLV-1) will develop adult T-cell leukemia/lymphoma (ATL), a disease frequently associated with hypercalcemia, bone destruction, and a fatal course refractory to current therapies. Overexpression of the HTLV-1–encoded Tax oncoprotein under the human granzyme B promoter causes large granular lymphocytic leukemia/lymphomas in mice. We found that Tax+ mice spontaneously developed hypercalcemia, high-frequency osteolytic bone metastases, and enhanced osteoclast activity. We evaluated Tax tumors for the production of osteoclast-activating factors. Purification of Tax+ tumor cells and nonmalignant tumor-infiltrating lymphocytes demonstrated that each of these populations expressed transcripts for distinct osteoclast-activating factors. We then evaluated the effect of osteoclast inhibition on tumor formation. Mice doubly transgenic for Tax and the osteoclast inhibitory factor, osteoprotegerin, were protected from osteolytic bone disease and developed fewer soft-tissue tumors. Likewise, osteoclast inhibition with bone-targeted zoledronic acid protected Tax+ mice from bone and soft-tissue tumors and prolonged survival. Tax+ mice represent the first animal model of high-penetrance spontaneous osteolytic bone metastasis and underscore the critical role of nonmalignant host cells recruited by tumor cells in the process of cancer progression and metastasis.

The ARF tumor suppressor regulates p53 as well as basic developmental processes independent of p53, including osteoclast activation, by controlling ribosomal biogenesis. Here we provide evidence that ARF is a master regulator of bone remodeling and osteosarcoma (OS) development in mice. Arf(-/-) mice displayed increased osteoblast (OB) and osteoclast (OC) activity with a significant net increase in trabecular bone volume. The long bones of Arf(-/-) mice had increased expression of OB genes while Arf(-/-) OB showed enhanced differentiation in vitro. Mice transgenic for the Tax oncogene develop lymphocytic tumors with associated osteolytic lesions, while Tax+Arf(-/-) mice uniformly developed spontaneous OS by 7 months of age. Tax+Arf(-/-) tumors were well differentiated OS characterized by an abundance of new bone with OC recruitment, expressed OB markers and displayed intact levels of p53 mRNA and reduced Rb transcript levels. Cell lines established from OS recapitulated characteristics of the primary tumor, including the expression of mature OB markers and ability to form mineralized tumors when transplanted. Loss of heterozygosity in OS tumors arising in Tax+Arf(+/-) mice emphasized the necessity of ARF-loss in OS development. Hypothesizing that inhibition of ARF-regulated bone remodeling would repress development of OS, we demonstrated that treatment of Tax+Arf(-/-) mice with zoledronic acid, a bisphosphonate inhibitor of OC activity and repressor of bone turnover, prevented or delayed the onset of OS. These data describe a novel role for ARF as a regulator of bone remodeling through effects on both OB and OC. Finally, these data underscore the potential of targeting bone remodeling as adjuvant therapy or in patients with genetic predispositions to prevent the development of OS.