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 (manuscript in preparation) from the parental SUM1315 breast cancer cell line (Forozan et al., 1999). 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 (Figure S4B-G). 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.
Chemotherapy induces Jagged1 expression in osteoblast lineage 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., 2008) (Figure 5A). There was no significant induction of Jagged1 in endothelial cells, RAW 264.7 preosteoclasts, 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. Hind limb 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 stress (ER stress) and oxidative stress. Some of the stress conditions have been associated with the regulation of Jagged1 expression (Paul et al., 2014). We utilized several treatments to mimic or inhibit these stress responses and then analyzed Jagged1 mRNA and protein levels within the cells. Among these treatments, we detected that ascorbate, which induces oxidative stress when used at high concentration, and H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} induced strong Jagged1 expression in MSCs, while ER stress inducers Brefeldin A (BFA) and Tunicamycin did not induce Jagged1 expression (Figure S5A and S5B). Consistent with this observation, cisplatin and docetaxel induced ROS production in MSCs, which could be completely blocked by N-Acetyl Cysteine (NAC), an ROS inhibitor (Figure S5C). Administration of NAC also completely blocked cisplatininduced Jagged1 expression in MSCs (Figure S5D). These results indicate that Jagged1 is induced in osteoblastic lineage cells during chemotherapy, likely through the ROS pathway.
mu CT\boldsymbol{\mu C T} 分析--在新泽西癌症研究所临床前成像共享资源中心使用INVEON PET/CT(西门子医疗集团)对股骨和胫骨进行扫描。X射线管设置为80千伏和 500 muA500 \mu \mathrm{~A} ,以最高分辨率采集图像,不进行CCD分档,因此体素大小为 9.44 mum9.44 \mu \mathrm{~m} 。在 195^(@)195^{\circ} 角度范围内使用 0.66^(@)0.66^{\circ} 旋转步长,曝光时间为 6500 毫秒。在使用 INVEON Research Workplace 软件(西门子医疗集团)进行分析之前,用光束硬化校正和 Hounsfield 校正对图像进行重建。使用三维高斯滤波器进行处理以减少噪音后,人工分割出与皮质和骨小梁区域相对应的 ROI。
X 射线成像和骨溶解病变定量--通过 X 射线射线照相术评估小鼠的骨溶解病变。将麻醉小鼠放在独立包装的胶片(BIOMAX XAR 胶片,Cat#: F5763-50EA, Sigma-Aldrich)上,使用 MX-20 Faxitron 仪器在 35 千伏电压下照射 X 射线 15 秒。胶片用柯尼卡 SRX-101A 冲洗机冲洗。使用 Adobe Photoshop 软件(Adobe Systems Inc.
Western Blot 分析-SDS 裂解缓冲液(0.05 mM Tris-HCl、50 mM BME、2% SDS、0.1% 溴酚蓝、10% 甘油)用于收集细胞中的蛋白质。样品经加热变性后,在 10%SDS-page凝胶上进行等量加载、分离,然后转移到 PVDF 膜(Millipore)上,并用 5%牛奶进行阻断。用于免疫印迹的一抗包括:抗 beta\beta -肌动蛋白(1:10,000 稀释,Abcam,cat#ab6276,克隆 AC-15)、抗裂解 Caspase 3(1:1000 稀释,Cell Signaling,Cat# 9661S)、抗 IL6
结果以平均值 +-SD\pm \mathrm{SD} (标准偏差)或平均值 +-SE\pm \mathrm{SE} (平均值的标准误差)报告,如图例所示。统计比较采用非配对双侧学生 t 检验(不等方差假设)和 Mann-Whitney U 检验。所有体外实验重复三次,动物实验重复一次。所有实验的代表性图像(包括 Western 印迹和
免疫荧光)至少重复两次,并显示代表性图像(骨髓样本除外)。图 8 中的免疫荧光图像是同类骨髓细胞标本(治疗前与治疗后)的代表性图像。使用 ImageJ 软件对这些 IF 图像进行量化。
The table highlights the genetically modified organisms and strains, cell lines, reagents, software, and source data essential to reproduce results presented in the manuscript. Depending on the nature of the study, this may include standard laboratory materials (i.e., food chow for metabolism studies), but the Table is not meant to be comprehensive list of all materials and resources used (e.g., essential chemicals such as SDS, sucrose, or standard culture media don’t need to be listed in the Table). Items in the Table must also be reported in the Method Details section within the context of their use. The number of primers and RNA sequences that may be listed in the Table is restricted to no more than ten each. If there are more than ten primers or RNA sequences to report, please provide this information as a supplementary document and reference this file (e.g., See Table S1 for XX) in the Key Resources Table.
Please note that ALL references cited in the Key Resources Table must be included in the References list. Please report the information as follows:
REAGENT or RESOURCE: Provide full descriptive name of the item so that it can be identified and linked with its description in the manuscript (e.g., provide version number for software, host source for antibody, strain name). In the Experimental Models section, please include all models used in the paper and describe each line/strain as: model organism: name used for strain/line in paper: genotype. (i.e., Mouse: OXTR ^("fl/fl "){ }^{\text {fl/fl }} : B6.129(SJL)-Oxtr ^("tm ")1.1Wsy//J{ }^{\text {tm }} 1.1 \mathrm{Wsy/J} ). In the Biological Samples section, please list all samples obtained from commercial sources or biological repositories. Please note that software mentioned in the Methods Details or Data and Software Availability section needs to be also included in the table. See the sample Table at the end of this document for examples of how to report reagents.
SOURCE: Report the company, manufacturer, or individual that provided the item or where the item can obtained (e.g., stock center or repository). For materials distributed by Addgene, please cite the article describing the plasmid and include “Addgene” as part of the identifier. If an item is from another lab, please include the name of the principal investigator and a citation if it has been previously published. If the material is being reported for the first time in the current paper, please indicate as “this paper.” For software, please provide the
company name if it is commercially available or cite the paper in which it has been initially described.
IDENTIFIER: Include catalog numbers (entered in the column as “Cat#” followed by the number, e.g., Cat#3879S). Where available, please include unique entities such as RRIDs, Model Organism Database numbers, accession numbers, and PDB or CAS IDs. For antibodies, if applicable and available, please also include the lot number or clone identity. For software or data resources, please include the URL where the resource can be downloaded. Please ensure accuracy of the identifiers, as they are essential for generation of hyperlinks to external sources when available. Please see the Elsevier list of Data Repositories with automated bidirectional linking for details. When listing more than one identifier for the same item, use semicolons to separate them (e.g. Cat#3879S; RRID: AB_2255011). If an identifier is not available, please enter “N/A” in the column.
A NOTE ABOUT RRIDs: We highly recommend using RRIDs as the identifier (in particular for antibodies and organisms, but also for software tools and databases). For more details on how to obtain or generate an RRID for existing or newly generated resources, please visit the RII or search for RRIDs.
请使用后面的空表,按小标题定义的部分组织信息,跳过与您的研究无关的部分。请勿添加小标题。要添加一行,请将光标放在要添加行的上方行尾处,即表格右边界外。然后按 ENTER 键添加行。无需删除空行。每个条目必须在单独一行中;不要在一个表格单元格中列出多个项目。请参阅本文档末尾的示例表格,了解如何引用试剂。
关键资源表
试剂或资源
SOURCE
IDENTIFIER
抗体
碱性磷酸酶/ALP。
in 1:100, Rat
Alkaline Phosphatase/ALPL
in 1:100, Rat| Alkaline Phosphatase/ALPL |
| :--- |
| in 1:100, Rat |
研发系统
Cat# MAB29091 RRID:AB_11129451
β Actin [AC-15] in
1:10,000, 鼠标
beta Actin [AC-15] in
1:10,000, Mouse| beta Actin [AC-15] in |
| :--- |
| 1:10,000, Mouse |
Abcam
Cat# ab6276 RRID:AB_2223210
Cleaved Caspase-3 (Asp175)
Cell Signaling Technology
Cat# 9661 RRID:AB_2341188
GFP in 1:1,000, Chicken
Abcam
Cat# ab13970 RRID:AB_300798
Jagged1(H114) in 1:1,000,
Rabbit
Jagged1(H114) in 1:1,000,
Rabbit| Jagged1(H114) in 1:1,000, |
| :--- |
| Rabbit |
Santa Cruz Biotechnology
Cat# sc-8303 RRID:AB_649685
Jagged1 in 1:100 (IF), Rabbit
Acris Antibodies
Cat# AP09127PU-N RRID:AB_2035312
Ki67 in 1:100, Rabbit
Abcam
Cat# ab15580 RRID:AB_443209
IL6 in 1:1,000, Rabbit
MBL International
Cat# JM-5144-100 RRID:AB_592035
1:1,000的PARP,兔子
细胞信号技术
Cat# 9542 RRID:AB_2160739
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
"Alkaline Phosphatase/ALPL
in 1:100, Rat" R & D Systems Cat# MAB29091 RRID:AB_11129451
"beta Actin [AC-15] in
1:10,000, Mouse" Abcam Cat# ab6276 RRID:AB_2223210
Cleaved Caspase-3 (Asp175) Cell Signaling Technology Cat# 9661 RRID:AB_2341188
GFP in 1:1,000, Chicken Abcam Cat# ab13970 RRID:AB_300798
"Jagged1(H114) in 1:1,000,
Rabbit" Santa Cruz Biotechnology Cat# sc-8303 RRID:AB_649685
Jagged1 in 1:100 (IF), Rabbit Acris Antibodies Cat# AP09127PU-N RRID:AB_2035312
Ki67 in 1:100, Rabbit Abcam Cat# ab15580 RRID:AB_443209
IL6 in 1:1,000, Rabbit MBL International Cat# JM-5144-100 RRID:AB_592035
PARP in 1:1,000, Rabbit Cell Signaling Technology Cat# 9542 RRID:AB_2160739| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
| :--- | :--- | :--- |
| Antibodies | | |
| Alkaline Phosphatase/ALPL <br> in 1:100, Rat | R & D Systems | Cat# MAB29091 RRID:AB_11129451 |
| beta Actin [AC-15] in <br> 1:10,000, Mouse | Abcam | Cat# ab6276 RRID:AB_2223210 |
| Cleaved Caspase-3 (Asp175) | Cell Signaling Technology | Cat# 9661 RRID:AB_2341188 |
| GFP in 1:1,000, Chicken | Abcam | Cat# ab13970 RRID:AB_300798 |
| Jagged1(H114) in 1:1,000, <br> Rabbit | Santa Cruz Biotechnology | Cat# sc-8303 RRID:AB_649685 |
| Jagged1 in 1:100 (IF), Rabbit | Acris Antibodies | Cat# AP09127PU-N RRID:AB_2035312 |
| Ki67 in 1:100, Rabbit | Abcam | Cat# ab15580 RRID:AB_443209 |
| IL6 in 1:1,000, Rabbit | MBL International | Cat# JM-5144-100 RRID:AB_592035 |
| PARP in 1:1,000, Rabbit | Cell Signaling Technology | Cat# 9542 RRID:AB_2160739 |
REAGENT or RESOURCE SOURCE IDENTIFIER
15D11 fully human antihuman Jagged1 antibody Amgen Inc. N/A
"Anti-Rabbit IgG -
Horseradish Peroxidase in 1:5,000, Goat" GE Healthcare Cat# RPN4301 RRID:AB_2650489
"Anti-Mouse IgG -
Horseradish Peroxidase in 1:5,000, Sheep" GE Healthcare Cat# NA931-1ml, RRID:AB_772210
Bacterial and Virus Strains
pMSCV-Puro Seth N., et al. 2011 NA
pMSCV-Jagged1 Seth N., et al. 2011 NA
pTyr-Co1a1-mJagged1 This paper NA
Biological Samples
14 pairs of bone marrow cytospin samples before and after Carboplatinum and paclitaxel treatment University Hospital Essen, Essen, Germany NA
Chemicals, Peptides, and Recombinant Proteins
OPG-Fc Amgen Inc. NA
Brefeldin A Sigma-Aldrich Cat# B6542-5MG
CCl (Carbon tetrachloride) Sigma-Aldrich Cat# 319961
Cisplatin Sigma-Aldrich Cat# 134357-100MG
Docetaxel Sigma-Aldrich Cat# 1224551-200MG
Hydrogen peroxide solution Sigma-Aldrich Cat# 216763
MRK003 Merck NA
N-Acetyl-L-cysteine Sigma-Aldrich Cat# A7250-5G
Paclitaxel Sigma-Aldrich Cat# T7191-25MG
(+)-Sodium L-ascorbate Sigma-Aldrich Cat# A4034
Tunicamycin Sigma-Aldrich Cat# T7765
Recombinant Mouse IL6 Protein R&D Biosystems Cat# 406-ML-005
Recombinant Mouse RANKL Protein R&D Biosystems Cat# 462-TEC-010
Critical Commercial Assays
Acid Phosphatase, Leukocyte (TRAP) Kit Sigma-Aldrich 387A-1KT
ALT Activity Assay Sigma-Aldrich MAK052
AST Activity Assay Sigma-Aldrich MAK055
Deposited Data
Gene expression microarray data GSE97997 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cpancgoafjcnxyv&acc=GSE979
Experimental Models: Cell Lines
Chinese hamster CHO ATCC Cat# CCL-61
Human embryonic kidney H29-Clone#7 Seth N., et al. 2011 NA| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
| :---: | :---: | :---: |
| 15D11 fully human antihuman Jagged1 antibody | Amgen Inc. | N/A |
| Anti-Rabbit IgG - <br> Horseradish Peroxidase in 1:5,000, Goat | GE Healthcare | Cat# RPN4301 RRID:AB_2650489 |
| Anti-Mouse IgG - <br> Horseradish Peroxidase in 1:5,000, Sheep | GE Healthcare | Cat# NA931-1ml, RRID:AB_772210 |
| Bacterial and Virus Strains | | |
| pMSCV-Puro | Seth N., et al. 2011 | NA |
| pMSCV-Jagged1 | Seth N., et al. 2011 | NA |
| pTyr-Co1a1-mJagged1 | This paper | NA |
| Biological Samples | | |
| 14 pairs of bone marrow cytospin samples before and after Carboplatinum and paclitaxel treatment | University Hospital Essen, Essen, Germany | NA |
| Chemicals, Peptides, and Recombinant Proteins | | |
| OPG-Fc | Amgen Inc. | NA |
| Brefeldin A | Sigma-Aldrich | Cat# B6542-5MG |
| CCl (Carbon tetrachloride) | Sigma-Aldrich | Cat# 319961 |
| Cisplatin | Sigma-Aldrich | Cat# 134357-100MG |
| Docetaxel | Sigma-Aldrich | Cat# 1224551-200MG |
| Hydrogen peroxide solution | Sigma-Aldrich | Cat# 216763 |
| MRK003 | Merck | NA |
| N-Acetyl-L-cysteine | Sigma-Aldrich | Cat# A7250-5G |
| Paclitaxel | Sigma-Aldrich | Cat# T7191-25MG |
| (+)-Sodium L-ascorbate | Sigma-Aldrich | Cat# A4034 |
| Tunicamycin | Sigma-Aldrich | Cat# T7765 |
| Recombinant Mouse IL6 Protein | R&D Biosystems | Cat# 406-ML-005 |
| Recombinant Mouse RANKL Protein | R&D Biosystems | Cat# 462-TEC-010 |
| Critical Commercial Assays | | |
| Acid Phosphatase, Leukocyte (TRAP) Kit | Sigma-Aldrich | 387A-1KT |
| ALT Activity Assay | Sigma-Aldrich | MAK052 |
| AST Activity Assay | Sigma-Aldrich | MAK055 |
| Deposited Data | | |
| Gene expression microarray data | GSE97997 | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cpancgoafjcnxyv&acc=GSE979 |
| Experimental Models: Cell Lines | | |
| Chinese hamster CHO | ATCC | Cat# CCL-61 |
| Human embryonic kidney H29-Clone#7 | Seth N., et al. 2011 | NA |
试剂或资源
SOURCE
IDENTIFIER
Mouse osteoblast MC3T3-E1 Clone #4
ATCC
Cat# CRL-2393
Mouse mesenchymal stromal cell (MSC)
Ren G., et al. 2008
NA
Mouse PyMT-A-FIG
Wan., et al., 2014
NA
Human breast cancer MCF7
ATCC
Cat# HTB-22
Human breast cancer SCP28
Kang Y., et al. 2003
NA
Human breast cancer SCP2
Kang Y., et al. 2003
NA
Human breast cancer SCP28Vector
Seth N., et al. 2011
NA
Human breast cancer SCP28Jagged1
Seth N., et al. 2011
NA
Human breast cancer SUM1315-M1B1
another manuscript in preparation, derivative from SUM1315
NA
Human embryonic kidney 293T
ATCC
Cat# CRL-3216
Mouse breast cancer 4T1.2
Eckhardt B.L., et al. 2005
RRID:CVCL_GR32
Experimental Models: Organisms/Strains
Mouse Bal/cJ
Jackson Laboratory
Cat# 000651
Mouse FVB/NJ
Jackson Laboratory
Cat# 001800
Mouse nu/nu
Jackson Laboratory
Cat#: 002019
Mouse Col1a1-Jag1 (FVB)
This paper
NA
XenoMouse XMG2KL
Kellermann S., et al. 2002
NA
XenoMouse XMG4KL
Kellermann S., et al. 2002
NA
Oligonucleotides
See Table S1 for real-time PCR primers
IDT
NA
Col1a1-Jag1-GT-F
IDT
CAACACCACGGAATTGTCAGT
Col1a1-Jag1-GT-R
IDT
GATGATGGGAACCCTGTCAA
Software and Algorithms
AxioVision software version 4.6.3
Zeiss
NA
GeneSpring 13 software
Agilent
NA
ImageJ
National Institute of Health
NA
INVEON Research Workplace software
Siemens Healthcare
NA
NIS-Elements Confocal
Nikon
NA
REAGENT or RESOURCE SOURCE IDENTIFIER
Mouse osteoblast MC3T3-E1 Clone #4 ATCC Cat# CRL-2393
Mouse mesenchymal stromal cell (MSC) Ren G., et al. 2008 NA
Mouse PyMT-A-FIG Wan., et al., 2014 NA
Human breast cancer MCF7 ATCC Cat# HTB-22
Human breast cancer SCP28 Kang Y., et al. 2003 NA
Human breast cancer SCP2 Kang Y., et al. 2003 NA
Human breast cancer SCP28Vector Seth N., et al. 2011 NA
Human breast cancer SCP28Jagged1 Seth N., et al. 2011 NA
Human breast cancer SUM1315-M1B1 another manuscript in preparation, derivative from SUM1315 NA
Human embryonic kidney 293T ATCC Cat# CRL-3216
Mouse breast cancer 4T1.2 Eckhardt B.L., et al. 2005 RRID:CVCL_GR32
Experimental Models: Organisms/Strains
Mouse Bal/cJ Jackson Laboratory Cat# 000651
Mouse FVB/NJ Jackson Laboratory Cat# 001800
Mouse nu/nu Jackson Laboratory Cat#: 002019
Mouse Col1a1-Jag1 (FVB) This paper NA
XenoMouse XMG2KL Kellermann S., et al. 2002 NA
XenoMouse XMG4KL Kellermann S., et al. 2002 NA
Oligonucleotides
See Table S1 for real-time PCR primers IDT NA
Col1a1-Jag1-GT-F IDT CAACACCACGGAATTGTCAGT
Col1a1-Jag1-GT-R IDT GATGATGGGAACCCTGTCAA
Software and Algorithms
AxioVision software version 4.6.3 Zeiss NA
GeneSpring 13 software Agilent NA
ImageJ National Institute of Health NA
INVEON Research Workplace software Siemens Healthcare NA
NIS-Elements Confocal Nikon NA| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
| :---: | :---: | :---: |
| Mouse osteoblast MC3T3-E1 Clone #4 | ATCC | Cat# CRL-2393 |
| Mouse mesenchymal stromal cell (MSC) | Ren G., et al. 2008 | NA |
| Mouse PyMT-A-FIG | Wan., et al., 2014 | NA |
| Human breast cancer MCF7 | ATCC | Cat# HTB-22 |
| Human breast cancer SCP28 | Kang Y., et al. 2003 | NA |
| Human breast cancer SCP2 | Kang Y., et al. 2003 | NA |
| Human breast cancer SCP28Vector | Seth N., et al. 2011 | NA |
| Human breast cancer SCP28Jagged1 | Seth N., et al. 2011 | NA |
| Human breast cancer SUM1315-M1B1 | another manuscript in preparation, derivative from SUM1315 | NA |
| Human embryonic kidney 293T | ATCC | Cat# CRL-3216 |
| Mouse breast cancer 4T1.2 | Eckhardt B.L., et al. 2005 | RRID:CVCL_GR32 |
| Experimental Models: Organisms/Strains | | |
| Mouse Bal/cJ | Jackson Laboratory | Cat# 000651 |
| Mouse FVB/NJ | Jackson Laboratory | Cat# 001800 |
| Mouse nu/nu | Jackson Laboratory | Cat#: 002019 |
| Mouse Col1a1-Jag1 (FVB) | This paper | NA |
| XenoMouse XMG2KL | Kellermann S., et al. 2002 | NA |
| XenoMouse XMG4KL | Kellermann S., et al. 2002 | NA |
| Oligonucleotides | | |
| See Table S1 for real-time PCR primers | IDT | NA |
| Col1a1-Jag1-GT-F | IDT | CAACACCACGGAATTGTCAGT |
| Col1a1-Jag1-GT-R | IDT | GATGATGGGAACCCTGTCAA |
| Software and Algorithms | | |
| AxioVision software version 4.6.3 | Zeiss | NA |
| GeneSpring 13 software | Agilent | NA |
| ImageJ | National Institute of Health | NA |
| INVEON Research Workplace software | Siemens Healthcare | NA |
| NIS-Elements Confocal | Nikon | NA |
SIGNIFICANCE
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 gamma\gamma-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 tumorprotective 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.
Highlights
Jagged1 antibody 15D11 reduces bone metastasis without significant side effects
quad\quad Chemotherapy induces tumor-protecting Jagged1 in osteoblasts
Transgenic expression of Jagged1 in osteoblasts promotes bone metastasis
quad15D11\quad 15 \mathrm{D} 11 sensitizes bone metastasis to chemotherapy
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank N. Sethi, B. Ell, R. Chakrabarti, T. Celia-Terrassa, L. Wan, H.A. Smith, Z. Li, W. Lu and other lab members for technical supports and helpful discussions, and J.J. Grady and C. DeCoste for assistance with flow cytometry, and T. Campbell at Rutgers Cancer Institute of New Jersey (RCINJ) Preclinical Imaging Facility for muCT\mu \mathrm{CT} imaging. We thank M. Alpern and V. Buynevich of the University Medical Center of Princeton at Plainsboro for assistance in blood sample analysis. We thank those key scientists, particularly L. Perkins and J. Ho, at Amgen Discovery Research that contributed to the development of the 15D11 antibody. This research was supported by a RCINJ Research Development Award, the Brewster Foundation, and grants from METAvivor Research and Support (AWD1004691), the U. S. Department of Defense (BC123187), the National Institute of Health (R01CA134519 and R01CA141062) and Amgen to Y.K., Cancer and Prevention Research Institute of Texas (CPRIT) grant RP170488 to B. L., and postdoctoral fellowships from Susan G. Komen to H.Z. and M.S., from DOD to G.R., and from NJCCR to M.S. This research was also supported by the Preclinical Imaging Facility and Pre-clinical Imaging and Flow Cytometry Shared Resources of the RCINJ (P30CA072720).
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Tumor-derived Jagged1 promote bone metastasis by engaging osteoblasts and osteoclasts (upper panel), while chemotherapy-induced Jagged1 in osteogenic cells promotes tumor cell survival under chemotherapy (lower panel). 15D11 targets both processes and synergizes with chemotherapy to strongly reduce bone metastasis.
Correspondence: Yibin Kang, Ph.D., Department of Molecular Biology, Washington Road, LTL 255, Princeton University, Princeton, NJ 08544, Phone: (609) 258-8834; Fax: (609) 258-2340, ykang@princeton.edu. Brendan Lee, M.D., Ph.D., Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Room R814 BCM225, Houston, TX 77030, Phone: (713) 798-5443, blee@bcm.edu. ^(10){ }^{10} Co-first author ^(11){ }^{11} Co-corresponding author ^(12){ }^{12} H.Z. current address: Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China Lead contact: Yibin Kang
SUPPLEMENTAL INFORMATION
Supplemental information, including seven Supplemental Figures, one Supplemental Table, and six Supplemental Movies, can be found with this article online.
CONFLICT OF INTEREST
Chadwick King, Jan Sun, Jodi Moriguchi, Helen Toni Jun, and Angela Coxon are either previous or current employees of Amgen Inc. This research was in part funded by Amgen.
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