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• W2775101460 abstract "•Jagged1 antibody 15D11 reduces bone metastasis without significant side effects•Chemotherapy induces tumor-protecting Jagged1 in osteoblasts•Transgenic expression of Jagged1 in osteoblasts promotes bone metastasis•15D11 sensitizes bone metastasis to chemotherapy Bone metastasis is a major health threat to breast cancer patients. Tumor-derived Jagged1 represents a central node in mediating tumor-stromal interactions that promote osteolytic bone metastasis. Here, we report the development of a highly effective fully human monoclonal antibody against Jagged1 (clone 15D11). In addition to its inhibitory effect on bone metastasis of Jagged1-expressing tumor cells, 15D11 dramatically sensitizes bone metastasis to chemotherapy, which induces Jagged1 expression in osteoblasts to provide a survival niche for cancer cells. We further confirm the bone metastasis-promoting function of osteoblast-derived Jagged1 using osteoblast-specific Jagged1 transgenic mouse model. These findings establish 15D11 as a potential therapeutic agent for the prevention or treatment of bone metastasis. Bone metastasis is a major health threat to breast cancer patients. Tumor-derived Jagged1 represents a central node in mediating tumor-stromal interactions that promote osteolytic bone metastasis. Here, we report the development of a highly effective fully human monoclonal antibody against Jagged1 (clone 15D11). In addition to its inhibitory effect on bone metastasis of Jagged1-expressing tumor cells, 15D11 dramatically sensitizes bone metastasis to chemotherapy, which induces Jagged1 expression in osteoblasts to provide a survival niche for cancer cells. We further confirm the bone metastasis-promoting function of osteoblast-derived Jagged1 using osteoblast-specific Jagged1 transgenic mouse model. These findings establish 15D11 as a potential therapeutic agent for the prevention or treatment of bone metastasis. Current treatments for bone metastasis often reduce skeletal-related events without significant reduction of tumor burden or survival benefit for patients. Furthermore, bone metastasis is particularly refractory to chemotherapy. While γ-secretase inhibitors have therapeutic efficacy against bone metastasis in preclinical models, the high gastrointestinal tract toxicity of these agents prevented their further clinical development. 15D11 is a fully human monoclonal antibody against Jagged1 with minimal toxicity and excellent therapeutic efficacy against bone metastasis. Importantly, by targeting tumor-protective osteoblastic Jagged1 induced by chemotherapy, 15D11 synergizes with chemotherapy to reduce bone metastasis burden by more than 100-fold and dramatically reduces metastatic relapse to bone from primary tumors. These results indicate broad potential applications of 15D11 for bone metastasis prevention or treatment. Breast cancer is the most common female malignancy and the second leading cause of cancer-related death in the United States. Among late-stage breast cancer patients, more than 70% suffer from bone metastasis, which is often accompanied by severe bone pain, fracture, and potentially lethal complications such as hypercalcemia (Weilbaecher et al., 2011Weilbaecher K.N. Guise T.A. McCauley L.K. Cancer to bone: a fatal attraction.Nat. Rev. Cancer. 2011; 11: 411-425Crossref PubMed Scopus (890) Google Scholar). Although radiotherapy, chemotherapy, and anti-osteolytic agents such as bisphosphonate and RANKL antibody denosumab can reduce morbidity associated with bone metastasis, these treatments often do not significantly extend the survival time of the patients or provide a cure (Weilbaecher et al., 2011Weilbaecher K.N. Guise T.A. McCauley L.K. Cancer to bone: a fatal attraction.Nat. Rev. Cancer. 2011; 11: 411-425Crossref PubMed Scopus (890) Google Scholar), as metastatic cancers often acquire resistance to these treatments. Tumor-stromal interaction plays a major role in promoting bone metastasis of breast cancer (Weilbaecher et al., 2011Weilbaecher K.N. Guise T.A. McCauley L.K. Cancer to bone: a fatal attraction.Nat. Rev. Cancer. 2011; 11: 411-425Crossref PubMed Scopus (890) Google Scholar). The bone microenvironment contains a great variety of stromal cell types, such as osteoblasts, osteoclasts, mesenchymal stem cells (MSCs), and hematopoietic cells. While previous research has focused on the cross-communication between breast cancer cells and bone-resorbing osteoclasts, the contributions of other stromal cell types to bone metastasis are less studied. Among the supporting stromal cells, bone-building osteoblasts have recently been shown to constitute an osteogenic niche that is critical for the survival and colonization of disseminated tumor cells (DTCs) in the bone (Shiozawa et al., 2011Shiozawa Y. Pedersen E.A. Havens A.M. Jung Y. Mishra A. Joseph J. Kim J.K. Patel L.R. Ying C. Ziegler A.M. et al.Human prostate cancer metastases target the hematopoietic stem cell niche to establish footholds in mouse bone marrow.J. Clin. Invest. 2011; 121: 1298-1312Crossref PubMed Scopus (553) Google Scholar, Wang et al., 2015Wang H. Yu C. Gao X. Welte T. Muscarella A.M. Tian L. Zhao H. Zhao Z. Du S. Tao J. et al.The osteogenic niche promotes early-stage bone colonization of disseminated breast cancer cells.Cancer Cell. 2015; 27: 193-210Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Despite this recent progress, our molecular understanding of the interaction between tumor cells and osteoblastic cells in the bone niche remain largely incomplete. For example, how such tumor-niche interactions contribute to the resistance of metastatic breast cancer to standard bone metastasis treatments, such as chemotherapy, remains poorly understood. In human breast cancer, elevated expression of Jagged1 and Notch1, but not other Notch pathway ligands or receptors, is significantly associated with poor prognosis (Reedijk et al., 2005Reedijk M. Odorcic S. Chang L. Zhang H. Miller N. McCready D.R. Lockwood G. Egan S.E. High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival.Cancer Res. 2005; 65: 8530-8537Crossref PubMed Scopus (618) Google Scholar, Sethi et al., 2011Sethi N. Dai X. Winter C.G. Kang Y. Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.Cancer Cell. 2011; 19: 192-205Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Our previous study identified tumor-derived Jagged1 as a bone metastasis-promoting factor by activating Notch signaling in osteoblasts to increase the production of interleukin-6 (IL6) and connective tissue growth factor (TGF), which feeds back to tumor cells to promote proliferation and survival. Meanwhile, Jagged1 stimulates osteoclastogenesis and bone degradation, leading to the release of bone-derived growth factors including TGF-β, a potent inducer of Jagged1 expression in tumor cells, thus forming a positive feedback cycle (Sethi et al., 2011Sethi N. Dai X. Winter C.G. Kang Y. Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.Cancer Cell. 2011; 19: 192-205Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Besides Jagged1's role in tumor-stromal interaction during bone metastasis progression, tumor- or stromal-derived Jagged1 has also been reported to induce angiogenesis, invasion, therapy resistance, and cancer stem cell renewal in lymphoma, colorectal cancer, and many other cancer types (Li et al., 2014Li D. Masiero M. Banham A.H. Harris A.L. The notch ligand JAGGED1 as a target for anti-tumor therapy.Front. Oncol. 2014; 4: 254Crossref PubMed Scopus (145) Google Scholar). Endothelium-derived Jagged1 promotes Notch activation in B cell lymphoma, leading to extra-nodal invasion and chemoresistance (Cao et al., 2014Cao Z. Ding B.S. Guo P. Lee S.B. Butler J.M. Casey S.C. Simons M. Tam W. Felsher D.W. Shido K. et al.Angiocrine factors deployed by tumor vascular niche induce B cell lymphoma invasiveness and chemoresistance.Cancer Cell. 2014; 25: 350-365Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). In colorectal cancer, a soluble form of Jagged1 derived from endothelial cells induces a cancer stem cell-like phenotype in colorectal cancer (Lu et al., 2013Lu J. Ye X. Fan F. Xia L. Bhattacharya R. Bellister S. Tozzi F. Sceusi E. Zhou Y. Tachibana I. et al.Endothelial cells promote the colorectal cancer stem cell phenotype through a soluble form of Jagged-1.Cancer Cell. 2013; 23: 171-185Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). These multi-functional roles of Jagged1 in different cancer types support the development of Jagged1 targeting therapeutics for cancer treatments. Several therapeutic strategies have been developed to target the Notch signaling pathway. Most of the inhibitors were designed to target γ-secretase, which mediates the proteolytic cleavage of Notch receptors to generate a signal-transducing Notch intracellular domain, a necessary step in Notch pathway activation upon ligand binding (Rizzo et al., 2008Rizzo P. Osipo C. Foreman K. Golde T. Osborne B. Miele L. Rational targeting of Notch signaling in cancer.Oncogene. 2008; 27: 5124-5131Crossref PubMed Scopus (337) Google Scholar). However, γ-secretase inhibitor (GSI) has been reported to induce severe gastrointestinal (GI) tract toxicity (Imbimbo, 2008Imbimbo B.P. Therapeutic potential of gamma-secretase inhibitors and modulators.Curr. Top. Med. Chem. 2008; 8: 54-61Crossref PubMed Scopus (125) Google Scholar), preventing further clinical development of this class of inhibitors. Targeting individual Notch receptor or ligand can potentially achieve therapeutic effect without causing severe GI tract toxicity. We utilized the XenoMouse technology to generate fully human monoclonal antibodies against Jagged1. These mice have the endogenous mouse immunoglobulin loci inactivated and large transgenes introduced, which are capable of recombination and fully human antibody repertoire development (Mendez et al., 1997Mendez M.J. Green L.L. Corvalan J.R. Jia X.C. Maynard-Currie C.E. Yang X.D. Gallo M.L. Louie D.M. Lee D.V. Erickson K.L. et al.Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice.Nat. Genet. 1997; 15: 146-156Crossref PubMed Scopus (330) Google Scholar). In brief, we immunized XenoMouse animals, including strains XMG2KL and XMG4KL (Kellermann and Green, 2002Kellermann S.A. Green L.L. Antibody discovery: the use of transgenic mice to generate human monoclonal antibodies for therapeutics.Curr. Opin. Biotechnol. 2002; 13: 593-597Crossref PubMed Scopus (81) Google Scholar), with Chinese hamster ovary cells transiently expressing human Jagged1 to generate large panels of antibodies. Cell-based fluorometric microvolume assay technology was used to analyze the specific binding of these antibodies to cell surface human Jagged1 protein. These antibodies were then counter screened to exclude cross-reactive binding to other major Notch ligands, including human Jagged2 and Dll4 (Figure S1A). To identify a suitable antibody antagonist that could be used for murine in vivo studies, the most potent blockers of the Notch receptor interaction were then further assayed for cross-reactive binding to murine Jagged1. Antibody clone 15D11 is of particularly high affinity to the murine Jagged1 protein, with an approximate dissociation constant (KD) of 23 pM in the enzyme kinetic exclusion assay (KinExA) (Figure 1A). Using a Notch luciferase reporter assay, we determined that 15D11 has a half maximal inhibitory concentration of 3.63 nM to human Jagged1 and 14.07 nM to murine Jagged1 (Figure 1B and data not shown). When administered in vivo (10 mg/kg, twice a week), 15D11 displayed minimal general toxicity based on body weight measurement, in contrast to significant weight loss of mice treated with GSI (Figure S1B). H&E staining and Alcian blue staining revealed no obvious GI tract toxicity after 15D11 treatment, compared with significant goblet cell metaplasia seen in GSI-treated animals (Figures S1C and S1D). To analyze liver toxicity, we measured the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the sera of these mice after the respective drug treatments. As a positive control, CCl4 treatment induced dramatic increase of ALT and AST activity (Figures S1E and S1F), while no change was observed after 15D11 or GSI treatment. A complete blood count test further indicated no significant hematologic toxicity of 15D11 (Figure S1G). Analysis of T cell population and activation status also did not reveal any difference between the immunoglobulin G (IgG) or 15D11 treatment groups (Figures S1H–S1K). Taken together, these results indicate that 15D11 is a specific Jagged1-targeting agent with excellent safety profile for in vivo application. Our previous study demonstrated that tumor-derived Jagged1 promotes the differentiation of monocytic pre-osteoclast into mature osteoclasts (Sethi et al., 2011Sethi N. Dai X. Winter C.G. Kang Y. Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.Cancer Cell. 2011; 19: 192-205Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). We developed an in vitro assay to test the potential inhibitory effect of 15D11 on Jagged1-dependent osteoclastogenesis. RAW264.7, a monocyte/macrophage cell line with the ability to differentiate into osteoclasts, was seeded on either Fc-coated or recombinant Jagged1 (rJagged1)-coated plates with a low level of RANKL (5 ng/mL), a concentration that is not sufficient to induce osteoclast differentiation (Ell et al., 2013Ell B. Mercatali L. Ibrahim T. Campbell N. Schwarzenbach H. Pantel K. Amadori D. Kang Y. Tumor-induced osteoclast miRNA changes as regulators and biomarkers of osteolytic bone metastasis.Cancer Cell. 2013; 24: 542-556Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Within 5 days, large multi-nuclear mature osteoclasts were generated on rJagged1-coated plates, but only limited numbers can be seen on Fc-coated plates. Furthermore, such Jagged1-dependent osteoclast differentiation was completely blocked by 15D11 (Figures 1C and 1D). Similar results were observed when using bone marrow-derived primary pre-osteoclasts in the assay (Figures 1E and S1L). To more closely mimic osteoclastogenesis induced by tumor-derived Jagged1, we co-cultured RAW264.7 cells with SCP28 cells, an MDA-MB-231 derivative cell line with moderate bone metastatic ability that has been stably labeled with luciferase and GFP (Kang et al., 2003Kang Y. Siegel P.M. Shu W. Drobnjak M. Kakonen S.M. Cordon-Cardo C. Guise T.A. Massague J. A multigenic program mediating breast cancer metastasis to bone.Cancer Cell. 2003; 3: 537-549Abstract Full Text Full Text PDF PubMed Scopus (2037) Google Scholar), with or without stable overexpression of Jagged1. Administration of 15D11 also inhibited osteoclast differentiation in this co-culture assay (Figures 1F and S1M). Another previously reported mechanism for tumor-derived Jagged1 to promote bone metastasis is through increasing IL6 production from osteoblasts (Sethi et al., 2011Sethi N. Dai X. Winter C.G. Kang Y. Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.Cancer Cell. 2011; 19: 192-205Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). As reported previously, co-culture of SCP28 with the mouse MC3T3 osteoblast cell line led to elevated expression of IL6 in a Jagged1-dependent manner (Figures 1G and 1H). Administration of 15D11 in tumor-osteoblast co-culture significantly repressed the expression of the Notch-induced gene Hes1 (detected by species-specific qRT-PCR) (Figure 1G), as well as IL6 expression at both the mRNA and protein levels (Figures 1G and 1H). Taken together, these in vitro experiments confirm that 15D11 effectively blocks Jagged1-Notch signaling activities that are important for bone metastasis development. To evaluate the therapeutic effect of 15D11 on bone metastasis, we turned to a well-established xenograft model for bone metastasis by using SCP28 cells. SCP28 has a low basal level of Jagged1 expression, and ectopic overexpression of Jagged1 in SCP28 has been demonstrated to enhance its bone metastatic ability (Sethi et al., 2011Sethi N. Dai X. Winter C.G. Kang Y. Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.Cancer Cell. 2011; 19: 192-205Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Tumor cells were inoculated via intracardiac (IC) injection into athymic nude mice to generate bone metastasis. We initiated the 15D11 antibody treatment 1 day before tumor cell injection and continued the treatment twice a week. Bone metastasis was monitored by weekly bioluminescence imaging (BLI) until mice were euthanized at the end of the experiment (∼5 weeks) for histopathological analysis (Figure 2A). We also used the decoy receptor for RANKL, OPG-Fc (equivalent to clinically approved RANKL blocking antibody denosumab), to block osteoclast differentiation as the positive control in the experiment. Jagged1 overexpression led to a significant increase in bone metastasis burden (Figures 2B and 2C), consistent with our previous report (Sethi et al., 2011Sethi N. Dai X. Winter C.G. Kang Y. Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.Cancer Cell. 2011; 19: 192-205Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). BLI analysis revealed an ∼5-fold decrease in SCP28-Jagged1 bone metastasis in mice treated with 15D11 compared with the IgG control group. As expected, OPG-Fc treatment also decreased the bone metastasis burden of mice (Figures 2B and 2C). X-ray imaging of the hindlimbs of mice injected with SCP28-Jagged1 revealed significantly more osteolytic areas compared with mice injected with the control SCP28 cells, a phenotype that can be reversed by 15D11 treatment (Figures 2B and 2D). While OPG-Fc treatment also protected bone from osteolysis, it also led to an abnormal increase of the trabecular bone density, as observed by X-ray and microcomputer tomography (μCT) analysis (Figures 2D and 2E; Movie S1. Representative μCT 3D Construction of Hindlimb Bone from SCP28-Vector Cell-Injected Mice, Related to Figure 2, Movie S2. Representative μCT 3D Construction of Hindlimb Bone from SCP28-Jagged1 Cell-Injected, IgG Antibody-Treated Mice, Related to Figure 2, Movie S3. Representative μCT 3D Construction of Hindlimb Bone from SCP28-Jagged1 Cell-Injected, 15D11 Antibody-Treated Mice, Related to Figure 2, Movie S4. Representative Micro-CT 3D Construction of Hindlimb Bone from SCP28-Jagged1 Cell-Injected, OPG-Treated Mice, Related to Figure 2). Consistently, tartrate-resistant acid phosphatase (TRAP) immunostaining of bone sections indicated an almost complete absence of osteoclasts in mice treated with OPG-Fc (Figure 2E). Interestingly, while Jagged1-overexpressing SCP28 promoted the recruitment of mature osteoclasts in the bone lesions, a considerable number of osteoclasts can still be observed in mice treated with 15D11 (Figures 2E and S2A). Importantly, there was no abnormal increase of trabecular bone density in 15D11-treated animals, in contrast to OPG-Fc-treated mice (Figure 2E). Taken together, these results suggest that 15D11 inhibits pathological activation of osteoclasts by tumor-derived Jagged1, but spares normal osteoclast activities needed to maintain normal bone homeostasis. To test the therapeutic effect of 15D11 on established bone metastasis, we used the SCP2 cell line, an extremely aggressive bone metastatic variant of MDA-MB-231 with a high level of endogenous Jagged1 expression (Sethi et al., 2011Sethi N. Dai X. Winter C.G. Kang Y. Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.Cancer Cell. 2011; 19: 192-205Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). The SCP2 cells rapidly generated multiple osteolytic bone metastases within 1 week after IC injection into nude mice, which often results in mortality within 4 weeks. We initiated twice-weekly injection of 15D11 1 week after IC injection of SCP2 cells when bone metastases were well established (Figure S2B). In this model of late treatment of aggressive bone metastasis, we detected a trend of reduced bone metastasis by either 15D11 or OPG-Fc treatment alone compared with the control group (Figures S2C and S2D), although neither of them reach statistical significance at the endpoint of the experiment (4 weeks) when the mice succumbed to metastatic cancers. Combinatory treatment of 15D11 and OPG-Fc resulted in a significant ∼5-fold decrease of bone metastasis 3 weeks after the initiation of treatments, indicating the benefit of targeting two different molecular mediators of bone metastasis (Figures S2C and S2D). Similar to the preventive treatment protocol, 15D11 treatment reduced the number of osteoclasts and decreased osteolytic bone areas, although some basal level of osteoclasts can still be observed, compared with the near complete absence of osteoclasts in mice treated with OPG-Fc (Figures S2E–S2G). Jagged1 has been extensively studied for its role in developmental angiogenesis (Benedito et al., 2009Benedito R. Roca C. Sorensen I. Adams S. Gossler A. Fruttiger M. Adams R.H. The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis.Cell. 2009; 137: 1124-1135Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar). Therefore, we tested the inhibitory effect of 15D11 on angiogenesis using two different angiogenesis models. In both neonatal mouse retina and subcutaneous xenograft tumor angiogenesis assays (Polverino et al., 2006Polverino A. Coxon A. Starnes C. Diaz Z. DeMelfi T. Wang L. Bready J. Estrada J. Cattley R. Kaufman S. et al.AMG 706, an oral, multikinase inhibitor that selectively targets vascular endothelial growth factor, platelet-derived growth factor, and kit receptors, potently inhibits angiogenesis and induces regression in tumor xenografts.Cancer Res. 2006; 66: 8715-8721Crossref PubMed Scopus (289) Google Scholar, Ridgway et al., 2006Ridgway J. Zhang G. Wu Y. Stawicki S. Liang W.C. Chanthery Y. Kowalski J. Watts R.J. Callahan C. Kasman I. et al.Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis.Nature. 2006; 444: 1083-1087Crossref PubMed Scopus (826) Google Scholar), 15D11 treatment showed no effect on angiogenesis (Figures S3A–S3D). Therefore, it is unlikely that 15D11 reduces bone metastasis through inhibiting angiogenesis. Chemotherapy is commonly used to manage bone metastasis, although bone lesions are usually more refractory to chemotherapy (Gu et al., 2004Gu B. Espana L. Mendez O. Torregrosa A. Sierra A. Organ-selective chemoresistance in metastasis from human breast cancer cells: inhibition of apoptosis, genetic variability and microenvironment at the metastatic focus.Carcinogenesis. 2004; 25: 2293-2301Crossref PubMed Scopus (27) Google Scholar). To evaluate the combinatorial effect of 15D11 with conventional chemotherapy, we treated bone metastasis generated by SCP28-Jagged1 cells with 15D11 and paclitaxel, using the late treatment protocol (treatment initiated 1 week after IC injection) (Figure 3A). The mouse group injected with SCP28-Jagged1 progressed slowly to the moribund state compared with the mouse group injected with SCP2 (7 weeks compared with 4 weeks with SCP2), which provided us with a sufficient time to observe the therapeutic effect of single or combinatory treatment of 15D11 and paclitaxel. Mice were treated with IgG, 15D11, paclitaxel, or both 15D11 and paclitaxel, 1 week after IC injection of SCP28-Jagged1 to establish bone metastases. Monotherapy using 15D11 resulted in a nearly 10-fold reduction of metastasis burden (Figure 3B). While paclitaxel alone significantly slowed down bone metastasis progression at early time points, resistance to chemotherapy emerged at later time points, resulting in only a mild reduction of tumor burden at the endpoint of the experiment (Figure 3B). Strikingly, when mice were treated with both 15D11 and paclitaxel, a synergistic >100-fold reduction of bone metastasis was observed compared with the IgG control group (Figures 3B and 3C). X-ray images and TRAP staining for osteoclasts confirmed significant decrease of osteoclast number and osteolytic lesions after 15D11 treatment alone or combined with chemotherapy (Figures 3C–3F). In particular, there is almost no osteolytic bone area in the combined treatment group, consistent with the BLI data. Taken together, these results demonstrate a potent synergistic effect of combined treatment of paclitaxel and 15D11 in diminishing bone metastasis generated by SCP28-Jagged1. This strong inhibitory effect of the combined treatment on bone metastasis could potentially be explained by the combined effect of blocking Jagged1-dependent stromal engagement by 15D11 and cytotoxicity of paclitaxel to tumor cells. If this is indeed the case, no synergistic therapeutic benefit should be expected when chemotherapy is combined with 15D11 for the treatment of bone metastasis generated by tumor cells with low or no Jagged1 expression. We thus used the parental SCP28 cells, which has very low endogenous Jagged1 level (Figure S4A), to test this directly. We performed the same bone metastasis and treatment experiments as we did with SCP28-Jagged1 (Figure 4A). Paclitaxel initially reduced bone metastasis burden, but the lesions quickly become refractory to chemotherapy and only slightly reduced metastatic burden (Figures 4B and 4C). As expected from the lack of Jagged1 expression in SCP28, 15D11 had no effect on reducing bone metastasis burden by SCP28 (Figures 4B and 4C). To our surprise, the combined treatment group displayed a dramatic reduction of bone metastasis burden, decreased osteolytic bone lesion areas, and reduced osteoclast number (Figures 4B–4F). Such a therapeutic response is far superior than single treatment of either paclitaxel or 15D11 alone, suggesting a synergy of targeting Jagged1 along with chemotherapy for bone metastasis, even in in tumor cells with low levels of Jagged1 expression. To validate whether the observed synergy of combined treatment can also be observed in other models of bone metastasis beyond the MDA-MB-231 series, we used the bone metastatic SUM1315-M1B1 cell line, which has been recently developed in our laboratory by in vivo selection for increased bone metastatic propensity (our unpublished data) from the parental SUM1315 breast cancer cell line (Forozan et al., 1999Forozan F. Veldman R. Ammerman C.A. Parsa N.Z. Kallioniemi A. Kallioniemi O.P. Ethier S.P. Molecular cytogenetic analysis of 11 new breast cancer cell lines.Br. J. Cancer. 1999; 81: 1328-1334Crossref PubMed Scopus (164) Google Scholar). SUM1315-M1B1 has a similarly low basal level of Jagged1 expression as SCP28 (Figure S4A). Again, a significant reduction of bone metastasis burden, osteolytic lesion area, and osteoclast numbers was only observed in mice treated with both paclitaxel and 15D11 (Figures S4B–S4G). Taken together, we identified a potent synergistic inhibitory effect on bone metastasis by combining chemotherapy and 15D11, and such therapeutic synergy is not dependent on high expression level of Jagged1 in tumor cells. We considered the possibility that chemotherapy agents may induce Jagged1 expression in either tumor cells or in the bone stromal cells. Such chemotherapy-induced Jagged1 might contribute to the resistance of bone metastasis to chemotherapy and can be targeted by 15D11, as we saw in the combined treatment. Key stromal cell types in bone metastasis include osteoclasts, osteoblasts and their progenitors (such as MSCs), and endothelial cells. We tested if Jagged1 could be induced in these cells and various breast cancer cell lines upon treatment of two different chemotherapy agents, paclitaxel and cisplatin, which are commonly used in the treatment of breast cancer. Jagged1 expression was significantly increased only in MC3T3-E1 pre-osteoblast cell and MSCs (Ren et al., 2008Ren G. Zhang L. Zhao X. Xu G. Zhang Y. Roberts A.I. Zhao R.C. Shi Y. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.Cell Stem Cell. 2008; 2: 141-150Abstract Full Text Full Text PDF PubMed Scopus (1550) Google Scholar) (Figure 5A). There was no significant induction of Jagged1 in endothelial cells, RAW 264.7 pre-osteoclasts, or in SCP28 and SUM1315-M1B1 tumor cells (Figure 5A). To confirm this finding in vivo, female nude mice were treated with either PBS or cisplatin. Hindlimb bones were dissected 48 hr later and immuno-stained with antibodies against Jagged1 and alkaline phosphatase (ALP), a marker for osteoblast cells. In the PBS control group, Jagged1+ cells were rarely detected and mostly co-localized with ALP+ osteoblasts. Cisplatin treatment induced much stronger Jagged1 expression based on immunostaining analysis, and these Jagged1+ cells again mostly overlapped with ALP+ cells (Figure 5B). This result thus indicates that chemotherapy induces Jagged1 expression in osteoblast cells in vivo. We next sought to understand which signaling pathway is responsible for Jagged1 induction after chemotherapy in osteoblast lineage cells. Chemotherapy generates many stress responses in cells, including endoplasmic reticulum (ER) stress and oxidative stress. Some of the stress conditions have been associated with the regulation of Jagged1 expression (Paul et al., 2014Paul M.K. Bis" @default.
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• W2775101460 date "2017-12-01" @default.
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• W2775101460 title "Therapeutic Antibody Targeting Tumor- and Osteoblastic Niche-Derived Jagged1 Sensitizes Bone Metastasis to Chemotherapy" @default.
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• W2775101460 doi "https://doi.org/10.1016/j.ccell.2017.11.002" @default.
• W2775101460 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5729937" @default.
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