Hydroxychloroquine and a low antiresorptive activity bisphosphonate conjugate prevent and reverse ovariectomy-induced bone loss in mice through dual antiresorptive and anabolic effects.

Osteoporosis remains incurable. The most widely used antiresorptive agents, bisphosphonates (BPs), also inhibit bone formation, while the anabolic agent, teriparatide, does not inhibit bone resorption, and thus they have limited efficacy in preventing osteoporotic fractures and cause some side effects. Thus, there is an unmet need to develop dual antiresorptive and anabolic agents to prevent and treat osteoporosis. Hydroxychloroquine (HCQ), which is used to treat rheumatoid arthritis, prevents the lysosomal degradation of TNF receptor-associated factor 3 (TRAF3), an NF-κB adaptor protein that limits bone resorption and maintains bone formation. We attempted to covalently link HCQ to a hydroxyalklyl BP (HABP) with anticipated low antiresorptive activity, to target delivery of HCQ to bone to test if this targeting increases its efficacy to prevent TRAF3 degradation in the bone microenvironment and thus reduce bone resorption and increase bone formation, while reducing its systemic side effects. Unexpectedly, HABP-HCQ was found to exist as a salt in aqueous solution, composed of a protonated HCQ cation and a deprotonated HABP anion. Nevertheless, it inhibited osteoclastogenesis, stimulated osteoblast differentiation, and increased TRAF3 protein levels in vitro. HABP-HCQ significantly inhibited both osteoclast formation and bone marrow fibrosis in mice given multiple daily PTH injections. In contrast, HCQ inhibited marrow fibrosis, but not osteoclast formation, while the HABP alone inhibited osteoclast formation, but not fibrosis, in the mice. HABP-HCQ, but not HCQ, prevented trabecular bone loss following ovariectomy in mice and, importantly, increased bone volume in ovariectomized mice with established bone loss because HABP-HCQ increased bone formation and decreased bone resorption parameters simultaneously. In contrast, HCQ increased bone formation, but did not decrease bone resorption parameters, while HABP also restored the bone lost in ovariectomized mice, but it inhibited parameters of both bone resorption and formation. Our findings suggest that the combination of HABP and HCQ could have dual antiresorptive and anabolic effects to prevent and treat osteoporosis.

Chimeric antigen receptor dendritic cells targeted delivery of a single tumoricidal factor for cancer immunotherapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells have been used to treat blood cancers by producing a wide variety of cytokines. However, they are not effective in treating solid cancers and can cause severe side-effects, including cytokine release syndrome. TNFα is a tumoricidal cytokine, but it markedly increases the protein levels of cIAP1 and cIAP2, the members of inhibitor of apoptosis protein (IAP) family of E3 ubiquitin ligase that limits caspase-induced apoptosis. Degradation of IAP proteins by an IAP antagonist does not effectively kill cancer cells but enables TNFα to strongly induce cancer cell apoptosis. It would be a promising approach to treat cancers by targeted delivery of TNFα through an inactive adoptive cell in combination with an IAP antagonist. METHODS: Human dendritic cells (DCs) were engineered to express a single tumoricidal factor, TNFα, and a membrane-anchored Mucin1 antibody scFv, named Mucin 1 directed DCs expressing TNFα (M-DCsTNF). The efficacy of M-DCsTNF in recognizing and treating breast cancer was tested in vitro and in vivo. RESULTS: Mucin1 was highly expressed on the surface of a wide range of human breast cancer cell lines. M-DCsTNF directly associated with MDA-MB-231 cells in the bone of NSG mice. M-DCsTNF plus an IAP antagonist, SM-164, but neither alone, markedly induce MDA-MB-231 breast cancer cell apoptosis, which was blocked by TNF antibody. Importantly, M-DCsTNF combined with SM-164, but not SM-164 alone, inhibited the growth of patient-derived breast cancer in NSG mice. CONCLUSION: An adoptive cell targeting delivery of TNFα combined with an IAP antagonist is a novel effective approach to treat breast cancer and could be expanded to treat other solid cancers. Unlike CAR-T cell, this novel adoptive cell is not activated to produce a wide variety of cytokines, except for additional overexpressed TNF, and thus could avoid the severe side effects such as cytokine release syndrome.

Hydroxychloroquine and a low activity bisphosphonate conjugate prevent and reverse ovariectomy-induced bone loss in mice through dual antiresorptive and anabolic effects.

Osteoporosis is incurable because there are no dual antiresorptive and anabolic therapeutic agents that can be administered long-term. The most widely used antiresorptive agents, bisphosphonates (BPs), also inhibit bone formation and thus have limited effect in preventing osteoporotic fracture. Hydroxychloroquine (HCQ), which is used to treat rheumatoid arthritis, prevents the lysosomal degradation of TNF receptor-associated factor 3 (TRAF3), an NF-κB adaptor protein that limits bone resorption and maintains bone formation. We attempted to covalently link HCQ to a hydroxyalklyl BP (HABP) with anticipated low antiresorptive activity, to target delivery of HCQ to bone to test if this targeting increases its efficacy to prevent TRAF3 degradation in the bone microenvironment and thus reduce bone resorption and increase bone formation, while reducing its systemic side effects. Unexpectedly, HABP-HCQ was found to exist as a salt in aqueous solution, composed of a protonated HCQ cation and a deprotonated HABP anion. Nevertheless, it inhibited osteoclastogenesis, stimulated osteoblast differentiation, and increased TRAF3 protein levels in vitro. HABP-HCQ significantly inhibited both osteoclast formation and bone marrow fibrosis in mice given multiple daily PTH injections. In contrast, HCQ inhibited fibrosis, but not osteoclast formation, while the HABP alone inhibited osteoclast formation, but not fibrosis, in the mice. HABP-HCQ, but not HCQ, prevented trabecular bone loss following ovariectomy in mice and, importantly, increased bone volume in ovariectomized mice with established bone loss because HABP-HCQ increased bone formation and decreased bone resorption parameters simultaneously. In contrast, HCQ increased bone formation, but did not decrease bone resorption parameters, while HABP also restored the bone lost in ovariectomized mice, but it inhibited parameters of both bone resorption and formation. Our findings suggest that the combination of HABP and HCQ could have dual antiresorptive and anabolic effects to prevent and treat osteoporosis.

Wettability and Frictional Studies of PEEK Composites against Co-Cr Alloys with Surface Textures.

With the aim of promoting the qualities for total hip joint replacement, the wettability and tribological behaviors of PEEK composites pins with two sets of different fillers (PEEK/CF or PEEK/CF/PTFE/graphite) against Co-Cr alloy discs with five categories of surface textures (polished, orthogonal, spiral, r-θ, and orthogonal combined with spiral) were explored. It is revealed that the existence of CF in PEEK matrix increases the hydrophilicity in addition to the strength of PEEK, while the addition of PTFE increases the hydrophobicity of PEEK. The Co-Cr alloy discs with hydrophilic properties can be adjusted as hydrophobic, with the depth of textured grooves exceeding the critical sag height determined by the contact angle and the groove width. It can be concluded that PEEK/CF/PTFE/graphite composite has a lower wear rate than PEEK only reinforced with CF against Co-Cr alloy, both without surface texture and with shallow or deep grooves. The existence of shallow grooves on the disc surface could help the PEEK blends to achieve a steady friction against Co-Cr alloy in addition to collecting the worn debris. PEEK blend pins with 10 vol% CF, 10 vol% PTFE and 10 vol% graphite can achieve a lower friction coefficient of no more than 0.2 against Co-Cr alloy discs with shallow grooves around 3.5 µm in orthogonal or spiral textures.

Nuclear Factor-Kappa B Regulation of Osteoclastogenesis and Osteoblastogenesis.

Maintenance of skeletal integrity requires the coordinated activity of multinucleated bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoclasts form resorption lacunae on bone surfaces in response to cytokines by fusion of precursor cells. Osteoblasts are derived from mesenchymal precursors and lay down new bone in resorption lacunae during bone remodeling. Nuclear factorkappa B (NF-κB) signaling regulates osteoclast and osteoblast formation and is activated in osteoclast precursors in response to the essential osteoclastogenic cytokine, receptor activator of NF-κB ligand (RANKL), which can also control osteoblast formation through RANK-RANKL reverse signaling in osteoblast precursors. RANKL and some pro-inflammatory cytokines, including tumor necrosis factor (TNF), activate NF-κB signaling to positively regulate osteoclast formation and functions. However, these cytokines also limit osteoclast and osteoblast formation through NF-κB signaling molecules, including TNF receptor-associated factors (TRAFs). TRAF6 mediates RANKL-induced osteoclast formation through canonical NF-κB signaling. In contrast, TRAF3 limits RANKL- and TNF-induced osteoclast formation, and it restricts transforming growth factor ß (TGFß)-induced inhibition of osteoblast formation in young and adult mice. During aging, neutrophils expressing TGFß and C-C chemokine receptor type 5 (CCR5) increase in bone marrow of mice in response to increased NF-κB-induced CC motif chemokine ligand 5 (CCL5) expression by mesenchymal progenitor cells and injection of these neutrophils into young mice decreased bone mass. TGFß causes degradation of TRAF3, resulting in decreased glycogen synthase kinase-3ß/ß-catenin-mediated osteoblast formation and age-related osteoporosis in mice. The CCR5 inhibitor, maraviroc, prevented accumulation of TGFß+/CCR5+ neutrophils in bone marrow and increased bone mass by inhibiting bone resorption and increasing bone formation in aged mice. This paper updates current understanding of how NF-κB signaling is involved in the positive and negative regulation of cytokine-mediated osteoclast and osteoblast formation and activation with a focus on the role of TRAF3 signaling, which can be targeted therapeutically to enhance bone mass.

TGFß1+CCR5+ neutrophil subset increases in bone marrow and causes age-related osteoporosis in male mice.

TGFß1 induces age-related bone loss by promoting degradation of TNF receptor-associated factor 3 (TRAF3), levels of which decrease in murine and human bone during aging. We report that a subset of neutrophils (TGFß1+CCR5+) is the major source of TGFß1 in murine bone. Their numbers are increased in bone marrow (BM) of aged wild-type mice and adult mice with TRAF3 conditionally deleted in mesenchymal progenitor cells (MPCs), associated with increased expression in BM of the chemokine, CCL5, suggesting that TRAF3 in MPCs limits TGFß1+CCR5+ neutrophil numbers in BM of young mice. During aging, TGFß1-induced TRAF3 degradation in MPCs promotes NF-κB-mediated expression of CCL5 by MPCs, associated with higher TGFß1+CCR5+ neutrophil numbers in BM where they induce bone loss. TGFß1+CCR5+ neutrophils decreased bone mass in male mice. The FDA-approved CCR5 antagonist, maraviroc, reduced TGFß1+CCR5+ neutrophil numbers in BM and increased bone mass in aged mice. 15-mon-old mice with TGFßRII specifically deleted in MPCs had lower numbers of TGFß1+CCR5+ neutrophils in BM and higher bone volume than wild-type littermates. We propose that pharmacologic reduction of TGFß1+CCR5+ neutrophil numbers in BM could treat or prevent age-related osteoporosis.

Tribological Properties of Carbon Fiber-Reinforced PEEK against 304 Stainless Steel with Reticulate Surface Texture.

With the aim of improving the durability and reliability of polyetheretherketone (PEEK) composites reinforced with carbon fiber (CF) as thrust bearings without lubricants, a reticulate surface texture was fabricated by plane honing on a stainless steel (SS) counterpart to promote its tribological properties. Pin-on-disk experiments were designed, with the results showing that the reticulate surface texture effectively reduces the friction coefficient from 0.40 to 0.20 compared with the polished SS surface, within the range of the pv value from 0.185 to 1.85 MPa∙m/s. The wear mechanism of the polished SS surface against CF-PEEK, proven with SEM and EDS observations as well as AE measurements, is revealed, falling into abrasive wear with SS particles embedded in the friction interface around the CF strips, causing three-body contact. The reduction in the friction coefficient of the textured SS disk against the CF-PEEK pin can be achieved due to diminution of the CF wear debris and SS particles, which are scraped off by the groove edges and trapped by the groove valleys, reducing the three-body abrasive wear, while the honed plateau is used as a flank surface like a cutting tool to scratch more soft PEEK particles as the transferred film, owing to adhesive wear. This investigation suggests that the SS disk with a honed surface structure can be used as the counterpart of CF-PEEK bearings with a low friction coefficient and wear rate under dry friction.

Investigation of Strain Fatigue Behavior for Inconel 625 with Laser Shock Peening.

With excellent creep resistance, high-temperature thermal strength and high-temperature fatigue strength, Inconel 625 is widely applied to fabricate structural components in the aerospace field, where fatigue life is a key point. Laser shock peening (LSP) is considered to improve the fatigue strength and fatigue crack growth resistance of metal materials. The present work was conducted to investigate the influence of LSP on strain-controlled fatigue behavior of Inconel 625. The surface microstructures of specimens before and after LSP were observed by transmission electron microscope (TEM). The strain-controlled fatigue loading tests with different strain amplitudes ranging from 0.4% to 1.2% were carried out on the specimens, and the topography of fracture appearance was examined by scanning electron microscope (SEM). The investigations showed that the specimens with LSP presented fewer crack initiations, shorter fatigue striations space and smaller dimples or micropores, which account for the enhancement of the fatigue life for the LSP specimens. Furthermore, the plastic deformation, ultra-fine grains, twins and dislocations caused by LSP could prevent crack initiation, crack propagation and ultimate fracture, hence prolonging the fatigue life of the Inconel 625. In addition, it was revealed that the cyclic strain hardening as well as cyclic strain softening remains almost the same to Inconel 625 with or without LSP.

Lipopolysaccharide sensitizes the therapeutic response of breast cancer to IAP antagonist.

Inhibitor of apoptosis protein (IAP) is a class of E3 ubiquitin ligases functioning to support cancer survival and growth. Many small-molecule IAP antagonists have been developed, aiming to degrade IAP proteins to kill cancer. We have evaluated the effect of lipopolysaccharide (LPS), a component of the bacterial outer membrane, on IAP antagonists in treating breast cancer in a mouse model to guide future clinical trials. We show that LPS promotes IAP antagonist-induced regression of triple-negative breast cancer (TNBC) from MDA-MB-231 cells in immunodeficient mice. IAP antagonists such as SM-164, AT-406, and BV6, do not kill MDA-MB-231 cells alone, but allow LPS to induce cancer cell apoptosis rapidly. The apoptosis caused by LPS plus SM-164 is blocked by toll-like receptor 4 (TLR4) or MyD88 inhibitor, which inhibits LPS-induced TNFα production by the cancer cells. Consistent with this, MDA-MB-231 cell apoptosis induced by LPS plus SM-164 is also blocked by the TNF inhibitor. LPS alone does not kill MDA-MB-231 cells because it markedly increases the protein level of cIAP1/2, which is directly associated with and stabilized by MyD88, an adaptor protein of TLR4. ER+ MCF7 breast cancer cells expressing low levels of cIAP1/2 undergo apoptosis in response to SM-164 combined with TNFα but not with LPS. Furthermore, TNFα but not LPS alone inhibits MCF7 cell growth in vitro. Consistent with these, LPS combined with SM-164, but not either of them alone, causes regression of ER+ breast cancer from MCF7 cells in immunodeficient mice. In summary, LPS sensitizes the therapeutic response of both triple-negative and ER+ breast cancer to IAP antagonist therapy by inducing rapid apoptosis of the cancer cells through TLR4- and MyD88-mediated production of TNFα. We conclude that antibiotics that can reduce microbiota-derived LPS should not be used together with an IAP antagonist for cancer therapy.

Plumbagin is a NF-κB-inducing kinase inhibitor with dual anabolic and antiresorptive effects that prevents menopausal-related osteoporosis in mice.

Osteoporosis is caused by enhanced bone resorption and relatively reduced bone formation. There is an unmet need to develop new agents with both antiresorptive and anabolic effects to treat osteoporosis, although drugs with either effect alone are available. A small molecular compound, plumbagin, was reported to inhibit receptor activator of nuclear factor kappa-B ligand-induced osteoclast (OC) differentiation by inhibiting IκBα phosphorylation-mediated canonical NF-κB activation. However, the key transcriptional factor RelA/p65 in canonical NF-κB pathway functions to promote OC precursor survival but not terminal OC differentiation. Here, we found that plumbagin inhibited the activity of NF-κB inducing kinase, the key molecule that controls noncanonical NF-κB signaling, in an ATP/ADP-based kinase assay. Consistent with this, plumbagin inhibited processing of NF-κB2 p100 to p52 in the progenitor cells of both OCs and osteoblasts (OBs). Interestingly, plumbagin not only inhibited OC but also stimulated OB differentiation in vitro. Importantly, plumbagin prevented trabecular bone loss in ovariectomized mice. This was associated with decreased OC surfaces on trabecular surface and increased parameters of OBs, including OB surface on trabecular surface, bone formation rate, and level of serum osteocalcin, compared to vehicle-treated mice. In summary, we conclude that plumbagin is a NF-κB-inducing kinase inhibitor with dual anabolic and antiresorptive effects on bone and could represent a new class of agent for the prevention and treatment of osteoporosis.

Processing and Profile Control of Microhole Array for PDMS Mask with Femtosecond Laser.

Polydimethylsiloxane (PDMS) is hailed as one of the foundational materials that have been applied to different products in various fields because of its chemical resistance, low cost, excellent flexibility, and high molding capability. With the aim to achieve surface texture with high efficiency by means of electrochemical micromachining with PDMS mask, a femtosecond laser is utilized to process a precision array of micro-through-holes on PDMS films as the molds. The ablation process of PDMS with a femtosecond laser was investigated via numerical simulation verified with experiments indicating a laser energy density of 4.865 mJ/mm2 as the ablation threshold of PDMS with the melting temperature of 930 K. The spiral scanning path with optimized radial offset was developed to ablate materials from the PDMS film to form the laminated profiles, and a tapered through hole was then formed with multilayer scanning. The profile dimension and accuracy were examined as control targets in terms of laser pulse energy and scanning speed, showing that a 12 µJ femtosecond laser pulse energy and 1000 mm/s scanning speed could bring about a nearly circular laminating profile with expected smaller exit diameter than the entry diameter. All the cross-section diameters of the microcone decreased with the increase of laser scanning speed, while the taper increased gradually and then saturated around a laser scanning speed of 800 mm/s due to the energy absorption resulting in smaller ablation in diameter and depth.

Material Removal Capability and Profile Quality Assessment on Silicon Carbide Micropillar Fabrication with a Femtosecond Laser.

Silicon carbide (SiC) has a variety of applications because of its favorable chemical stability and outstanding physical characteristics, such as high hardness and high rigidity. In this study, a femtosecond laser with a spiral scanning radial offset of 5 µm and a spot radius of 6 µm is utilized to process micropillars on a SiC plate. The influence of pulsed laser beam energies and laser translation velocities on the micropillar profiles, dimensions, surface roughness Ra, and material removal capability (MRC) of micropillars was investigated. The processing results indicate that the micropillar has the best perpendicularity, with a micropillar bottom angle of 75.59° under a pulsed beam energy of 50 µJ in the range of 10-70 µJ, with a pulsed repetition rate of 600 kHz and a translation velocity of 0.1 m/s. As the laser translation velocity increases between 0.2 m/s and 1.0 m/s under a fixed pulsed beam energy of 50 µJ and a constant pulsed repetition rate of 600 kHz, the micropillar height decreases from 119.88 µm to 81.79 µm, with the MRC value increasing from 1.998 µm3/µJ to 6.816 µm3/µJ, while the micropillar bottom angle increases from 68.87° to 75.59°, and the Ra value diminishes from 0.836 µm to 0.341 µm. It is suggested that a combination of a higher pulsed laser beam energy with a faster laser translation speed is recommended to achieve micropillars with the same height, as well as an improved processing efficiency and surface finish.

TNF-Polarized Macrophages Produce Insulin-like 6 Peptide to Stimulate Bone Formation in Rheumatoid Arthritis in Mice.

The risk of osteoporosis is increased in rheumatoid arthritis (RA). Anti-tumor necrosis factor (TNF) therapy has markedly improved the outcomes of RA patients but does not improve osteoporosis in some reports. This could be a combined result of disease severity and other therapeutic agents, such as glucocorticoids that accelerate osteoporosis progression. We evaluated the effects of anti-TNF therapy on osteoporosis in an animal model of RA and explored the possible mechanisms involved. Six-week-old TNF transgenic (TNF-Tg) mice with early stage erosive arthritis were treated with TNF antibody (Ab) or control immunoglobulin (IgG) weekly for 4 weeks. We found that TNF Ab completely blocked the development of erosive arthritis in TNF-Tg mice, but only slightly increased vertebral bone mass, associated with reduction in parameters of both bone resorption and formation. Similarly, TNF Ab slightly increased trabecular bone mass in tibias of 8-month-old TNF-Tg mice with advanced erosive arthritis. Interestingly, TNFα increased osteoblast differentiation from mouse bone marrow stromal cells (BMSCs) containing large number of macrophages but not from pure mesenchymal progenitor cells (MPCs). TNFα-polarized macrophages (TPMs) did not express iNos and Arginase 1, typical markers of inflammatory and resident macrophages. Interestingly, TPMs stimulated osteoblast differentiation, unlike resident and inflammatory macrophages polarized by IL-4 and interferon-λ, respectively. RNA-seq analysis indicated that TPMs produced several anabolic factors, including Jagged1 and insulin like 6 (INSL6). Importantly, inhibition of either Jagged1 or INSL6 blocked TNFα-induced osteoblast differentiation. Furthermore, INSL6 Ab significantly decreased the expansion of TNF-induced MPCs in BMSCs, and anti-TNF Ab reduced INSL6 expression by macrophages in vitro and in TNF-Tg mice in vivo. We conclude that TPMs produce INSL6 to stimulate bone formation and anti-TNF Ab blocks not only enhanced bone resorption but also the anabolic effect of TPMs on bone, limiting its effect to increase bone mass in this model of RA. © 2021 American Society for Bone and Mineral Research (ASBMR).

Regulation of TNF-Induced Osteoclast Differentiation.

Increased osteoclast (OC) differentiation and activity is the critical event that results in bone loss and joint destruction in common pathological bone conditions, such as osteoporosis and rheumatoid arthritis (RA). RANKL and its decoy receptor, osteoprotegerin (OPG), control OC differentiation and activity. However, there is a specific concern of a rebound effect of denosumab discontinuation in treating osteoporosis. TNFα can induce OC differentiation that is independent of the RANKL/RANK system. In this review, we discuss the factors that negatively and positively regulate TNFα induction of OC formation, and the mechanisms involved to inform the design of new anti-resorptive agents for the treatment of bone conditions with enhanced OC formation. Similar to, and being independent of, RANKL, TNFα recruits TNF receptor-associated factors (TRAFs) to sequentially activate transcriptional factors NF-κB p50 and p52, followed by c-Fos, and then NFATc1 to induce OC differentiation. However, induction of OC formation by TNFα alone is very limited, since it also induces many inhibitory proteins, such as TRAF3, p100, IRF8, and RBP-j. TNFα induction of OC differentiation is, however, versatile, and Interleukin-1 or TGFß1 can enhance TNFα-induced OC formation through a mechanism which is independent of RANKL, TRAF6, and/or NF-κB. However, TNFα polarized macrophages also produce anabolic factors, including insulin such as 6 peptide and Jagged1, to slow down bone loss in the pathological conditions. Thus, the development of novel approaches targeting TNFα signaling should focus on its downstream molecules that do not affect its anabolic effect.

Bisphosphonates for delivering drugs to bone.

Advances in the design of potential bone-selective drugs for the treatment of various bone-related diseases are creating exciting new directions for multiple unmet medical needs. For bone-related cancers, off-target/non-bone toxicities with current drugs represent a significant barrier to the quality of life of affected patients. For bone infections and osteomyelitis, bacterial biofilms on infected bones limit the efficacy of antibiotics because it is hard to access the bacteria with current approaches. Promising new experimental approaches to therapy, based on bone-targeting of drugs, have been used in animal models of these conditions and demonstrate improved efficacy and safety. The success of these drug-design strategies bodes well for the development of therapies with improved efficacy for the treatment of diseases affecting the skeleton. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

RANKL-Based Osteoclastogenic Assay from Murine Bone Marrow Cells.

The osteoclast is the unique type of cell that resorbs bone in vivo and it is required for normal skeletal development and postnatal homeostasis. Osteoclast deficiency impairs skeletal development during embryogenesis and results in osteopetrosis and impaired tooth eruption. In contrast, excessive osteoclast formation in adults results in bone loss in a number of conditions, including osteoporosis, rheumatoid arthritis, and metastatic bone disease. Osteoclasts are derived from monocytes/macrophages; they can be generated in vitro by treatment of these precursor cells with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). This chapter describes procedures for generating osteoclasts from mouse bone marrow cells in vitro using M-CSF and RANKL and assessing their ability to form resorption lacunae on thin bone slices.

Targeting anti-cancer agents to bone using bisphosphonates.

The skeleton is affected by numerous primary and metastatic solid and hematopoietic malignant tumors, which can cause localized sites of osteolysis or osteosclerosis that can weaken bones and increase the risk of fractures in affected patients. Chemotherapeutic drugs can eliminate some tumors in bones or reduce their volume and skeletal-related events, but adverse effects on non-target organs can significantly limit the amount of drug that can be administered to patients. In these circumstances, it may be impossible to deliver therapeutic drug concentrations to tumor sites in bones. One attractive mechanism to approach this challenge is to conjugate drugs to bisphosphonates, which can target them to bone where they can be released at diseased sites. Multiple attempts have been made to do this since the 1990s with limited degrees of success. Here, we review the results of pre-clinical and clinical studies made to target FDA-approved drugs and other antineoplastic small molecules to bone to treat diseases affecting the skeleton, including osteoporosis, metastatic bone disease, multiple myeloma and osteosarcoma. Results to date are encouraging and indicate that drug efficacy can be increased and side effects reduced using these approaches. Despite these successes, challenges remain: no drugs have gone beyond small phase 2 clinical trials, and major pharmaceutical companies have shown little interest in the approach to repurpose any of their drugs or to embrace the technology. Nevertheless, interest shown by smaller biotechnology companies in the technology suggests that bone-targeting of drugs with bisphosphonates has a viable future.

The IAP Antagonist SM-164 Eliminates Triple-Negative Breast Cancer Metastasis to Bone and Lung in Mice.

The most challenging issue for breast cancer (BC) patients is metastasis to other organs because current therapies do not prevent or eliminate metastatic BC. Here, we show that SM-164, a small molecule inhibitor, which degrades inhibitor of apoptosis proteins (IAPs), eliminated early-stage metastases and reduced progression of advanced BC metastasis from MDA-MB-231 BC cells in bones and lungs of nude mice. Mechanistically, SM-164-induced BC cell death is TNFα-dependent, with TNFα produced by IL-4-polarized macrophages triggering MDA-MB-231 cell apoptosis in combination with SM-164. SM-164 also inhibited expression of RANKL, which mediates interactions between metastatic BC and host microenvironment cells and induces osteoclast-mediated osteolysis. SM-164 did not kill adriamycin-resistant BC cells, while adriamycin inhibited SM-164-resistant BC cell growth, similar to parental cells. We conclude that SM-164 is a promising therapeutic agent for early stage bone and lung metastasis from triple-negative breast cancer that should be given prior to conventional chemotherapy.

TNF Receptor-Associated Factor 6 Mediates TNFα-Induced Skeletal Muscle Atrophy in Mice During Aging.

During aging, muscle mass decreases, leading to sarcopenia, associated with low-level chronic inflammation (inflammaging), which induces sarcopenia by promoting proteolysis of muscle fibers and inhibiting their regeneration. Patients with a variety of pathologic conditions associated with sarcopenia, including rheumatoid arthritis (RA), have systemically elevated TNFα serum levels, and transgenic mice with TNFα overexpression (TNF-Tg mice, a model of RA) develop sarcopenia between adolescence and adulthood before they age. However, if and how TNFα contributes to the pathogenesis of sarcopenia during the normal aging process and in RA remains largely unknown. We report that TNFα levels are increased in skeletal muscles of aged WT mice, associated with muscle atrophy and decreased numbers of satellite cells and Type IIA myofibers, a phenotype that we also observed in adult TNF-Tg mice. Aged WT mice also have increased numbers of myeloid lineage cells in their skeletal muscles, including macrophages and granulocytes. These cells have increased TNFα expression, which impairs myogenic cell differentiation. Expression levels of TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, which mediates signaling by some TNF receptor (TNFR) family members, are elevated in skeletal muscles of both aged WT mice and adult TNF-Tg mice. TRAF6 binds to TNFR2 in C2C12 myoblasts and mediates TNFα-induced muscle atrophy through NF-κB-induced transcription of the muscle-specific E3 ligases, Atrogen1 and Murf1, which promote myosin heavy-chain degradation. Haplo-deficiency of TRAF6 prevents muscle atrophy and the decrease in numbers of satellite cells, Type IIA myofibers, and myogenic regeneration in TRAF6+/- ;TNF-Tg mice. Our findings suggest that pharmacologic inhibition of TRAF6 signaling in skeletal muscles during aging could treat/prevent age- and RA-related sarcopenia by preventing TNFα-induced proteolysis and inhibition of muscle fiber regeneration. © 2020 American Society for Bone and Mineral Research.