Exploration of the Fluorescent Properties and the Modulated Activities against Sirtuin Fluorogenic Assays of Chromenone-Derived Natural Products.

Chromenone-derived natural products include chromones (flavone, isoflavone) and coumarins. Chromenone compounds not only exhibit impressive biological activities, but also are an important resource of experimentally used fluorophores, such as, 7-amino-4-methylcoumarin (AMC). Various chromenone compounds have reported to have weak fluorescence, and this has the potential to interfere with the measurements during AMC fluorogenic assays and result in non-robust assay readouts. Several flavones and isoflavones were found as SIRT1 activators, while fluorogenic sirtuin assays utilized AMC labelled peptides as the substrates. In this study we investigated whether the fluorescent properties of chromenone-derived natural products interrupt the measurement of SIRT1/2 modulated activities. We found that the reported SIRT1 activators: flavones were detected with the SIRT1 activation activity, but isoflavones were not detected with SIRT1 activation activity, and instead that they were found to be fluorogenic compounds. Another chromenone compound, osthole, exhibited a moderate SIRT2 inhibitory activity with an IC50 of 10 µM. In conclusion, the fluorescent properties of these chromenone compounds do affect the measurement of the sirtuin activities of both inhibitors and activators. However, if the possible fluorescence properties are mitigated in the assay readout, these fluorogenic assays enable the screening of activity modulators.

Genistein induces apoptosis of colon cancer cells by reversal of epithelial-to-mesenchymal via a Notch1/NF-κB/slug/E-cadherin pathway.

BACKGROUND: Genistein has been known to inhibit proliferation and induce apoptosis in several kinds of cancer cells. While knowledge of genistein in regulating epithelial mesenchymal transition (EMT) of colon cancer cells is unknown. METHODS: To investigate the effects and mechanisms of genistein on EMT of colon cancer cells, HT-29 cells were used and treated by genistein and TNF-α in this paper. EMT was determined by cell invasion assays using a transwell chamber and the expression changes of EMT-related markers were confirmed by RT-PCR, Western blotting, and immunofluorescence staining. RESULTS: Genistein inhibited cell migration at 200 µmol/L. Genistein reversed the EMT of colon cancer cells by upregulation of E-cadherin and downregulation of N-cadherin, accompanied by the suppression of EMT related makers, such as Snail2/slug, ZEB1, ZEB2, FOXC1, FOXC2 and TWIST1. Moreover, genistein can inhibit the expression of notch-1, p-NF-κB and NF-κB, while promote the expression of Bax/Bcl-2 and caspase-3 in HT-29 cells. CONCLUSION: The present study demonstrated that genistein suppressed the migration of colon cancer cells by reversal the EMT via suppressing the Notch1/NF-κB/slug/E-cadherin pathway. Genistein may be developed as a potential antimetastasis agent to colon cancer.

Ad5/48 hexon oncolytic virus expressing sTGFßRIIFc produces reduced hepatic and systemic toxicities and inhibits prostate cancer bone metastases.

We are interested in developing oncolytic adenoviruses for the treatment of prostate cancer (PCa) bone metastases. A key limitation of Adenovirus 5 (Ad5) is that upon systemic administration, it produces major liver and systemic toxicities. To address this issue, a chimaeric Ad5/48 adenovirus mHAd.sTßRFc was created. Seven hypervariable regions of Ad5 hexon present in Ad5-based Ad.sTßRFc expressing soluble transforming growth factor beta receptor II-Fc fusion protein (sTGßRIIFc), were replaced by those of Ad48. mHAd.sTßRFc, like Ad.sTßRFc, was replication competent in the human PCa cells, and produced high levels of sTGßRIIFc expression. Compared to Ad.sTßRFc, the systemic delivery of mHAd.sTßRFc in nude mice resulted in much reduced systemic toxicity, and reduced liver sequestration. Ad.sTßRFc produced significant liver necrosis, and increases in alanine transaminase, aspartate transaminase, lactate dehydrogenase, tumor necrosis factor-α, and interleukin-6 levels, while mHAd.sTßRFc produced much reduced responses of these markers. Intravenous delivery of Ad.sTßRFc or mHAd.sTßRFc (5 × 10(10) viral particles/mouse) in nude mice bearing PC-3-luc PCa bone metastases produced inhibition of bone metastases. Moreover, a larger dose of the mHAd.sTßRFc (4 × 10(11) viral particles /mouse) was also effective in inhibiting bone metastases. Thus, mHAd.sTßRFc could be developed for the treatment of PCa bone metastases.

Sustainable and ultrafast antibiotics removal, self-cleaning and disinfection with electroactive metal-organic frameworks/carbon nanotubes membrane.

Although conventional nanofiltration (NF) membrane is widely applied in water treatment, it faces the challenges of insufficient selectivity toward emerging contaminants, low permeability and non-sustainable fouling control. Herein, a novel electroactive metal-organic frameworks/carbon nanotubes membrane was constructed by facile and green nanobubbles-mediated non-solvent-induced phase separation (NIPS) strategy for ultrafast antibiotics removal. It presented 3-fold to 100-fold higher permeability (101.3-105.7 L·h-1·m-2·bar-1) without compromising rejection (71.8 %-99.3 %) of common antibiotics (tetracycline, norfloxacin, sulfamethoxazole, sulfamethazine) than most commercial and state-of-the-art NF membranes. The separation mechanism was due to the synergy of loose selective layer with three-dimensional interconnected networks and UiO-66/CNTs with unique pore sieving and charge property. It also presented excellent antibiotics selectivity with high NaCl/tetracycline separation factor of 194 and CuCl2/tetracycline separation factor of 316 for remediation of antibiotics and heavy metal combined pollution. Meanwhile, it possessed efficient anti-fouling, antibacterial and electro-driven self-cleaning ability, which enabled sustainable fouling control and disinfection with short process, low energy and chemical consumption. Furthermore, potential application of UiO-66/CNTs membrane in wastewater reclamation was demonstrated by stable antibiotics rejection, efficient flux recovery and long-term stability over 260 h. This study would provide useful insights into removal of emerging contaminants from water by advanced NF membrane.

Homology Identification and Cross-Contamination Analysis: A Method for Evaluating the Quality of Biological Samples Stored in a Biobank Using the Advanta Sample ID Genotyping Panel.

Biological samples are important resources for scientific research. These samples are stored in biobanks over years until needed, and some of them can never be retrieved if they are improperly stored, causing them to be wasted. Thus, they are priceless, and they should be used correctly and effectively. Sample quality substantially affects biomedical research results. However, sample misidentification or mix-up is common. It is necessary to establish quality standards for sample identification. In this study, we used the Advanta Sample ID genotyping panel to detect homology identification and cross-contamination. We compared the single-nucleotide polymorphism (SNP) typing results of two different samples and calculated the similarity score of homologous sample pairs and nonhomologous sample pairs. Through analysis, we obtained a similarity score cutoff point of 0.8620, which was an effective way to distinguish homology and nonhomology. Cross-contamination was detected in two sets of mixtures (STD8:STD6 and jj3:1-P) mixed at a series of special ratios. Sensitivity was dependent on the sample characteristics and mixing ratios. Finally, we assessed the effect of sample degradation degree on SNP genotyping and found that degraded samples with a minimal DNA integrity number of 1.9 had complete genotyping results. On the whole, this study shows that the Sample ID panel is reliable for homology identification and cross-contamination analysis. Moreover, this technology has promising further applications in biological sample quality control.

The safety and efficacy of systemic delivery of a new liver-de-targeted TGFß signaling inhibiting adenovirus in an immunocompetent triple negative mouse mammary tumor model.

Aberrant TGFß signaling is linked to metastasis and tumor immune escape of many cancers including metastatic triple negative breast cancer (mTNBC). Previously, we have found that oncolytic adenoviruses expressing a TGFß signaling inhibitory protein (sTGFßRIIFc) induced immune activation in a mouse TNBC (4T1) immunocompetent subcutaneous model with intratumoral injection. Systemic administration of adenoviruses can be a superior route to treat mTNBC but faces the challenges of increased toxicity and viral clearance. Thus, we created a liver-de-targeted sTGFßRIIFc- and LyP-1 peptide-expressing adenovirus (mHAdLyp.sT) with enhanced breast cancer cell tropism. Its safety and immune response features were profiled in the 4T1 model. Our data showed that the systemic administration of mHAdLyp.sT resulted in reduced hepatic and systemic toxicity. mHAdLyp.sT was also effective in increasing Th1 cytokines and anti-tumor cell populations by cytokine analysis, spleen/tumor qRT-PCR, and flow cytometry. We further tested the therapeutic effects of mHAdLyp.sT alone and in combination with immune checkpoint inhibitors (ICIs). mHAdLyp.sT alone and with all ICI combinations elicited significant inhibition of lung metastasis by histological analysis. When mHAdLyp.sT was combined with both anti-PD-1 and anti-CTLA-4 antibodies, primary 4T1 tumor growth was also significantly inhibited. We are confident in advancing this new treatment option for mTNBC.

The Safety and Efficacy of Systemic Delivery of a New Liver-de-targeted TGFß Signaling Inhibiting Adenovirus in an Immunocompetent Triple Negative Mouse Mammary Tumor Model.

Aberrant TGFß signaling is linked to metastasis and tumor immune escape of many cancers including metastatic triple negative breast cancer (mTNBC). Previously, we have found that oncolytic adenoviruses expressing a TGFß signaling inhibitory protein (sTGFßRIIFc) induced immune activation in a mouse TNBC (4T1) immunocompetent subcutaneous model with intratumoral injection. Systemic administration of adenoviruses can be a superior route to treat mTNBC but faces the challenges of increased toxicity and viral clearance. Thus, we created a liver-de-targeted sTGFßRIIFc- and LyP-1 peptide-expressing adenovirus (mHAdLyp.sT) with enhanced breast cancer cell tropism. Its safety and immune response features were profiled in the 4T1 model. Our data showed that the systemic administration of mHAdLyp.sT resulted in reduced hepatic and systemic toxicity. mHAdLyp.sT was also effective in increasing Th1 cytokines and anti-tumor cell populations by cytokine analysis, spleen/tumor qRT-PCR, and flow cytometry. We further tested the therapeutic effects of mHAdLyp.sT alone and in combination with immune checkpoint inhibitors (ICIs). mHAdLyp.sT alone and with all ICI combinations elicited significant inhibition of lung metastasis by histological analysis. When mHAdLyp.sT was combined with both anti-PD-1 and anti-CTLA-4 antibodies, primary 4T1 tumor growth was also significantly inhibited. We are confident in advancing this new treatment option for mTNBC.

Oncolytic adenovirus expressing soluble TGFß receptor II-Fc-mediated inhibition of established bone metastases: a safe and effective systemic therapeutic approach for breast cancer.

In recent years, oncolytic adenoviruses have shown some promise as a novel class of antitumor agents. However, their utility in targeting bone metastases is relatively less studied. We have examined whether the systemic therapy of oncolytic adenoviruses expressing the soluble form of transforming growth factor-ß (TGFß) receptor II fused with human immunoglobulin G1 can be developed for the treatment of established breast cancer bone metastases. MDA-MB-231-luc2 human breast cancer cells were injected in the left heart ventricle of nude mice to establish bone metastasis. Mice with hind limb tumors were administered (on days 8 and 11) oncolytic adenoviruses-Ad.sTßRFc or mhTERTAd.sTßRFc. Skeletal tumor growth was monitored weekly by bioluminescence imaging (BLI) and radiography. At the termination time on day 28, hind limb bones were analyzed for tumor burden, synchrotron micro-computed tomography, and osteoclast activation. Intravenous delivery of Ad.sTßRFc and mhTERTAd.sTßRFc induced significant inhibition of tumor growth, reduction of tumor burden, osteoclast activation, and increased animals' survival. Oncolytic adenoviruses were safer than dl309, a wild-type virus. A slight elevation of liver enzyme activity was observed after Ad.sTßRFc administration; this subsided with time. Based on these studies, we believe that Ad.sTßRFc and mhTERTAd.sTßRFc can be developed as a safe and effective approach for the treatment of established bone metastasis.

LyP-1-Modified Oncolytic Adenoviruses Targeting Transforming Growth Factor ß Inhibit Tumor Growth and Metastases and Augment Immune Checkpoint Inhibitor Therapy in Breast Cancer Mouse Models.

We report here the development of oncolytic adenoviruses (Ads) that have reduced toxicity, enhanced tumor tropism, produce strong antitumor response, and can overcome resistance to immune checkpoint inhibitor therapy in breast cancer. We have shown that LyP-1 receptor (p32) is highly expressed on the surface of breast cancer cells and tumors from cancer patients, and that increased stromal expression of transforming growth factor ß-1 (TGFß-1) is associated with triple-negative breast cancer. Therefore, we constructed oncolytic Ads, AdLyp.sT and mHAdLyp.sT, in which the p32-binding LyP-1 peptide was genetically inserted into the adenoviral fiber protein. Both AdLyp.sT and mHAdLyp.sT express sTGFßRIIFc, a TGFß decoy that can inhibit TGFß pathways. mHAdLyp.sT is an Ad5/48 chimeric hexon virus in which hypervariable regions (HVRs 1-7) of Ad5 are replaced with the corresponding Ad48 HVRs. AdLyp.sT and mHAdLyp.sT exhibited better binding, replication, and produced higher sTGFßRIIFc protein levels in breast cancer cell lines compared with Ad.sT or mHAd.sT control viruses without LyP-1 peptide modification. Systemic delivery of mHAdLyp.sT in mice resulted in reduced hepatic/systemic toxicity compared with Ad.sT and AdLyp.sT. Intravenous delivery of AdLyp.sT and mHAdLyp.sT elicited a strong antitumor response in a human MDA-MB-231 bone metastasis model in mice, as indicated by bioluminescence imaging, radiographic tumor burden, serum TRACP 5b and calcium, and body weight analyses. Furthermore, intratumoral delivery of AdLyp.sT in 4T1 model in immunocompetent mice inhibited tumor growth and metastases, and augmented anti-PD-1 and anti-CTLA-4 therapy. Based on these studies, we believe that AdLyp.sT and mHAdLyp.sT can be developed as potential targeted immunotherapy agents for the treatment of breast cancer.

Genistein and daidzein induce apoptosis of colon cancer cells by inhibiting the accumulation of lipid droplets.

AIM: The purpose of this study was to investigate the possible mechanisms of genistein (GEN) and daidzein (DAI) in inducing apoptosis of colon cancer cells by inhibition of lipid droplets (LDs) accumulation. METHODS: HT-29 cells were used and treated by GEN or DAI in this paper. LDs accumulation was induced and inhibited by oleic acid (OA) and C75, respectively. The expression changes of LDs-related markers were confirmed by semiquantitative real time-PCR (RT-PCR), Western blotting, and immunofluorescence staining. RESULTS: GEN and DAI effectively reduced the LDs accumulation and downregulated the expression of Perilipin-1, ADRP and Tip-47 family proteins and vimentin levels. GEN and DAI significantly induced the mRNA expression of PPAR-γ, Fas, FABP, glycerol-3-phosphate acyltransferase (GPAT3), and microsomal TG transfer protein (MTTP), and reduced the mRNA expression of UCP2. Furthermore, the results showed a decrease of PI3K expression by GEN and DAI when compared with OA treatment, and both GEN and DAI can increase the expression of FOXO3a and caspase-8 significantly when these proteins were decreased by OA treatment. GEN is more effective than DAI in inducing cell apoptosis. CONCLUSION: Our results demonstrated that GEN and DAI inhibit the accumulation of LDs by regulating LDs-related factors and lead to a final apoptosis of colon cancer cells. These results may provide important new insights into the possible molecular mechanisms of isoflavones in anti-obesity and anti-tumor functions.

Efficient gene transfer into hematopoietic cells by a retargeting adenoviral vector system with a chimeric fiber of adenovirus serotype 5 and 11p.

OBJECTIVE: Adenoviral vectors (Ad) were widely used in gene therapy and study of gene function, but the commonly used serotype 5 adenovirus-based vectors (Ad5) could poorly transduce hematopoietic cells because of low expression of viral receptors on these cells. To overcome this limitation, we developed a retargeting adenovector with a chimeric fiber of Ad5 and Ad11p (Ad5F11p) and evaluated its gene transfer ability in hematopoietic cells. MATERIALS AND METHODS: An Ad11p fiber pseudotyped Ad5 vector was generated by modifying the fiber gene of pAdEasy-1 backbone plasmid. Ad5F11p-GFP encoding enhanced green fluorescence protein (GFP) gene was transferred into human leukemic cell lines, primary leukemic cells, and CD34(+) hematopoietic cells. The gene transduction efficiency was determined by fluorescence-activated cell sorting assay. RESULTS: More than 90% of U937 or K562 cells could be infected by Ad5F11p-GFP at a moderate multiplicity of infection (MOI). Ad5F11p-GFP is also significantly more effective than control Ad5-GFP in infection of primary myeloid leukemic cells. At 200 MOI, GFP-positive percentages of Ad5F11p-GFP transduced myeloid leukemic cells range from 10.58% to 92.63% with a median of 28.65%. Ad5F11p-GFP could transduce about 50% human hematopoietic stem/progenitor (CD34(+)) cells, while Ad5-GFP could transduce <15% at 200 MOI. CD46 was reported to be the receptor of Ad11p. Our data suggest that CD46 participates in the process of Ad5F11p-GFP infection but is not the unique molecule determining its gene transfer efficiency of host cells. CONCLUSION: We established a retargeting adenovector system, which could infect hematopoietic cells effectively and would benefit research work on Ad tropism.

Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, ß-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases.

Systemic delivery of oncolytic adenoviruses targeting transforming growth factor-ß inhibits established bone metastasis in a prostate cancer mouse model.

We have examined whether Ad.sTßRFc and TAd.sTßRFc, two oncolytic viruses expressing soluble transforming growth factor-ß receptor II fused with human Fc (sTGFßRIIFc), can be developed to treat bone metastasis of prostate cancer. Incubation of PC-3 and DU-145 prostate tumor cells with Ad.sTßRFc and TAd.sTßRFc produced sTGFßRIIFc and viral replication; sTGFßRIIFc caused inhibition of TGF-ß-mediated SMAD2 and SMAD3 phosphorylation. Ad(E1-).sTßRFc, an E1(-) adenovirus, produced sTGFßRIIFc but failed to replicate in tumor cells. To examine the antitumor response of adenoviral vectors, PC-3-luc cells were injected into the left heart ventricle of nude mice. On day 9, mice were subjected to whole-body bioluminescence imaging (BLI). Mice bearing hind-limb tumors were administered viral vectors via the tail vein on days 10, 13, and 17 (2.5×10(10) viral particles per injection per mouse, each injection in a 0.1-ml volume), and subjected to BLI and X-ray radiography weekly until day 53. Ad.sTßRFc, TAd.sTßRFc, and Ad(E1-).sTßRFc caused significant inhibition of tumor growth; however, Ad.sTßRFc was the most effective among all the vectors. Only Ad.sTßRFc and TAd.sTßRFc inhibited tumor-induced hypercalcemia. Histomorphometric and synchrotron micro-computed tomographic analysis of isolated bones indicated that Ad.sTßRFc induced significant reduction in tumor burden, osteoclast number, and trabecular and cortical bone destruction. These studies suggest that Ad.sTßRFc and TAd.sTßRFc can be developed as potential new therapies for prostate cancer bone metastasis.

Systemic delivery of a novel liver-detargeted oncolytic adenovirus causes reduced liver toxicity but maintains the antitumor response in a breast cancer bone metastasis model.

We are interested in developing oncolytic adenoviruses for the treatment of bone metastasis of cancer. A key limitation of systemic delivery of oncolytic adenovirus type 5 (Ad5) is that the majority of the virus is taken up by the liver, causing liver damage and systemic toxicity. Given that Ad5 hexon binding with blood coagulation factor X is a key factor in liver sequestration, and that a rare serotype, Ad48, has a diminished capacity to bind with factor X, we have generated mHAd.luc2, a novel hexon-chimeric oncolytic adenovirus. To create mHAd.luc2, seven hypervariable regions of Ad5 hexon were substituted with the corresponding regions from Ad48. Compared with Ad5-based oncolytic virus Ad.luc2, intravenous injection of mHAd.luc2 into nude mice resulted in significantly reduced liver uptake. A single high dose (1.0×10(11) viral particles/mouse) of Ad.luc2 resulted in 100% animal death by day 3; whereas none of the mice died in the mHAd.luc2 group. Liver enzyme and liver pathology studies indicated that mHAd.luc2 induced significantly less liver toxicity compared with Ad.luc2. Both mHAd.luc2 and Ad.luc2 exhibited similar binding with breast tumor cells, whereas in the presence of factor X, mHAd.luc2 binding was reduced. Both mHAd.luc2 and Ad.luc2 had nearly equal replication potential in breast cancer cells in vitro. Intravenous injection of mHAd.luc2 and Ad.luc2 into nude mice bearing bone metastases resulted in uptake of the viruses into skeletal tumors, and induced significant inhibition of established bone metastases. Thus, liver-detargeted oncolytic adenovirus can be developed for the treatment of breast cancer bone metastasis.

Systemic delivery of an oncolytic adenovirus expressing soluble transforming growth factor-ß receptor II-Fc fusion protein can inhibit breast cancer bone metastasis in a mouse model.

We have investigated whether systemic delivery of an oncolytic adenovirus, Ad.sTßRFc, expressing the soluble form of transforming growth factor-ß receptor II fused with human immunoglobulin Fc fragment (sTGFßRIIFc), could inhibit breast cancer bone metastasis in a mouse model. MDA-MB-231 (human breast cancer) cells were inoculated into the left heart ventricles of nude mice. Once the skeletal tumors were visible by X-rays, mice were intravenously injected with either buffer, Ad.sTßRFc, Ad(E1⁻).sTßRFc (a replication-deficient adenovirus expressing sTGFßRIIFc), or Ad.luc2 (a replicating adenovirus expressing firefly luciferase gene). On days 2 and 7 after viral injections, viral replication and sTGFßRIIFc expression were detected in the skeletal tumors in Ad.sTßRFc-treated group; only viral replication in Ad.luc2 group, and sTGFßRIIFc expression in the Ad(E1⁻).sTßRFc group, were detected. To examine the therapeutic effects, buffer or various viral vectors were administered on days 4 and 7 after intracardiac injection of MDA-MB-231 cells. On day 28, X-ray radiography showed a highly significant reduction in lesion size by Ad.sTßRFc, a significant reduction by Ad.luc2, and some reduction by Ad(E1⁻).sTßRFc. Goldner's trichrome and hematoxylin-eosin staining of the bone sections revealed a significant reduction of tumor burden in the Ad.sTßRFc group, but not in the Ad(E1⁻).sTßRFc or Ad.luc2 group. There were significant reductions in free calcium levels by Ad.sTßRFc, Ad(E1⁻).sTßRFc, and Ad.luc2; however, only in the Ad.sTßRFc group were calcium levels reduced to the normal values. These results suggest that concomitant viral replication and sTGFßRIIFc production are important to inhibit bone metastasis and osteolysis, and that Ad.sTßRFc could be developed for targeting breast cancer bone metastases.