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靶向肿瘤和成骨细胞小体生成的 Jagged1 的治疗性抗体能使骨转移对化疗敏感


Hanqiu Zheng 1 , 10 , 12 1 , 10 , 12 ^(1,10,12){ }^{1,10,12} , Yangjin Bae 2 , 10 2 , 10 ^(2,10){ }^{2,10} , Sabine Kasimir-bauer 3 3 ^(3){ }^{3} , Rebecca Tang 1 1 ^(1){ }^{1} , Jin Chen 1 1 ^(1){ }^{1} 、任广文 1 1 ^(1){ }^{1} , 袁敏 1 1 ^(1){ }^{1} , 马克-埃斯波西托 1 1 ^(1){ }^{1} , 李文阳 1 1 ^(1)^{1} , 魏勇 1 1 ^(1){ }^{1} , 沈敏红 1 1 ^(1){ }^{1} 、张兰静 4 4 ^(4){ }^{4} , 尼古拉-图皮岑 5 5 ^(5){ }^{5} , 克劳斯-潘特尔 6 6 ^(6){ }^{6} , 查德威克-金 7 7 ^(7){ }^{7} , 孙扬 7 7 ^(7){ }^{7} , 乔迪-森口 7 7 ^(7)^{7} , 海伦-托尼-君 8 8 ^(8){ }^{8} , 安吉拉-考克森 7 7 ^(7){ }^{7} , 布伦丹-李 2 , 11 2 , 11 ^(2,11){ }^{2,11} , 和康一斌 1 , 9 , 11 1 , 9 , 11 ^(1,9,11){ }^{1,9,11} .

1 1 ^(1){ }^{1} 分子生物学系,普林斯顿大学,美国新泽西州普林斯顿 08544

2 2 ^(2){ }^{2} 贝勒医学院分子与人类遗传学系,美国德克萨斯州休斯顿 77030

3 3 ^(3){ }^{3} 埃森大学医院妇产科,德国埃森

4 4 ^(4){ }^{4} 新泽西州普林斯伯勒普林斯顿大学医学中心病理学系;新泽西州新不伦瑞克罗格斯癌症研究所

5 5 ^(5){ }^{5} 俄罗斯,莫斯科,癌症研究中心,造血免疫学实验室

6 6 ^(6){ }^{6} 汉堡-埃彭多夫大学医学中心肿瘤生物学系,实验医学中心,德国汉堡

7 7 ^(7){ }^{7} 肿瘤研究,安进公司,美国加利福尼亚州千橡市91320号

8MabVax Therapeutics Holdings, Inc.

9 9 ^(9){ }^{9} 癌症代谢与生长项目,罗格斯新泽西癌症研究所,新泽西州新不伦瑞克,08903,美国

 摘要


骨转移是乳腺癌患者的主要健康威胁。肿瘤来源的 Jagged1 是介导肿瘤与基质相互作用的中心节点,而这种相互作用会促进溶骨性骨转移。在此,我们报告了针对 Jagged1 的高效全人源单克隆抗体(克隆 15D11)的开发情况。除了对表达 Jagged1 的肿瘤细胞的骨转移有抑制作用外,15D11 还能显著提高骨转移对化疗的敏感性,因为化疗会诱导成骨细胞中 Jagged1 的表达,从而为癌细胞提供一个生存位点。我们利用成骨细胞特异性 Jagged1 转基因小鼠模型进一步证实了成骨细胞来源的 Jagged1 促进骨转移的功能。这些发现确立了 15D11 作为预防或治疗骨转移的潜在治疗药物的地位。

 简介


Zheng等人开发了一种针对Jagged1的全人源单克隆抗体15D11,它能抑制乳腺癌细胞上的Jagged1,并阻断成骨细胞衍生的Jagged1的转移促进作用。在乳腺癌小鼠模型中,15D11 能减少骨转移并使转移灶对化疗敏感。

 引言


在美国,乳腺癌是最常见的女性恶性肿瘤,也是导致癌症相关死亡的第二大原因。在晚期乳腺癌患者中,70%以上患有骨转移,通常伴有严重的骨痛、骨折和潜在的致命并发症,如高钙血症(Weilbaecher 等人,2011 年)。虽然放疗、化疗和抗骨质溶解剂(如双磷酸盐和 RANKL 抗体地诺单抗)可以降低骨转移相关的发病率,但这些治疗方法往往不能显著延长患者的生存时间或提供治愈(Weilbaecher 等人,2011 年),因为转移性癌症往往会对这些治疗方法产生抗药性。

肿瘤与基质的相互作用在促进乳腺癌骨转移方面发挥着重要作用(Weilbaecher 等人,2011 年)。骨微环境包含多种基质细胞类型,如成骨细胞、破骨细胞、间充质干细胞(MSCs)和造血细胞。以往的研究主要关注乳腺癌细胞与骨吸收破骨细胞之间的交叉交流,而对其他基质细胞类型对骨转移的贡献研究较少。支持性基质细胞包括


最近的研究表明,造骨成骨细胞构成了一个成骨生态位,它对扩散的肿瘤细胞在骨中的生存和定植至关重要(Shiozawa 等人,2011 年;Wang 等人,2015 年)。尽管最近取得了这些进展,但我们对肿瘤细胞与骨小龛中成骨细胞之间相互作用的分子认识在很大程度上仍不完整。例如,这种肿瘤-骨龛相互作用是如何导致转移性乳腺癌对化疗等标准骨转移治疗产生耐药性的,我们仍然知之甚少。

在人类乳腺癌中,Jagged1和Notch1(而非其他Notch通路配体或受体)的高表达与预后不良密切相关(Reedijk等人,2005年;Sethi等人,2011年)。我们之前的研究发现,肿瘤衍生的Jagged1是一种骨转移促进因子,它通过激活成骨细胞中的Notch信号来增加白细胞介素-6(IL6)和结缔组织生长因子(CTGF)的产生,并反馈给肿瘤细胞以促进增殖和存活。同时,Jagged1刺激破骨细胞生成和骨降解,导致骨源性生长因子(包括TGF- β β beta\beta )的释放,TGF- β β beta\beta 是肿瘤细胞中Jagged1表达的有效诱导因子,从而形成正反馈循环(Sethi等人,2011年)。

除了Jagged1在骨转移进展过程中肿瘤与基质相互作用的作用外,据报道,肿瘤或基质衍生的Jagged1还能诱导淋巴瘤、结直肠癌和许多其他癌症类型的血管生成、侵袭、耐药性和癌症干细胞更新(Li等人,2014年)。内皮衍生的Jagged1可促进B细胞淋巴瘤中的Notch活化,导致结外侵袭和化疗耐药性(Cao等人,2014年)。在结直肠癌中,来自内皮细胞的可溶性Jagged1可诱导结直肠癌的癌症干细胞样表型(Lu等人,2013年)。Jagged1在不同癌症类型中的这些多功能作用为开发Jagged1靶向疗法治疗癌症提供了支持。

针对Notch信号通路开发了多种治疗策略。大多数抑制剂都是针对 γ γ gamma\gamma -分泌酶设计的,分泌酶介导Notch受体的蛋白水解,生成信号转导Notch胞内结构域(NICD),这是配体结合后激活Notch通路的必要步骤(Rizzo等人,2008年)。然而,据报道 γ γ gamma\gamma 分泌酶抑制剂(GSI)会引起严重的胃肠道毒性(Imbimbo,2008年),阻碍了这类抑制剂的进一步临床开发。靶向单个 Notch 受体或配体有可能在不引起严重胃肠道毒性的情况下达到治疗效果。

 结果


针对 Jagged1 的全人源单克隆抗体的生成和特性分析


我们利用 XenoMouse 技术生成了针对 Jagged1 的全人源单克隆抗体。这些小鼠的内源性小鼠免疫球蛋白基因座被灭活,并引入了大型转基因,这些转基因能够进行重组并产生全人源抗体(Mendez 等人,1997 年)。简而言之,我们用瞬时表达人 Jagged1 的中国仓鼠细胞(CHO)免疫 XenoMouse 动物,包括 XMG2KL 和 XMG4KL 品系(Kellermann 和 Green,2002 年),以产生大量的人 Jagged1 抗体。


抗体。基于细胞的荧光微量检测技术(FMAT)被用来分析这些抗体与细胞表面人类 Jagged1 蛋白的特异性结合。然后对这些抗体进行了反筛选,以排除与其他主要 Notch 配体(包括人类 Jagged2 和 Dll4)的交叉反应结合(图 S1A)。为了找出可用于小鼠体内研究的合适抗体拮抗剂,我们进一步检测了 Notch 受体相互作用的最强阻断剂与小鼠 Jagged1 的交叉反应结合。抗体克隆 15D11 与小鼠 Jagged1 蛋白的亲和力特别高,在酶动力学排阻试验(KinExA)中的解离常数 ( K d ) K d (K_(d))\left(\mathrm{K}_{\mathrm{d}}\right) 约为 23 pM(图 1A)。利用 Notch 荧光素酶报告分析,我们确定 15D11 对人 Jagged1 的半最大抑制浓度(IC50)为 3.63 nM,对鼠 Jagged1 的半最大抑制浓度(IC50)为 14.07 nM(图 1B 和数据未显示)。

在体内给药时( 10 mg / kg 10 mg / kg 10mg//kg10 \mathrm{mg} / \mathrm{kg} ,每周两次),根据体重测量,15D11显示出最小的全身毒性,与之形成对比的是,接受GSI治疗的小鼠体重显著下降(图S1B)。血红素和伊红染色(H&E)以及阿尔新蓝染色显示,15D11处理后没有明显的消化道毒性,而GSI处理后的动物则出现了明显的上皮细胞增生(图S1C和S1D)。为了分析肝脏毒性,我们测量了小鼠在接受相应药物治疗后血清中丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)的活性。作为阳性对照, CCl 4 CCl 4 CCl_(4)\mathrm{CCl}_{4} 处理会导致ALT和AST活性急剧升高(图S1E和S1F),而15D11或GSI处理后则未观察到任何变化。全血细胞计数(CBC)检测进一步表明,15D11没有明显的血液学毒性(图S1G)。对 T 细胞群和活化状态的分析也没有发现 IgG 或 15D11 治疗组之间有任何差异(图 S1H-K)。综上所述,这些结果表明 15D11 是一种特异性 Jagged1 靶向药物,在体内应用时具有极佳的安全性。

我们之前的研究表明,肿瘤来源的 Jagged1 可促进单核细胞前破骨细胞分化为成熟的破骨细胞(Sethi 等人,2011 年)。我们开发了一种体外试验来测试 15D11 对 Jagged1 依赖性破骨细胞生成的潜在抑制作用。RAW264.7是一种具有分化成破骨细胞能力的单核细胞/巨噬细胞系,我们将其播种在Fc涂层或重组Jagged1(rJagged1)涂层的平板上,并施以低浓度的RANKL( 5 ng / ml 5 ng / ml 5ng//ml5 \mathrm{ng} / \mathrm{ml} ),这一浓度不足以诱导破骨细胞分化(Ell等人,2013年)。在 5 天内,rJagged1 包被的平板上产生了大量多核成熟破骨细胞,但在 Fc 包被的平板上只能看到有限的数量。此外,这种依赖 Jagged1 的破骨细胞分化被 15D11 完全阻断(图 1C 和 1D)。在试验中使用骨髓衍生的原发性前破骨细胞时也观察到了类似的结果(图 1E 和图 S1L)。为了更接近地模拟肿瘤来源的 Jagged1 诱导的破骨细胞生成,我们将 RAW264.7 细胞与 SCP28 细胞(一种具有中度骨转移能力的 MDA-MB-231 衍生细胞系,已用荧光素酶和 GFP 稳定标记(Kang 等,2003 年))共培养,无论是否稳定过表达 Jagged1。在这种共培养试验中,施用 15D11 还能抑制破骨细胞的分化(图 1F 和图 S1M)。

之前报道的另一种肿瘤源 Jagged1 促进骨转移的机制是通过增加成骨细胞产生 IL6(Sethi 等人,2011 年)。正如之前所报道的,SCP28 与小鼠 MC3T3 成骨细胞系共培养会导致 IL6 以 Jagged1 依赖性方式表达升高(图 1G 和 1H)。在肿瘤-成骨细胞共培养中施用 15D11 能显著抑制 Notch 诱导基因 Hes1 的表达(通过物种特异性 qRT-PCR 检测)(图 1G),以及 IL6 在 mRNA 和蛋白水平上的表达(图 1G 和 1H)。综上所述,这些体外实验证实 15D11 能有效阻断 Jagged1Notch 信号活动,而 Jagged1Notch 信号活动对骨转移的发展非常重要。


15D11 抗体可减轻表达 Jagged1 的肿瘤细胞的骨转移


为了评估15D11对骨转移的治疗效果,我们利用SCP28细胞建立了一个成熟的骨转移异种移植模型。SCP28 的 Jagged1 基础表达水平较低,异位过表达 Jagged1 可增强其骨转移能力(Sethi 等人,2011 年)。通过心内注射(IC)将肿瘤细胞接种到无胸腺裸鼠体内以产生骨转移。我们在肿瘤细胞注射前一天开始 15D11 抗体治疗,每周治疗两次。每周通过生物发光成像(BLI)监测骨转移,直到实验结束( 5 5 ∼5\sim 5 周)将小鼠安乐死,进行组织病理学分析(图2A)。我们还使用 RANKL 诱饵受体 OPG-Fc(相当于临床批准的 RANKL 阻断抗体地诺单抗)阻断破骨细胞分化作为实验的阳性对照。Jagged1 过表达导致骨转移负荷显著增加(图 2B 和 2C),这与我们之前的报告(Sethi 等,2011 年)一致。BLI分析显示,与IgG对照组相比,用15D11治疗的小鼠SCP28-Jagged1骨转移量减少了 5 5 ∼5\sim 5 倍。正如预期的那样,OPG-Fc 治疗也会减少小鼠的骨转移负荷(图 2B 和 2C)。注射了 SCP28-Jagged1 的小鼠后肢的 X 射线成像显示,与注射了对照组 SCP28 细胞的小鼠相比,溶骨区明显增多,而 15D11 治疗可逆转这种表型(图 2B 和 2D)。 虽然 OPG-Fc 治疗也能保护骨骼免受骨溶解,但通过 X 射线和微计算机断层扫描( μ CT μ CT muCT\mu \mathrm{CT} )分析观察到,它也导致骨小梁密度异常增加(图 2D-E 和影片 S1-4)。同样,对骨切片进行耐酒石酸磷酸酶(TRAP)免疫染色表明,用 OPG-Fc 治疗的小鼠几乎完全没有破骨细胞(图 2E)。有趣的是,虽然过表达 SCP28 的 Jagged1 促进了骨病变中成熟破骨细胞的募集,但在用 15D11 处理的小鼠中仍可观察到相当数量的破骨细胞(图 2E 和图 S2A)。重要的是,与 OPG-Fc 处理的小鼠相比,15D11 处理的动物骨小梁密度没有异常增加(图 2E)。综上所述,这些结果表明,15D11 可抑制肿瘤衍生的 Jagged1 对破骨细胞的病理性激活,但却保留了维持正常骨稳态所需的正常破骨细胞活性。

为了测试 15D11 对已形成的骨转移的治疗效果,我们使用了 SCP2 细胞系,这是 MDA-MB-231 的一种极具侵袭性的骨转移变体,具有高水平的内源性 Jagged1 表达(Sethi 等人,2011 年)。SCP2 细胞能迅速生成


在裸鼠体内注射 SCP2 细胞后 1 周内出现多发性溶骨性骨转移,这通常会导致裸鼠在 4 周内死亡。我们在 SCP2 细胞 IC 注射一周后,即骨转移成熟时开始每周注射两次 15D11(图 S2B)。在这个晚期治疗侵袭性骨转移的模型中,我们发现与对照组相比,单独使用 15D11 或 OPG-Fc 治疗有减少骨转移的趋势(图 S2C 和 S2D),尽管在实验终点(4 周)小鼠死于转移性癌症时,这两种治疗方法都没有达到统计学意义。15D11和OPG-Fc的联合治疗使骨转移在治疗开始3周后显著减少 5 5 ∼5\sim 5 倍,这表明靶向两种不同的骨转移分子介质是有益的(图S2C和S2D)。与预防性治疗方案类似,15D11治疗减少了破骨细胞的数量并减少了溶骨性骨面积,尽管仍可观察到一些破骨细胞的基础水平,但与OPG-Fc治疗的小鼠相比,破骨细胞几乎完全消失(图S2E-G)。

Jagged1 在发育性血管生成中的作用已被广泛研究(Benedito 等人,2009 年)。因此,我们使用两种不同的血管生成模型测试了 15D11 对血管生成的抑制作用。在新生小鼠视网膜和皮下异种移植肿瘤血管生成实验中(Polverino 等人,2006 年;Ridgway 等人,2006 年),15D11 处理对血管生成没有影响(图 S3A-S3D)。因此,15D11不太可能通过抑制血管生成来减少骨转移。


15D11 使骨转移瘤对化疗敏感


化疗通常用于治疗骨转移瘤,但骨转移瘤通常对化疗较为难治(Gu 等人,2004 年)。为了评估 15D11 与传统化疗的联合作用,我们采用晚期治疗方案(注射 IC 1 周后开始治疗),用 15D11 和紫杉醇治疗 SCP28-Jagged1 细胞产生的骨转移瘤(图 3A)。与注射SCP2的小鼠组相比,注射SCP28-Jagged1的小鼠组进展缓慢(7周比注射SCP2的4周),这为我们观察15D11和紫杉醇单一或联合治疗的疗效提供了充足的时间。小鼠在 IC 注射 SCP28-Jagged1 确诊骨转移一周后,接受 IgG、15D11、紫杉醇或两者治疗。使用 15D11 进行单药治疗可使转移负荷减少近 10 倍(图 3B)。虽然单用紫杉醇可在早期时间点明显减缓骨转移的进展,但在后期时间点出现了对化疗的耐药性,导致实验结束时肿瘤负荷仅轻微减少(图 3B)。令人震惊的是,当小鼠同时接受 15D11 和紫杉醇治疗时,与 IgG 对照组相比,骨转移量协同减少了 >100 倍(图 3B 和 3C)。X 射线图像和破骨细胞 TRAP 染色证实,15D11 单独或与化疗联合治疗后,破骨细胞数量和溶骨病变显著减少(图 3C-F)。尤其是联合治疗组几乎没有溶骨区,这与 BLI 数据一致。 综上所述,这些结果表明紫杉醇和 15D11 的联合治疗在减少 SCP28-Jagged1 产生的骨转移方面具有强大的协同作用。

15D11阻断Jagged1依赖性基质参与和紫杉醇对肿瘤细胞的细胞毒性的共同作用可能解释了联合治疗对骨转移的强烈抑制作用。如果情况确实如此,那么当化疗与 15D11 联合用于治疗由低表达或无 Jagged1 表达的肿瘤细胞产生的骨转移时,预计不会产生协同治疗效果。因此,我们使用内源性 Jagged1 含量很低的亲本 SCP28 细胞(图 S4A)来直接测试这一点。我们进行了与 SCP28-Jagged1 相同的骨转移和治疗实验(图 4A)。紫杉醇起初能减轻骨转移负荷,但病变很快对化疗产生耐药性,只能轻微减轻转移负荷(图 4B 和 4C)。正如在 SCP28 中缺乏 Jagged1 表达所预期的那样,15D11 对减少 SCP28 的骨转移负荷没有影响(图 4B 和 4C)。出乎我们意料的是,联合治疗组显示出骨转移负荷的显著减少、溶骨性骨病变面积的减少和破骨细胞数量的减少(图 4B-F)。这种治疗反应远远优于单独使用紫杉醇或 15D11 的治疗,表明即使在 Jagged1 表达水平较低的肿瘤细胞中,靶向 Jagged1 与化疗一起治疗骨转移也能产生协同作用。
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 H 2 O 2 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. 


来源于成骨细胞的Jagged1促进骨转移播种和化疗抗性


为了直接测试成骨细胞中 Jagged1 表达的增加对骨转移播种和进展的影响,我们利用了两种模型。在第一个模型中,无胸腺裸鼠接受 PBS 或顺铂治疗,顺铂的半衰期比紫杉醇短得多。一天后,我们采用髂内动脉注射法(IIA)将肿瘤细胞接种到小鼠后肢(Wang 等,2015 年),以专门研究化疗后基质对骨转移播种的影响,因为在这种实验环境下肿瘤细胞不会暴露于临床剂量的顺铂。在测试的所有三种不同癌细胞系(MCF7、SCP28 和 SUM1315-M1B1)中,骨转移播种都显著增加(图 5CE)。免疫染色证实,经顺铂预处理的小鼠骨中GFP阳性肿瘤细胞明显增多,这些肿瘤细胞通常位于 ALP + ALP + ALP^(+)\mathrm{ALP}^{+} 骨细胞附近(图5F和影片S5-6)。

由于化疗可能会对小鼠产生广泛的全身性影响,从而直接或间接地影响上述实验的结果,因此我们决定采用另一种方法,在不使用化疗的情况下特异性地增加成骨细胞中 Jagged1 的表达。为此,我们产生了一种由 Col1a1 启动子驱动的成骨细胞特异性 Jagged1 过表达的转基因小鼠品系(图 S6A 和 S6B)。Col1a1Jag1小鼠总体上健康且能育(详细报告见另一篇正在准备的手稿),但股骨和胫骨中部区域的骨明显缩短和肿胀(图S6C)。 μ μ mu\mu CT成像和骨组织学分析表明,皮质骨区域的骨密度降低,骨小梁高度细化,骨小梁区域的骨密度也显著降低(图6A和图6B)。


数据未显示)。我们还在 Col1a1-Jag1 小鼠的皮质骨中发现了过多 TRAP 阳性的破骨细胞(图 6B),这与之前报道的 Jagged1 促进破骨细胞生成的功能一致(Sethi 等人,2011 年)。用 15D11 治疗 Col1a1-Jag1 小鼠三个月后,Col1a1-Jag1 小鼠中过多的 TRAP 阳性破骨细胞显著减少,证实了该小鼠品系的表型确实是由成骨细胞衍生的 Jagged1 引起的(图 6B)。

接下来,我们测试了这些小鼠的长期骨转移进展和短期转移播种。在长期骨转移试验中,我们使用了本实验室先前从 MMTV-PyMT 乳腺肿瘤(Wan 等人,2014 年)中建立的合成 PyMT-A-FIG 细胞系,该细胞系稳定地标记了萤火虫-IRES-GFP(FIG)报告基因,以便于体内追踪。PyMT-A-FIG细胞通过胫内注射接种到野生型(WT)或Col1a1-Jag1小鼠体内以产生骨转移。BLI 分析显示,Col1a1-Jag1 小鼠的骨转移肿瘤负荷比 WT 小鼠增加了近 20 倍(图 6C)。为了测试短期骨转移播种,我们通过 IIA 向 Col1a1-Jag1 小鼠或 WT 小鼠注射 PyMT-A-FIG 细胞。肿瘤接种四天后,Col1a1-Jag1 小鼠骨中存活的肿瘤细胞已经 3 3 3~ 3 倍于 WT 小鼠(图 6D)。免疫染色显示,Col1a1-Jag1 小鼠骨中的 GFP 阳性肿瘤细胞播种更多,通常与 ALP + + ^(+){ }^{+} 成骨细胞密切接触(图 6E)。综上所述,这些结果证实成骨细胞衍生的Jagged1促进了骨转移的播种和生长。

为了在体外模拟成骨细胞系细胞的化疗抗性和肿瘤播种效应,我们改编了一种体外三维肿瘤-基质共培养试验(Wang 等,2015)。与之前的报道一致,当肿瘤细胞与成骨细胞系细胞在低附着板中共培养时,它们生成了异型球体,肿瘤细胞形成外层壳状结构,成骨细胞形成内核球体(图S7A)。在用顺铂或多西他赛(紫杉醇的一种强效形式)处理时,共培养肿瘤球的存活率比纯肿瘤球高(图 7A),这表明成骨细胞系细胞在化疗过程中促进了肿瘤细胞的存活。为了检测细胞凋亡通路的状态,收集了单独培养 SCP28 或与 MC3T3 细胞共培养的细胞裂解物。顺铂治疗72小时后,SCP28单培养细胞中的裂解PARP和裂解Caspase-3(CC3)CC3被强烈诱导,而在共培养样本中,这些凋亡通路相关蛋白的水平仍然很低(图7B)。为了确定观察到的化疗抗性是否需要细胞-细胞直接接触,我们收集了顺铂处理过的间充质干细胞的条件培养基(CM)并进行了相同的检测。我们发现,与与间充质干细胞共培养的肿瘤细胞对化疗的较高耐受性相比,在有CM存在的情况下,肿瘤细胞仅受到部分化疗保护(图S7B)。这一结果表明,Jagged1介导的最佳化疗抗性需要肿瘤细胞与间质细胞的直接接触,而这种接触是Notch信号转导所必需的。 事实上,当我们分析顺铂或多西他赛治疗后共培养肿瘤细胞中Notch下游基因的表达时,我们发现包括人HES2、HEY1和HEY2在内的多个Notch下游基因在化疗后的共培养肿瘤细胞中显著上调(图7C)。将肿瘤细胞与内皮细胞共培养,对肿瘤生长没有保护作用。


图 S7C-D),这表明只有成骨细胞系细胞而非内皮细胞能保护肿瘤细胞免受骨中化疗的影响。

据报道,Notch 激活可通过影响 p53 调控的凋亡途径来规避凋亡(Dotto,2009 年)。我们比较了SUM1315-M1B1肿瘤细胞在顺铂处理前后、有无成骨细胞共培养情况下的基因表达谱。我们重点分析了 161 个与细胞凋亡相关的基因(详见 STAR 方法)。在顺铂处理条件下,共培养与肿瘤单独培养的凋亡相关基因表达差异超过 2 倍,其中 10 个是抗凋亡基因,在肿瘤单独培养中顺铂处理后表达减少,但在共培养条件下顺铂处理后基础表达水平升高并进一步增加。同样,6 个促凋亡基因在顺铂单独处理肿瘤细胞时被诱导,但这种变化在共培养条件下被抑制(图 7D)。值得注意的是,在这 16 个基因中,至少有 5 个基因是已知受 p53 通路调控的。MCL1、CCNA1、GADD45和BAX是p53的直接转录靶标,而CCND2是p53通路的间接基因。综上所述,这些结果表明,成骨细胞介导的肿瘤细胞化疗抗性至少部分是由Notch激活及其抑制p53调控的凋亡通路的作用介导的。

为了直接分析Jagged1是否能保护肿瘤细胞免受化疗诱导的凋亡,我们对两轮紫杉醇和15D11单药或联合治疗后的小鼠骨切片的GFP + + ^(+){ }^{+} 肿瘤细胞和CC3进行了免疫荧光染色,如图4A中的实验。在 IgG 或 15D11 处理组中,几乎检测不到 CC3 染色。虽然紫杉醇处理组有大量肿瘤细胞的CC3呈阳性,但紫杉醇和15D11联合处理组的 CC + CC + CC^(+)\mathrm{CC}^{+} 肿瘤细胞数量最多(图7E和7F)。

为了检测Jagged1抗体15D11是否会阻断间充质干细胞在化疗期间带来的生存益处,我们将SCP28和SUM1315-M1B1细胞与间充质干细胞进行了联合培养。在对细胞进行化疗的同时,还用 IgG 或 15D11 抗体对细胞进行孵育。当肿瘤细胞与间充质干细胞共培养时,给予 15D11 能明显减少存活肿瘤球的数量(图 S7E 和 S7F)。在与 MC3T3 细胞的共培养实验中也观察到了类似的结果。为了在体内证实这一结果,我们在肿瘤细胞注射前一天用顺铂处理裸鼠。就在注射 SUM1315-M1B1 肿瘤细胞 IIA 时,我们还用 IgG 或 15D11 抗体处理了小鼠。注射 4 天后,通过 BLI 和 IF 染色跟踪骨转移播种情况。与之前的实验类似(图 5C-5E),更多的肿瘤细胞播种到了用顺铂预处理的小鼠骨中(图 7G)。15D11 治疗在很大程度上消除了化疗预处理骨中转移播种的增加。使用 ER + + ^(+){ }^{+} MCF7 乳腺癌细胞也得到了类似的结果(图 S7G-H)。对安乐死动物的骨切片进行共焦 IF 成像进一步证实,在化疗小鼠中,ALP + + ^(+){ }^{+} 成骨细胞旁的肿瘤细胞播种增加,而用 15D11 治疗后肿瘤细胞播种减少(图 7H)。总之,我们的研究结果表明,尽管化疗对肿瘤细胞具有细胞毒性,但由于化疗导致成骨细胞系细胞中Jagged1的表达增加,化疗的全部治疗潜力受到了影响。成骨细胞衍生的 Jagged1 可激活

肿瘤细胞中的Notch信号转导可促进肿瘤细胞对化疗诱导的细胞凋亡产生耐药性。将化疗与 15D11 治疗相结合,可以消除这种由基质介导的化疗耐药机制,在骨转移治疗中取得最佳疗效。


15D11 和化疗的联合治疗可协同降低自发性骨转移的发生率


我们的小鼠模型研究揭示了化疗诱导的成骨细胞中的Jagged1在促进骨转移的化疗耐药性中的作用。为了证实接受化疗的人类癌症患者的成骨细胞中是否也会诱导 Jagged1,我们采集了患者在接受卡铂和紫杉醇辅助化疗前后的配对骨髓细胞标本,并进行了免疫染色分析。化疗前,Jagged1的表达水平较低,主要在ALP + + ^(+){ }^{+} 成骨细胞上表达(图8A)。化疗后,成骨细胞上的Jagged1染色显著增加(图8A和8B),这与我们在体外和小鼠模型中的观察结果一致(图5A和5B)。

在早期乳腺癌的临床治疗中,通常采用新辅助化疗和辅助化疗来预防未来复发。化疗固然能减少局部复发和骨转移,但化疗诱导的成骨细胞Jagged1在促进骨转移播种方面的作用可能会减弱。因此,结合化疗和 15D11 治疗可能会进一步降低骨复发的风险。为了验证这一观点,我们利用了 4T1.2 乳腺肿瘤细胞,这种肿瘤细胞可以自发地从乳腺转移到骨骼(Eckhardt 等人,2005 年)。将 4T1.2 肿瘤细胞注射到雌性 Balb/c 小鼠的乳腺脂肪垫中,以建立原发性肿瘤。当原发性肿瘤的大小达到 5 毫米时,小鼠被随机分组,分别接受 IgG、紫杉醇、15D11 或紫杉醇和 15D11 的治疗(图 8C)。根据 X 射线图像,我们发现 IgG 对照组中超过 50%的小鼠后肢骨出现骨转移。15D11或紫杉醇治疗可使骨转移发生率降低 15 30 % 15 30 % 15-30%15-30 \% ,而紫杉醇和15D11联合治疗可有效抑制骨转移,只有10%的小鼠发生骨转移(图8D和8E)。骨病变面积在联合治疗中也显著减少(图 8F)。总之,这些结果表明,对癌症患者进行辅助化疗联合 15D11 有可能降低未来骨转移复发的发生率。

 讨论


作为Notch的主要配体之一,Jagged1最近被证明是癌症进展(包括骨转移)的重要驱动因素(Sethi等人,2011年)。为了开发针对 Jagged1 的治疗性抗体,我们利用 XenoMouse 技术开发了全人 Jagged1 抗体,并根据阻断效果、亲和力和特异性选择了 15D11 进行进一步测试。15D11 在体内没有显示出可检测到的副作用,同时在体外保持了抑制 Jagged1 介导的信号转导的强大能力,并减少了表达 Jagged1 的乳腺癌细胞在体内的骨转移。与 OPG-Fc 不同的是


作为 RANKL 的诱饵受体,15D11 只能抑制病理性破骨细胞的生成,但却能维持正常骨平衡所需的破骨细胞的基本数量,避免了通常在使用 OPG-Fc、地诺单抗或双膦酸盐治疗后观察到的骨密度异常增加。因此,15D11 是一种很有希望作为骨转移治疗药物进行进一步临床开发的候选药物。

虽然化疗能有效控制甚至治愈不同类型的癌症,但化疗耐药性的产生,尤其是在转移性癌症中,往往会导致癌症患者最终死亡(Anampa 等人,2015 年)。了解化疗耐药性的机制对于制定使化疗耐药性肿瘤重新敏感的策略至关重要。以往对化疗耐药性的研究大多集中于肿瘤内在机制,如药物吸收减少或药物外流增加、药物与靶点相互作用的改变、细胞反应的改变,特别是细胞修复 DNA 损伤或耐受应激条件的能力增强,以及细胞凋亡途径的缺陷。然而,肿瘤微环境,尤其是转移器官部位独特的基质壁龛,也可能对化疗耐药性的形成产生重要影响。在目前的研究中,我们发现了肿瘤与成骨细胞之间的相互作用,这种相互作用促进了骨转移的化疗耐药性。在这里,我们证明了包括紫杉醇和顺铂在内的各种化疗药物会诱导成骨细胞和间充质干细胞中Jagged1的表达,而Jagged1会反馈给肿瘤细胞,激活Notch信号,促进化疗耐药性。在我们的体外三维共培养系统或各种骨转移小鼠模型中施用 15D11 能显著提高肿瘤细胞和骨转移对化疗的敏感性。最引人注目的是,当化疗与 15D11 联合使用时,在我们的骨转移小鼠模型中观察到骨转移负荷减少了近 100 倍。 因此,15D11 是一种独特的治疗药物,能够同时靶向肿瘤衍生的 Jagged1 和化疗诱导的成骨细胞中的 Jagged1,从而抑制多种下游 Notch 信号事件,而这些信号事件对于溶骨病变的扩大和骨中癌细胞的化疗抗性都很重要(图 8G)。

Jagged1在一部分原发性乳腺肿瘤中过表达,这些肿瘤容易发生骨转移(Li等人,2014年;Sethi等人,2011年)。然而,肿瘤细胞中 Jagged1 的表达并不能诱导其自身 Notch 信号的激活(Sethi 等人,2011 年)。在我们目前的研究中,只有当肿瘤细胞在化疗过程中与成骨细胞 Jagged1 相互作用时,Notch 信号才会被激活。这一观察结果与已知的 Notch 反式激活和顺式抑制机制一致。例如,在果蝇眼睛的发育过程中,虽然相邻细胞都表达 Notch 配体和受体,但 Notch 信号激活只发生在一个方向,而不是两个细胞,从而形成了组织内的发育模式(Miller 等人,2009 年)。更重要的是,Notch 配体也会对同一细胞产生顺式抑制信号(Sprinzak 等人,2010 年)。未来的研究应探索肿瘤衍生的 Jagged1 无法诱导自身 Notch 激活的确切原因,以及化疗和氧化应激如何特异性地诱导成骨细胞系细胞中的 Jagged1,而不是肿瘤细胞和其他基质细胞。其他基质细胞类型,包括血管内皮细胞、破骨细胞、神经元和骨髓细胞也可能通过不同的分子机制导致耐药性(Duan等人,2014;Hanoun等人,2014;Pitt等人,2015),这将是一个有趣的进一步研究方向。

切除原发肿瘤后,许多乳腺癌患者会接受辅助化疗,以降低未来复发的风险。尽管辅助化疗在减少复发方面的疗效已得到证实,但相当一部分乳腺癌患者最终仍会出现骨和其他器官的转移性复发(Anampa 等人,2015 年)。事实上,在手术切除原发肿瘤和辅助化疗后的很长一段时间内,乳腺癌患者体内经常会观察到播散性肿瘤细胞(DTCs),这些 DTCs 的存在与转移性复发的风险相关(Braun 等人,2005 年)。这些发现强调了提高辅助化疗疗效的重要性。我们目前的研究表明,化疗诱导的成骨细胞中的 Jagged1 可能为 DTCs 提供了一个有利于生存的生态位,而针对 Jagged1 介导的生态位可能会增加化疗对 DTCs 的清除并减少骨转移。作为这种联合治疗策略的概念验证,小鼠 4T1.2 乳腺肿瘤在联合化疗和 15D11 治疗后,其自发性骨转移显著减少。鉴于 15D11 极佳的安全性,它是进一步临床开发的理想药物,既可用于治疗已确立的骨转移,也可用于预防经常骨转移的早期乳腺癌和其他癌症的复发。

 星级方法


试剂和资源共享联系人


更多信息以及资源和试剂需求,请通过ykang@princeton.edu与主要联系人康一斌联系。

15D11 抗体是安进公司的财产,根据 MTA 用于本研究。如需 15D11 抗体,需直接与安进公司协商。


实验模型和受试者详情


动物模型-所有涉及小鼠的程序和实验方案均已获得普林斯顿大学和安进公司的机构动物护理和使用委员会(IACUC)批准。小鼠购自杰克逊实验室,详细信息见关键资源表。雌性BALB/c小鼠(4-6周大)被麻醉后,做一个小切口以显露乳腺,进行正位原发肿瘤形成和自发性骨转移实验。将悬浮在 10 μ l 10 μ l 10 mul10 \mu \mathrm{l} PBS中的 10 5 10 5 10^(5)10^{5} GFP标记的4 T 1.2肿瘤细胞直接注射到乳腺脂肪垫中。通过 X 射线成像监测自发性骨转移。当原发肿瘤达到 20 × 20 mm 20 × 20 mm 20 xx20mm20 \times 20 \mathrm{~mm} 时,小鼠被安乐死。实验性骨转移使用了心内注射(IC)、髂动脉内注射(IIA)和胫骨内注射(IT)三种方法。所有这些注射方法都是从亚融合细胞培养中收获肿瘤细胞,用 PBS 冲洗,在 IC 和 IIA 注射时将 10 6 10 6 10^(6)10^{6} 细胞 / ml / ml //ml/ \mathrm{ml} 悬浮于 PBS 中,在 IT 注射时将 2.5 × 10 7 2.5 × 10 7 2.5 xx10^(7)2.5 \times 10^{7} 细胞 / ml / ml //ml/ \mathrm{ml} 悬浮于 PBS 中。注射前用氯胺酮( 100 mg / kg 100 mg / kg 100mg//kg100 \mathrm{mg} / \mathrm{kg} 体重)和甲苯噻嗪( 10 mg / kg 10 mg / kg 10mg//kg10 \mathrm{mg} / \mathrm{kg} 体重)麻醉小鼠。IC注射时,用26G针头将0.1毫升细胞注入4-6周大的雌性无胸腺裸鼠的左心室。通过 BLI 成像确认注射成功,生物发光信号在全身均匀分布。注射 IIA 时,在股骨和髂骨中线的皮肤上切开一个约 1.5 厘米的小切口。肌肉


解剖髂动脉。在解剖显微镜下,用 31G 超细胰岛素针注射 0.1 毫升 PBS 中的癌细胞。然后按压创面 10 分钟止血,并在 BLI 成像前用伤口夹缝合。IT 注射使用 4-6 周大的雌性 FVB 小鼠。小鼠腿部弯曲,用 70 % 70 % 70%70 \% 乙醇擦拭皮肤区域。将 28G 针头插入髌骨下,穿透胫骨生长板,缓慢按压活塞,注入 10 μ l 10 μ l 10 mul10 \mu \mathrm{l} 细胞液。每周通过 BLI 和 X 射线成像监测骨转移负荷。在实验终点,小鼠安乐死,进行骨组织学分析,一半的骨样本用于 μ CT μ CT muCT\mu \mathrm{CT} 成像和骨密度定量。

人类 Jagged1 cDNA 经 PCR 扩增后,用 XhoI 和 EcoRI 限制位点克隆到逆转录病毒表达载体 pMSCVpuro 中。为了产生 Col1a1-Jag1 小鼠品系,我们使用了 pTyr-Co1a1 骨干载体。小鼠 cDNA 文库是用 FVB 背景小鼠的乳腺 mRNA 生成的,并使用用于 RT-PCR 的 SuperScript III First Strand Synthesis System(118080-051 Life Technologies CA)反转录为 cDNA。用一对带有 SwaI 限制性酶悬垂位点的引物(CATT ATTTAAATgccaccatgcggtccccacgga 和 CATT ATTTAAAT ctgctatacgatgtattccatccgg)对该文库中的 Jagged1 cDNA 进行 PCR 扩增,然后插入 pTyr-Col1a1 质粒的 SwaI 位点并进行测序确认。使用引物对 CAACACCACGGAATTGTCAGT 和 GATGATGGGAACCCTGTCAA 进行基因分型,最终 PCR 产物为 1012 bps。

临床骨髓细胞浆分析--从德国埃森的埃森大学医院获得了 14 对癌症患者在使用卡铂和紫杉醇前后的骨髓细胞浆样本(不含任何 DTC)。普林斯顿大学的机构审查委员会认为,患者身份已被去除了,并被认为是豁免的。染色前,细胞载玻片在室温下干燥 1 小时。玻片解冻 3 分钟,用 PBS 中的 4 % 4 % 4%4 \% PFA 固定 10 分钟,然后用 PBS 中的 0.2% Triton X-100 通透 10 分钟,并用 10% 山羊血清阻断 20 分钟。然后用 Jagged1 一抗孵育 1 小时,二抗孵育 45 分钟。样品在与下一组一抗(碱性磷酸酶抗体)和二抗孵育前阻断 20 分钟。所有抗体均用 10%山羊血清稀释。载玻片用 Hoechst 染色,并用 Prolong gold antifade 试剂装入盖玻片。

细胞系-293T、4T1.2、SCP28、SCP28-Vector 和 -Jagged1 细胞、SCP28、RAW264.7 在添加 10% FBS、1% 青霉素-链霉素和 0.2% 两性霉素 B 的 DMEM 培养液中培养;MCF7 细胞在添加 10% FBS、 1 % 1 % 1%1 \% 青霉素-链霉素和 0.01 mg / ml 0.01 mg / ml 0.01mg//ml0.01 \mathrm{mg} / \mathrm{ml} 胰岛素的 DMEM 培养液中培养。SUM1315-M1B1 用 F12 培养基培养,其中添加 5% FBS、1% 青霉素-链霉素、 0.2 % 0.2 % 0.2%0.2 \% 两性霉素 B、 5 μ g / ml 5 μ g / ml 5mug//ml5 \mu \mathrm{~g} / \mathrm{ml} 胰岛素和 10 ng / ml 10 ng / ml 10ng//ml10 \mathrm{ng} / \mathrm{ml} 表皮生长因子。H29-Clone#7 在 DMEM 中培养,DMEM 补充有 10% FBS、1% 青霉素-链霉素、 0.2 % 0.2 % 0.2%0.2 \% 两性霉素 B、 0.5 μ g / ml 0.5 μ g / ml 0.5 mug//ml0.5 \mu \mathrm{~g} / \mathrm{ml} 强力霉素、 2 μ g / ml 2 μ g / ml 2mug//ml2 \mu \mathrm{~g} / \mathrm{ml} 嘌呤霉素和 300 μ g / ml 300 μ g / ml 300 mug//ml300 \mu \mathrm{~g} / \mathrm{ml} G418。H29-Clone#7 用于生产逆转录病毒时,不使用所有抗生素。MC3T3-E1 克隆 #4(以下简称 MC3T3)在 a-MEM 中培养,加入 10 % 10 % 10%10 \% FBS、 1 % 1 % 1%1 \% 青霉素-链霉素和 0.2 % 0.2 % 0.2%0.2 \% G418。

小鼠间充质基质细胞(MSC,分离自 Balb/c)在添加 10%FBS、1%青霉素-链霉素、0.2%两性霉素 B 和 20 ng / ml 20 ng / ml 20ng//ml20 \mathrm{ng} / \mathrm{ml} FGF 的 DMEM 中培养。使用 Lipofectamine 2000 将所有质粒转染到不同的细胞系中,具体操作请参照制造商的手册(Life technologies, CA, USA)。为了生成稳定的细胞系,我们的研究使用了基于 pLEX-MCS 的慢病毒载体或基于 pMSCV-hygro 的逆转录病毒载体。慢病毒在 HEK293T 细胞中进行包装,而逆转录病毒则使用 H29 细胞系进行包装。转染 2 天和 3 天后,从这些含有病毒的包装细胞中收集条件培养基。用嘌呤霉素或百日咳霉素筛选感染细胞,以产生稳定表达的细胞系。293T 和 H29clone#7 是来自女性人类的细胞系。所有其他癌症细胞系都是来自雌性人类或小鼠的乳腺癌细胞系。

 方法细节


破骨细胞分化试验--为了进行破骨细胞分化试验,将 RAW264.7 细胞接种到 Fc- 或重组 Jagged1 蛋白板上,或与指定细胞系共培养,浓度为 0.2 百万细胞/孔,置于 12 孔板中。然后在 DMEM 加 10% FBS 中用 5 ng / ml 5 ng / ml 5ng//ml5 \mathrm{ng} / \mathrm{ml} RANKL 处理细胞,每两天更换一次培养基,从第 5 天起每天更换两次培养基。从 6 周大 WT Balb/c 小鼠胫骨冲洗的骨髓细胞中分离出原代前破骨细胞,并通过 70 μ m μ m mum\mu \mathrm{m} 细胞过滤器过滤,然后在含有 10% FBS 的 a-MEM 中培养过夜。翌日,将不粘附的细胞培养并补充 50 ng / mL 50 ng / mL 50ng//mL50 \mathrm{ng} / \mathrm{mL} M-CSF 2 天。然后将细胞重新接种到Fc-或重组Jagged1蛋白平板上,或在DMEM加10% FBS中加入5ng/ml RANKL,每2天更换一次培养基(从第5天开始每天更换两次培养基),在12孔板中与指定细胞系共培养。使用白细胞酸性磷酸酶试剂盒(387A-1KT Sigma)对成熟破骨细胞进行 TRAP 染色,将 TRAP 阳性和多核细胞定量为成熟破骨细胞。

二维和三维肿瘤-基质共培养--对于二维肿瘤-成骨细胞共培养,将 SCP28vector 或 SCP28-Jagged1 肿瘤细胞与 MC3T3 细胞培养在 10 cm 平板中,培养基为含 10% FBS 的 DMEM。共培养一天后,将培养基更换为无血清 DMEM,再培养 24 小时,然后收集条件培养基。收集的条件培养基使用 Amicon Ultra-15 (3K) 离心过滤器(UFC900324,EMD Millipore,美国马萨诸塞州)在室温下以 4000 rpm 离心浓缩,以备进一步使用。在肿瘤-间质细胞三维共培养试验中,将 10000 个肿瘤细胞和 MC3T3 细胞(或间充质干细胞)按 1:1 的比例放入无血清乳球形成培养基的低附着力平板(Corning, Corning NY, USA)中培养。培养基由 1 毫升 B27(Life Technologies,CA,USA)、 20 ng / ml 20 ng / ml 20ng//ml20 \mathrm{ng} / \mathrm{ml} bFGF(Novoprotein,NJ,USA)、 20 ng / ml 20 ng / ml 20ng//ml20 \mathrm{ng} / \mathrm{ml} EGF(Novoprotein,NJ,USA)、 100 μ g / ml 100 μ g / ml 100 mug//ml100 \mu \mathrm{~g} / \mathrm{ml} 庆大霉素(Life Technologies,CA,USA)和 0.25 毫升非必需氨基酸溶液(Life Technologies,CA,USA)新鲜配制而成,总共有 50 毫升 DMEM/F12 培养基。在使用相应的化疗药物之前,先将细胞培养成三维球体。

MC3T3和间充质干细胞的标记--为了生成标记的间充质干细胞和MC3T3细胞用于共培养研究,我们用pLEX-mCherry慢病毒感染这两种细胞系两天,并让细胞扩增5天。培养细胞经胰蛋白酶消化后重新悬浮于 FACS 缓冲液(PBS,添加 5% 的新生小牛血清)中,并通过 70 mm 尼龙细胞过滤器过滤,然后在 FACS 分选仪(BD Biosciences)上进行流式细胞分析。未标记的亲代细胞用作阴性对照。DAPI 用于核反染色,以去除死细胞。


15D11 抗体的生成与特性分析


免疫:全人源的Jagged1抗体是利用XenoMouse技术产生的,这种转基因小鼠能表达不同的全人源 IgG κ IgG κ IgG kappa\operatorname{IgG\kappa } IgG λ IgG λ IgG lambda\operatorname{IgG} \boldsymbol{\lambda} 抗体的相应异型(Kellermann和Green,2002;Mendez等人,1997)。用表达全长人类 Jagged1 的 CHO 转染子免疫 XMG2-KL 和 XMG4-KL 株系小鼠。细胞免疫原的剂量为 4.0 × 10 6 4.0 × 10 6 4.0 xx10^(6)4.0 \times 10^{6} Jagged1转染细胞/小鼠,随后的增强剂量为 2.0 × 10 6 2.0 × 10 6 2.0 xx10^(6)2.0 \times 10^{6} Jagged1转染细胞/小鼠。注射部位为尾基部皮下注射和腹腔注射。使用的佐剂是明矾(E.M. Sergent Pulp and Chemical Co., Clifton, NJ, cat. # 1452-250)。小鼠免疫期为 8 周至 12 周。

抗体特异性测定:培养 14 天后,用荧光微量检测技术(FMAT)(Applied Biosystems, Foster City, CA)对杂交瘤上清液进行人 Jagged1 特异性单克隆抗体筛选。上清针对瞬时转染人 Jagged1 的 293T 细胞进行筛选,并针对瞬时转染相同表达质粒但不含 J A G 1 J A G 1 JAG1J A G 1 基因的 293T 细胞进行反筛选。

配体结合亲和力测试:配体结合竞争法用于鉴定(杂交瘤上清液中的)能与 Jagged1 配体结合并阻止三种受体结合的抗体:Notch-3、Notch 2 和 Notch-1。将 20 μ l 20 μ l 20 mul20 \mu \mathrm{l} 杂交瘤上清与50000个以 4 C 4 C 4^(@)C4^{\circ} \mathrm{C} 瞬时表达的细胞培养1小时,然后用PBS/BSA洗涤两次。然后用 5 μ g / ml 5 μ g / ml 5mug//ml5 \mu \mathrm{~g} / \mathrm{ml} 花色素标记的 Notch-3(#1559-NT,R&D Systems 公司)在 4 C 4 C 4^(@)C4^{\circ} \mathrm{C} 浓度下处理细胞,再用 PBS/BSA 洗涤两次。将细胞重新悬浮在 1 毫升 PBS/BSA 中,使用 FACS Calibur TM TM  ^("TM "){ }^{\text {TM }} 仪器分析抗体结合情况。使用 Notch-2(#3735-NT,R&D Systems)和 Notch-1(#3647-TK,R&D Systems)进行了类似的检测。

实验包括阴性对照杂交瘤上清液。在这些阴性对照实验中观察到的平均信号被用作检测的最大可能信号。将实验上清与最大信号进行比较,计算出每孔的抑制率(抑制率 = (1-(抗 BCMA 杂交瘤上清的 FL1/最大 FL1 信号)))。

其他结合特征:进行 FACS 结合试验,以评估抗 Jagged1 特异性抗体与鼠 Jagged1 以及相关 Notch 配体(人 Jagged2 和人 Dll4)的结合情况。FACS 检测是通过孵育


杂交瘤上清液与 50000 个细胞在 4 C 4 C 4^(@)C4^{\circ} \mathrm{C} 下混合 1 小时,然后用 PBS/H2O 洗两次。


BSA。然后用 4 C 4 C 4^(@)C4^{\circ} \mathrm{C} 浓度的氟铬标记二抗处理细胞,再清洗两次。将细胞重新悬浮在 1 毫升 PBS/BSA 中,使用 FACSCalibur TM TM ^(TM){ }^{\mathrm{TM}} 仪器分析抗体结合情况。


K d K d K_(d)\mathbf{K}_{\mathbf{d}} KinExA测定:在KinExA上测试抗Jagged1抗体15D11与293T/ muJagged1 clone1细胞的结合。简言之,UltraLink Biosupport(Pierce cat# 53110)预涂山羊抗-huFc(Jackson Immuno Research cat# 109-005-098)并用 BSA 阻断。在 1 % 1 % 1%1 \% FBS、 0.05 % 0.05 % 0.05%0.05 \% 叠氮化钠、DMEM 中,将 10 pM 和 100 pM 的 15D11 抗体与不同密度的 ( 1.5 × 10 2 9.0 × 10 6 1.5 × 10 2 9.0 × 10 6 (1.5 xx10^(2)-9.0 xx10^(6):}\left(1.5 \times 10^{2}-9.0 \times 10^{6}\right. 细胞/毫升)表达 muJagged1 的 293T 细胞孵育。含有 15D11 抗体和全细胞的样品在室温下摇动 4 小时。使用 Beckman GS-6R 离心机以约 220 g 离心 5 分钟,将全细胞和抗体-细胞复合物与未结合的游离抗体分离。上清液通过 0.22 μ m 0.22 μ m 0.22 mum0.22 \mu \mathrm{~m} 过滤器过滤,然后再通过山羊抗-huFc包被珠。用荧光(Cy5)标记的山羊抗人IgG(H+L)抗体(Jackson Immuno Research cat# 109-175-088)定量检测珠子结合的 Ab 15D11 的量。结合信号与各细胞密度下溶液中游离 Ab 15D11 的浓度成正比。平衡解离常数 ( K d ) K d (K_(d))\left(\mathrm{K}_{\mathrm{d}}\right) 是在 KinExA TM TM ^(TM){ }^{\mathrm{TM}} Pro 软件中使用未知配体模型估计的,用于 n 曲线分析。

体内治疗时间表和剂量--初始治疗在肿瘤细胞注射前一天或肿瘤细胞注射后一周进行,具体见各实验描述。对于 IgG 或 15D11 抗体治疗,小鼠按 10 mg / kg 10 mg / kg 10mg//kg10 \mathrm{mg} / \mathrm{kg} 剂量腹腔注射,每周两次。对于 OPG-Fc 治疗,小鼠按 3 mg / kg 3 mg / kg 3mg//kg3 \mathrm{mg} / \mathrm{kg} 剂量进行腹腔注射,每周两次。紫杉醇治疗时,先将紫杉醇粉末溶解在 95% 的乙醇中,超声振荡过夜,然后与 cremophor(Sigma 目录号:769193-1KG)按 1:1 的比例混合,使最终浓度达到 20 mg / ml 20 mg / ml 20mg//ml20 \mathrm{mg} / \mathrm{ml} 。紫杉醇在使用前用 PBS 稀释 5 倍。小鼠第一次静脉注射 25 mg / kg 25 mg / kg 25mg//kg25 \mathrm{mg} / \mathrm{kg} ,然后每次 20 mg / kg 20 mg / kg 20mg//kg20 \mathrm{mg} / \mathrm{kg} ,每周两次,共注射6次。在图 5D 的实验中,顺铂的剂量为 2 mg / kg 2 mg / kg 2mg//kg2 \mathrm{mg} / \mathrm{kg} 。在使用 GSI 抑制剂(MRK003)进行治疗时,小鼠口服 GSI(每次使用前新鲜溶解在 0.5% 甲基纤维素中)或载体对照( 0.5 % 0.5 % 0.5%0.5 \% 甲基纤维素),每周两次,剂量为 100 mg / kg 100 mg / kg 100mg//kg100 \mathrm{mg} / \mathrm{kg} 。用药时间为 "开机 2 天,关机 5 天"。

15D11 抗体对血管生成的影响--15D11 抗体对血管生成的影响通过两种方法确定:1)小鼠新生儿视网膜研究。按照先前研究(Ridgway 等人,2006 年)中的类似方法采集、染色和固定小鼠视网膜;2)肿瘤血管生成模型。 2 × 10 6 2 × 10 6 2xx10^(6)2 \times 10^{6} 将 Colo 205 细胞植入雌性无胸腺裸鼠皮下。小鼠在第 11 天随机接受 IgG 或 15D11 抗体治疗,剂量为 300 μ g / mouse 300 μ g / mouse 300 mug//mouse300 \mu \mathrm{~g} / \mathrm{mouse} ,每周两次。通过触诊监测肿瘤体积。为确定肿瘤血管生成效果,收集了用 500 μ g 500 μ g 500 mug500 \mu \mathrm{~g} IgG或15D11抗体治疗96小时的小鼠Colo205肿瘤。通过 CD31 染色和苏木精反染观察瘤内血管。(每组 n = 3 n = 3 n=3\mathrm{n}=3 )。未观察到组间血管差异。

骨组织学分析--在每个实验的终点,即最后一个 BLI 时间点之后,立即切除小鼠后肢骨。之后,将带瘤后肢骨固定在 10%中性缓冲福尔马林中,在 10%乙二胺四乙酸脱钙 2 周,并用石蜡包埋,进行苏木精和伊红(H&E)、酒石酸磷酸酶(TRAP)(Kos 等人,2003 年)或免疫组化染色。使用蔡司 Axiovert 200 显微镜和 AxioVision 软件 4.6.3 SP1 版对 H&E 染色的骨转移样本进行组织形态计量分析。为了定量分析病灶面积,使用 10 倍物镜聚焦于感兴趣的肿瘤区域,并使用 AxioCamICc3 相机采集图像,曝光时间设定为 100 毫秒左右。破骨细胞数量以多核TRAP + + ^(+){ }^{+} 细胞评估,并以数量/视野报告。为了对骨样本进行免疫荧光染色,在每次实验的终点切除小鼠后肢骨骼,然后立即用 PBS 和 4% 新鲜多聚甲醛染色。然后将未脱钙的骨骼样本冷冻在包埋介质中,用 Leica CM3050S 研究型低温恒温器在 20 μ M 20 μ M 20 muM20 \mu \mathrm{M} 温度下用 IIIC 型 Cryofilm(Section-Lab,日本)进行切片,并用相应的一抗和荧光团标记的二抗进行染色。GFP 抗体:Cat# ab13970(Abcam)。Jagged1 抗体:Cat#AP09127PU-N(Acris Antibodies)。ALP 抗体Cat# MAB29091 (R&D Systems)。裂解的 Caspase-3 抗体:Cat# 9661S (Cell Signaling)。Ki67 抗体Cat# ab15580 (Abcam).使用普林斯顿大学分子生物学共聚焦显微镜核心设施的尼康 A1 共聚焦显微镜拍摄图像。


μ C T μ C T 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 μ A 500 μ A 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 μ m 9.44 μ m 9.44 mum9.44 \mu \mathrm{~m} 。在 195 195 195^(@)195^{\circ} 角度范围内使用 0.66 0.66 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 β β 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


(1:1000稀释,MLB International Cooperation,Cat#JM-5144-100)、抗 PARP(1:1000 稀释,Cell Signaling,Cat#9532S)、抗 Jagged1(1:1,000 稀释,Santa Cruz Biotechnology,Cat#SC8303)。用辣根过氧化物酶(HRP)结合的抗小鼠、兔或大鼠二抗(1:2,000 稀释,GE Healthcare)孵育膜 1 小时,用 ECL 底物(GE Healthcare)检测化学发光信号。

RNA 分离和 qRT-PCR 分析--按照生产商的说明使用 RNeasy 试剂盒(Qiagen)从细胞中分离总 RNA。使用 Superscript III 反转录试剂盒(Invitrogen)将 RNA 反转录为 cDNA。实时 RTPCR 在 ABI 790096 HT 系列 PCR 仪(Applied Biosystem)上使用 SYBR Green Supermix(Bio-Rad Laboratories)进行。基因特异性引物组的最终浓度为 0.5 μ M 0.5 μ M 0.5 muM0.5 \mu \mathrm{M} 。所有实时 RT-PCR 检测均在至少两个独立实验中重复进行。各目标基因的相对表达值与 GAPDH/Gapdh mRNA 水平进行了归一化处理。所用引物见补充表 S1。

芯片和热图生成-SUM1315-M1B1乳腺癌细胞(GFP标记)单独培养或与MC3T3-E1克隆4号骨成骨细胞共培养,并用对照PBS或 10 μ M 10 μ M 10 muM10 \mu \mathrm{M} 顺铂处理48小时。使用 FACS 根据 GFP 标记对肿瘤细胞进行分类。

根据生产商的说明,使用 RNAeasy Mini Kit(Qiagen,Valencia VA)从这些样本中收集 RNA。按照生产商的说明,使用安捷伦全人类基因组芯片 4×44K G4112F 测定基因表达谱。简而言之,使用 Agilent Quick Amp Labeling Kit 对 RNA 样品和通用人类参考 RNA(Agilent 740000)分别进行 CTP-cy5 和 CTP-cy3 标记。将标记的测试和参考 RNA 样品按等比例混合,并与人类 GE 4 × 44 K 4 × 44 K 4xx44K4 \times 44 \mathrm{~K} 阵列杂交。使用安捷伦 G2505C 扫描仪扫描阵列,并使用安捷伦特征提取软件(v11.0)提取原始数据。使用 GeneSpring 13 软件(安捷伦)分析数据。单个探针的表达值是指 Log2(Cy5/Cy3)比值。微阵列数据集存入 GEO 数据库,登录号为 GSE97997。

微阵列数据的热图显示了从MsigDB(http://software.broadinstitute.org/ gsea/msigdb/cards/HALLMARK_APOPTOSIS.html )中的Hallmark-apoptosis特征中选出的促凋亡和抗凋亡基因。这些基因的表达值是根据生理盐水和顺铂处理中成对培养条件(单独培养与联合培养)样本的中位数进一步转换而来的。


量化和统计分析


结果以平均值 ± SD ± SD +-SD\pm \mathrm{SD} (标准偏差)或平均值 ± SE ± SE +-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。

DATA AND SOFTWARE AVAILABILITY 

All microarray data generated in this study have been deposited as a superseries at the NCBI Gene Expression Omnibus with the accession code GSE97997, and can be accessed at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cpancgoafjcnxyv&acc=GSE97997. 

ADDITIONAL RESOURCES (None) KEY RESOURCES TABLE 

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 fl/fl  ^("fl/fl "){ }^{\text {fl/fl }} : B6.129(SJL)-Oxtr tm 1.1 Wsy / J tm  1.1 Wsy / J ^("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 |
 试剂或资源 SOURCE IDENTIFIER

15D11 全人抗人 Jagged1 抗体
Amgen Inc.  N/A 
 
Anti-Rabbit IgG -
Horseradish Peroxidase in 1:5,000, Goat
Anti-Rabbit IgG - Horseradish Peroxidase in 1:5,000, Goat| 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
Anti-Mouse IgG - Horseradish Peroxidase in 1:5,000, Sheep| 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 
 中国仓鼠 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 - 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 γ γ 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
  • 15 D 11 15 D 11 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 μ CT μ CT 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).

References

Anampa J, Makower D, Sparano JA. Progress in adjuvant chemotherapy for breast cancer: an overview. BMC Med. 2015; 13:195. [PubMed: 26278220]
Benedito R, Roca C, Sorensen I, Adams S, Gossler A, Fruttiger M, Adams RH. The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell. 2009; 137:1124-1135. [PubMed: 19524514]
Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC, Schlimok G, Diel IJ, Gerber B, Gebauer G, et al. A pooled analysis of bone marrow micrometastasis in breast cancer. The New England journal of medicine. 2005; 353:793-802. [PubMed: 16120859]
Cao Z, Ding BS, Guo P, Lee SB, Butler JM, Casey SC, Simons M, Tam W, Felsher DW, Shido K, et al. Angiocrine factors deployed by tumor vascular niche induce B cell lymphoma invasiveness and chemoresistance. Cancer cell. 2014; 25:350-365. [PubMed: 24651014]
Dotto GP. Crosstalk of Notch with p53 and p63 in cancer growth control. Nat Rev Cancer. 2009; 9:587-595. [PubMed: 19609265]
Duan CW, Shi J, Chen J, Wang B, Yu YH, Qin X, Zhou XC, Cai YJ, Li ZQ, Zhang F, et al. Leukemia propagating cells rebuild an evolving niche in response to therapy. Cancer cell. 2014; 25:778-793. [PubMed: 24937459]
Eckhardt BL, Parker BS, van Laar RK, Restall CM, Natoli AL, Tavaria MD, Stanley KL, Sloan EK, Moseley JM, Anderson RL. Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix. Molecular cancer research : MCR. 2005; 3:1-13. [PubMed: 15671244]
Ell B, Mercatali L, Ibrahim T, Campbell N, Schwarzenbach H, Pantel K, Amadori D, Kang Y. Tumorinduced osteoclast miRNA changes as regulators and biomarkers of osteolytic bone metastasis. Cancer cell. 2013; 24:542-556. [PubMed: 24135284]
Forozan F, Veldman R, Ammerman CA, Parsa NZ, Kallioniemi A, Kallioniemi OP, Ethier SP. Molecular cytogenetic analysis of 11 new breast cancer cell lines. Br J Cancer. 1999; 81:1328-1334. [PubMed: 10604729]
Gu 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; 25:2293-2301. [PubMed: 15347599]
Hanoun M, Zhang D, Mizoguchi T, Pinho S, Pierce H, Kunisaki Y, Lacombe J, Armstrong SA, Duhrsen U, Frenette PS. Acute myelogenous leukemia-induced sympathetic neuropathy promotes malignancy in an altered hematopoietic stem cell niche. Cell Stem Cell. 2014; 15:365-375. [PubMed: 25017722]
Imbimbo BP. Therapeutic potential of gamma-secretase inhibitors and modulators. Current topics in medicinal chemistry. 2008; 8:54-61. [PubMed: 18220933]
Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen SM, Cordon-Cardo C, Guise TA, Massague J. A multigenic program mediating breast cancer metastasis to bone. Cancer cell. 2003; 3:537-549. [PubMed: 12842083]
Kellermann SA, Green LL. Antibody discovery: the use of transgenic mice to generate human monoclonal antibodies for therapeutics. Current opinion in biotechnology. 2002; 13:593-597. [PubMed: 12482519]
Kos CH, Karaplis AC, Peng JB, Hediger MA, Goltzman D, Mohammad KS, Guise TA, Pollak MR. The calcium-sensing receptor is required for normal calcium homeostasis independent of parathyroid hormone. The Journal of clinical investigation. 2003; 111:1021-1028. [PubMed: 12671051]
Li D, Masiero M, Banham AH, Harris AL. The notch ligand JAGGED1 as a target for anti-tumor therapy. Frontiers in oncology. 2014; 4:254. [PubMed: 25309874]
Lu 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; 23:171-185. [PubMed: 23375636]
Mendez MJ, Green LL, Corvalan JR, Jia XC, Maynard-Currie CE, Yang XD, Gallo ML, Louie DM, Lee DV, Erickson KL, et al. Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nature genetics. 1997; 15:146-156. [PubMed: 9020839]
Miller AC, Lyons EL, Herman TG. cis-Inhibition of Notch by endogenous Delta biases the outcome of lateral inhibition. Curr Biol. 2009; 19:1378-1383. [PubMed: 19631544]
Paul MK, Bisht B, Darmawan DO, Chiou R, Ha VL, Wallace WD, Chon AT, Hegab AE, Grogan T, Elashoff DA, et al. Dynamic changes in intracellular ROS levels regulate airway basal stem cell homeostasis through Nrf2-dependent Notch signaling. Cell Stem Cell. 2014; 15:199-214. [PubMed: 24953182]
Pitt LA, Tikhonova AN, Hu H, Trimarchi T, King B, Gong Y, Sanchez-Martin M, Tsirigos A, Littman DR, Ferrando AA, et al. CXCL12-Producing Vascular Endothelial Niches Control Acute T Cell Leukemia Maintenance. Cancer cell. 2015; 27:755-768. [PubMed: 26058075]
Polverino 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; 66:8715-8721. [PubMed: 16951187]
Reedijk M, Odorcic S, Chang L, Zhang H, Miller N, McCready DR, Lockwood G, Egan SE. Highlevel coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival. Cancer Res. 2005; 65:8530-8537. [PubMed: 16166334]
Ren G, Zhang L, Zhao X, Xu G, Zhang Y, Roberts AI, Zhao RC, Shi Y. Mesenchymal stem cellmediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell. 2008; 2:141-150. [PubMed: 18371435]
Ridgway J, Zhang G, Wu Y, Stawicki S, Liang WC, Chanthery Y, Kowalski J, Watts RJ, Callahan C, Kasman I, et al. Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature. 2006; 444:1083-1087. [PubMed: 17183323]
Rizzo P, Osipo C, Foreman K, Golde T, Osborne B, Miele L. Rational targeting of Notch signaling in cancer. Oncogene. 2008; 27:5124-5131. [PubMed: 18758481]
Sethi N, Dai X, Winter CG, Kang Y. Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells. Cancer cell. 2011; 19:192-205. [PubMed: 21295524]
Shiozawa Y, Pedersen EA, Havens AM, Jung Y, Mishra A, Joseph J, Kim JK, Patel LR, Ying C, Ziegler AM, et al. Human prostate cancer metastases target the hematopoietic stem cell niche to establish footholds in mouse bone marrow. The Journal of clinical investigation. 2011; 121:12981312. [PubMed: 21436587]
Sprinzak D, Lakhanpal A, Lebon L, Santat LA, Fontes ME, Anderson GA, Garcia-Ojalvo J, Elowitz MB. Cis-interactions between Notch and Delta generate mutually exclusive signalling states. Nature. 2010; 465:86-90. [PubMed: 20418862]
Wan L, Lu X, Yuan S, Wei Y, Guo F, Shen M, Yuan M, Chakrabarti R, Hua Y, Smith HA, et al. MTDH-SND1 interaction is crucial for expansion and activity of tumor-initiating cells in diverse oncogene- and carcinogen-induced mammary tumors. Cancer cell. 2014; 26:92-105. [PubMed: 24981741]
Wang H, Yu C, Gao X, Welte T, Muscarella AM, 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
Weilbaecher KN, Guise TA, McCauley LK. Cancer to bone: a fatal attraction. Nat Rev Cancer. 2011; 11:411-425. [PubMed: 21593787]
Figure 1. Characterization of fully human monoclonal antibodies against Jagged1 (A) Kinetic exclusion assay for binding of 15D11 with murine Jagged1 protein. Increasing number ( 1.5 × 10 2 1.5 × 10 2 1.5 xx10^(2)1.5 \times 10^{2} to 9 × 10 6 9 × 10 6 9xx10^(6)9 \times 10^{6} ) of 293 T cells with murine Jagged1 expression (Clone #1) were added into the solution with fixed amount of 15D11 antibody ( 10 or 100 pM ) and remaining amount of antibody in the liquid was determined after 4 hr . (B) A Notch reporter luciferase assay was used to determine IC50 for 15D11 in Jagged1-induced Notch activation. Human TE671 expressing a multimerized 7XCSL-responsive element upstream of a minimal promoter (Notch reporter) were co-cultured with HEK293 expressing Jagged1. 15D11 ( 0.07 1333.4 nM 0.07 1333.4 nM 0.07-1333.4nM0.07-1333.4 \mathrm{nM} ) was added to the cells for 2 days. Luciferase signal was quantified. The percent inhibition was calculated relative to signals of reactions lacking 15D11 and 293T coculture. © RAW264.7 pre-osteoclast cells were seeded on Fc- or recombinant Jagged1 coated plates in the presence of 5 ng / ml 5 ng / ml 5ng//ml5 \mathrm{ng} / \mathrm{ml} RANKL and indicated concentrations of IgG or 15D11. Cells were cultured for 5-7 days before fixing for TRAP staining to visualize multinucleated mature osteoclasts. (D) Quantification of TRAP + + ^(+){ }^{+}osteoclasts per field from C. Data is presented as mean ± ± +-\pm SEM. n = 3 . p < 0.01 n = 3 . p < 0.01 n=3.^(****)p < 0.01\mathrm{n}=3 .{ }^{* *} \mathrm{p}<0.01 by Student’s t-test. n.s., not significant. (E) SCP28-Vector or SCP28-Jagged1 cells were co-cultured with RAW264.7 cells in the presence of 5 ng / ml 5 ng / ml 5ng//ml5 \mathrm{ng} / \mathrm{ml} RANKL and 1 μ g / ml 1 μ g / ml 1mug//ml1 \mu \mathrm{~g} / \mathrm{ml} IgG or 15D11. Cells were cultured for 5-7 days
before fixing for TRAP staining to visualize multi-nucleated mature osteoclasts. (F) Primary bone marrow cells were flushed from tibias of 4-6 week old wild-type FVB mice and plated for 24 hr , after which the non-adherent cells were collected and cultured in M-CSF (50 ng / mL ng / mL ng//mL\mathrm{ng} / \mathrm{mL} ) for 2 days and then co-cultured with SCP28-Vector or SCP28-Jagged1 in the presence of RANKL ( 20 ng / mL 20 ng / mL 20ng//mL20 \mathrm{ng} / \mathrm{mL} ) and 1 μ g / ml 1 μ g / ml 1mug//ml1 \mu \mathrm{~g} / \mathrm{ml} IgG or 15D11. Cells were allowed to differentiate into osteoclasts for additional 4-5 days before fixing and TRAP staining. (G) mRNA expression of mouse II6 and Hes1 in MC3T3 cells co-cultured with SCP28-vector control or SCP28-Jagged1 cells, treated with either IgG control or 15D11. Primers used in this experiment were specifically designed to only detect mouse genes, but not human genes. n = 3 . p < 0.01 n = 3 . p < 0.01 n=3.^(****)p < 0.01\mathrm{n}=3 .{ }^{* *} \mathrm{p}<0.01 by Student’s t-test. Data is presented as mean ± ± +-\pm SEM. (H) Expression of IL6 protein in cell lysate and conditioned media (CM) taken from MC3T3 cells co-cultured with SCP28-Vector or SCP28-Jagged1 cells (left). Recombinant IL6 served as positive control. IL6 protein expression in CM from MC3T3 cells co-cultured with SCP28-Vector or SCP28-Jagged1 cells, treated with either IgG control or 15D11 antibody (right). β β beta\beta-actin served as loading control. Bars below the IL6 immunoblots represent relative band density of IL6 as quantified by ImageJ software. Scale bars in C, E, and F: 100 μ m 100 μ m 100 mum100 \mu \mathrm{~m}. See also Figure S1.
Figure 2. 15D11 inhibits tumor-derived Jagged1 dependent bone metastasis (A) Schematic representation of the experiment. 10 5 10 5 10^(5)10^{5} tumor cells were IC injected into each mouse. Mice were treated one day before the tumor cell injection and continued twice a week at the dosage of 10 mg / kg 10 mg / kg 10mg//kg10 \mathrm{mg} / \mathrm{kg} for IgG/15D11, and 3 mg / kg 3 mg / kg 3mg//kg3 \mathrm{mg} / \mathrm{kg} for OPG-Fc. (B) Representative BLI and X-ray images on Day 1 immediately after the IC injection and on Day 35 at the end point of the experiment. © Quantification of bone metastasis burden each week based on BLI imaging from the experiment. n = 10 n = 10 n=10\mathrm{n}=10 per group; data presented as mean ± ± +-\pm SEM. * < 0.05 < 0.05 ^(**) < 0.05{ }^{*}<0.05 by Mann-Whitney test. (D) Quantification of osteolytic lesions on Day 35. n = 8 n = 8 n=8\mathrm{n}=8 per group; data presented as mean ± ± +-\pm SEM. p < 0.01 p < 0.01 ^(****)p < 0.01{ }^{* *} \mathrm{p}<0.01 by Student’s t-test. AU: arbitrary units. (E) Representative μ CT , H & E μ CT , H & E muCT,H&E\mu \mathrm{CT}, \mathrm{H} \mathrm{\& E} staining and TRAP staining images from the experiment. T: tumor; B: bone. Scale bars: 100 μ m 100 μ m 100 mum100 \mu \mathrm{~m} for H&E and 25 μ m 25 μ m 25 mum25 \mu \mathrm{~m} for TRAP. See also Figure S2-3 and Movies S1-4.
Figure 3. Synergistic inhibition of SCP28-Jagged1 bone metastasis with combined treatment of 15D11 and chemotherapy
(A) Schematic representation of the experiment. 10 5 10 5 10^(5)10^{5} tumor cells were IC injected into each mouse. Treatments were initiated one week after the injection and continued twice a week at the dosage of 10 mg / kg 10 mg / kg 10mg//kg10 \mathrm{mg} / \mathrm{kg} for IgG / 15 D 11 IgG / 15 D 11 IgG//15D11\mathrm{IgG} / 15 \mathrm{D} 11, and 20 mg / kg 20 mg / kg 20mg//kg20 \mathrm{mg} / \mathrm{kg} for paclitaxel. (B) Quantification of bone metastasis burden each week based on BLI imaging. n = 10 n = 10 n=10\mathrm{n}=10 mice per group; data presented as mean ± ± +-\pm SEM. p < 0.05 p < 0.05 ^(**)p < 0.05{ }^{*} \mathrm{p}<0.05 and p < 0.01 p < 0.01 ^(****)p < 0.01{ }^{* *} \mathrm{p}<0.01 by Mann-Whitney test. © Representative BLI and X-ray images of two mice per group at week 7. (D) Representative H&E and TRAP staining images from the experiment at the end point. Scale bar: 100 μ m 100 μ m 100 mum100 \mu \mathrm{~m} for H&E and 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m} for TRAP. (E) Quantification of osteolytic lesions based on X-ray images from D. n = 10 n = 10 n=10\mathrm{n}=10 mice per group; data presented mean ± ± +-\pm SEM. p < 0.01 p < 0.01 ^(****)p < 0.01{ }^{* *} \mathrm{p}<0.01 by Student’s t-test. AU: arbitrary units. (F) Quantification of TRAP + + ^(+){ }^{+}osteoclasts from decalcificed histological
bone sections of hind limbs from mice in D , n = 10 D , n = 10 D,n=10\mathrm{D}, \mathrm{n}=10 per group. Data is presented as mean ± ± +-\pm SEM. p < 0.01 p < 0.01 ^(****)p < 0.01{ }^{* *} \mathrm{p}<0.01 by Student’s t-test.
Figure 4. Synergistic inhibition of SCP28 bone metastasis with combined treatment of 15D11 and chemotherapy
(A) Schematic illustration of the experimental procedure. 10 5 10 5 10^(5)10^{5} tumor cells were IC injected into each mouse. Treatments were initiated one week after the injection and continued twice a week at the dosage of 10 mg / kg 10 mg / kg 10mg//kg10 \mathrm{mg} / \mathrm{kg} for IgG/15D11, and 20 mg / kg 20 mg / kg 20mg//kg20 \mathrm{mg} / \mathrm{kg} for paclitaxel. (B) Quantification of bone metastasis burden based on BLI imaging. n = 10 n = 10 n=10\mathrm{n}=10 mice per group; data presented as mean ± ± +-\pm SEM. p < 0.05 p < 0.05 ^(**)p < 0.05{ }^{*} \mathrm{p}<0.05 and p < 0.01 p < 0.01 ^(****)p < 0.01{ }^{* *} \mathrm{p}<0.01 by Mann-Whitney test. © Representative BLI images and X-ray images at week 6 of two mice per group. (D) Representative H&E and TRAP staining images from the experiment at the end point. Scale bar: 100 μ m 100 μ m 100 mum100 \mu \mathrm{~m} for H&E and 50 μ m 50 μ m 50 mum50 \mu \mathrm{~m} for TRAP. (E) Quantification of osteolytic lesions based on X-ray images from D. n = 10 n = 10 n=10\mathrm{n}=10 mice per group. Data is presented as mean ± ± +-\pm SEM. ^(****){ }^{* *} p < 0.01 < 0.01 < 0.01<0.01 by Student’s t-test. AU: arbitrary units. (F) Quantification of TRAP + + ^(+){ }^{+}osteoclasts from decalcified histological bone sections of hind limbs from mice in D , n = 10 D , n = 10 D,n=10\mathrm{D}, \mathrm{n}=10 per group. Data presented as mean ± ± +-\pm SEM. p < 0.01 p < 0.01 ^(****)p < 0.01{ }^{* *} \mathrm{p}<0.01 by Student’s t-test. See also Figure S4.
Figure 5. Chemotherapy induces Jagged1 expression in osteoblast lineage cells
(A) Jagged1 mRNA expression in osteoblast (OB) MC3T3-E1-Clone#4 osteoblast cells and mesenchymal stem cells (MSC), HPMECL endothelial cells (endo), RAW264.7 preosteoclasts (pre-OC), and SCP28 and SUM1315-M1B1 breast tumor cells 48 hr posttreatment with 25 nM paclitaxel or 10 μ M 10 μ M 10 muM10 \mu \mathrm{M} cisplatin. n = 3 n = 3 n=3\mathrm{n}=3 per group; data presented as mean ± ± +-\pm SEM. * << 0.05 << 0.05 <<0.05<<0.05 by Student’s t-test. (B) Athymic nude mice were intravenously (i.v.) injected with PBS or cisplatin ( 2 mg / kg 2 mg / kg 2mg//kg2 \mathrm{mg} / \mathrm{kg} ) and euthanized 48 hr later. Hind limb bones were collected for IF staining against ALP (green) and Jagged1 (red). Nuclei were counter-stained with DAPI (blue). (C-E) Athymic nude mice were treated with either PBS or cisplatin (2 mg / kg mg / kg mg//kg\mathrm{mg} / \mathrm{kg} ) on Day 0. One day after the pre-treatment, MCF7 ©, SUM1315-M1B1 (D) or SCP28 (E) cells were delivered locally to the hind limbs of these mice by IIA injection. BLI imaging and quantification of BLI signal at Day 4. n = 5 n = 5 n=5n=5; data presented as mean ± ± +-\pm SEM. * p < 0.05 p < 0.05 p < 0.05\mathrm{p}<0.05 by Student’s t-test. (F) Mice from experiment in E were euthanized after BLI imaging at Day 4. Hind limbs were fixed and processed for IF imaging of GFP-expressing tumor cells (green) and ALP + + ^(+){ }^{+}osteoblasts (red). Scale bar: 80 μ m 80 μ m 80 mum80 \mu \mathrm{~m}. See also Figure S5 and Movies S5-6.
Figure 6. Osteoblast-derived Jagged1 promotes bone metastasis seeding and progression (A) Representative μ CT μ CT muCT\mu \mathrm{CT} imaging and H&E staining images from FVB WT and Col1a1-Jag1 mice. Yellow box: Trabecular bone areas of decreased bone density (in μ CT μ CT muCT\mu \mathrm{CT} imaging) that were filled with bone marrow cells (in H&E staining). Scale bar: 0.5 mm in μ CT μ CT muCT\mu \mathrm{CT} and 200 μ m μ m mum\mu \mathrm{m} in H&E. (B) Col1a1-Jag1 mice were treated with IgG or 15D11 (10 mg/kg) twice a week for 3 months. Mice were then euthanized for bone sample collection and histology analysis. Representative TRAP staining images from IgG or 15D11 group were presented. Scale bar: 200 μ m 200 μ m 200 mum200 \mu \mathrm{~m}. © PyMT-A-FIG cells were delivered locally to the hind limbs of WT and Col1a1Jag1 mice by intra-tibial injection. Three weeks after injection, the engraftment of breast cancer cells in the bone was assessed by BLI. Each data point represents an individual mouse ( n = 4 n = 4 n=4\mathrm{n}=4 ); horizontal line represents mean ± ± +-\pm SEM. p < 0.01 p < 0.01 ^(****)p < 0.01{ }^{* *} \mathrm{p}<0.01 by Student’s t -test. (D) PyMT-A-FIG cells were delivered locally to the hind limbs of WT and Col1a1-Jag1 mice by IIA injection. Tumor cells in the bone were quantified based on BLI imaging at Day 1 and Day 4 (right panel). n = 5 n = 5 n=5\mathrm{n}=5; data presented mean ± ± +-\pm SEM. p < 0.05 p < 0.05 ^(**)p < 0.05{ }^{*} \mathrm{p}<0.05 by Student’s t-test. (E) IF staining of bone samples at Day 4 from experiment in C, showing GFP-expressing tumor cells (green) and ALP + + ^(+){ }^{+}osteoblasts (red). Scale bar: 80 μ m 80 μ m 80 mum80 \mu \mathrm{~m}. See also Figure S6.
Figure 7. Pro-survival effect of osteoblast Jagged1 on cancer cells can be blocked by 15 D 11 (A) 3-D tumorsphere culture of MCF7, SCP28, and SUM1315-M1B1 cells, alone or in coculture with osteoblast cells, were treated with indicated concentrations of cisplatin or docetaxel. Two days after the chemotherapy, the surviving tumor spheres were counted under the fluorescent microscope. n = 3 n = 3 n=3\mathrm{n}=3 per group; data presented as mean ± ± +-\pm SEM. * < 0.05 < 0.05 < 0.05<0.05 by Student’s t-test. (B) Cell culture of SCP28 cells alone or in co-culture with MC3T3-E1 pre-osteoblast cells were treated with PBS or cisplatin. Protein samples were collected at 6 hr , 24 hr hr , 24 hr hr,24hr\mathrm{hr}, 24 \mathrm{hr}, and 72 hr later. Cleaved PARP and cleaved caspase-3 were determined by immunoblotting. Full length PARP is 116 kD and cleaved PARP is 89 kD . © Real-time PCR analysis of Notch target genes (HEY1, HEY2, and HES2) in tumor cells co-cultured with MSC and treated with indicated chemotherapeutic agents. n = 3 n = 3 n=3\mathrm{n}=3 per group; data presented as mean ± ± +-\pm SEM. * p < 0.05 p < 0.05 p < 0.05\mathrm{p}<0.05 by Student’s t-test. (D) Microarray analysis was performed with mRNA from cell culture of SUM1315-M1B1cells alone or in co-culture with MC3T3-E1 osteoblasts, with or without cisplatin treatment. Heatmap shows 16 apoptosis-related genes with at least 2-fold change in the cisplatin treatment condition (tumor cell culture alone vs. co-culture). Five p53 pathway-related genes were highlighted in
red. (E-F) 10 5 10 5 10^(5)10^{5} SCP28 cells were IC injected into each mouse. Treatments were initiated one week after the injection for twice at the dosage of 10 mg / kg 10 mg / kg 10mg//kg10 \mathrm{mg} / \mathrm{kg} for IgG/15D11, and 20 mg / kg 20 mg / kg 20mg//kg20 \mathrm{mg} / \mathrm{kg} for paclitaxel. Hind limb bones were collected, fixed and cryo-sectioned for IF staining against CC3 for apoptotic cells and GFP for tumor cells (E), and CC3 positive apoptotic cells among GFP-positive tumor cells were quantified in F. Scale bar: 25 μ m . n = 5 25 μ m . n = 5 25 mum.n=525 \mu \mathrm{~m} . \mathrm{n}=5 mice per group; data presented as mean ± ± +-\pm SEM. ^(****){ }^{* *} p < 0.01 < 0.01 < 0.01<0.01 by Student’s t-test. (G) Athymic nude mice were treated with either PBS or cisplatin via i.v. injection on Day 0. On Day 1, PBS pretreated mice were treated with IgG, while cisplatin pre-treated mice were treated with either IgG or 15D11. SUM1315-M1B1 cells were delivered locally to the hind limbs of these nude mice by IIA injection. Tumor cells in the bone were quantified based on BLI imaging on Day 1 and Day 4. BLI signal at Day 4 was normalized to BLI signal intensity at Day 1 (lower panel). n = 5 n = 5 n=5\mathrm{n}=5; data presented as mean ± ± +-\pm SEM. p < 0.05 p < 0.05 ^(**)p < 0.05{ }^{*} \mathrm{p}<0.05 by Student’s t-test. (H) Mice from experiment in G were euthanized after BLI imaging at Day 4. Hind limbs were fixed and processed for IF imaging of GFP-expressing tumor cells (green) and ALP + + ^(+){ }^{+}osteoblasts (red). Scale bars: 40 μ m 40 μ m 40 mum40 \mu \mathrm{~m}. See also Figure S7.
Figure 8. Combination of 15D11 and chemotherapy strongly inhibits spontaneous bone metastasis
(A) Paired bone marrow cytospin samples were collected from cancer patients before and after cisplatin + paclitaxel chemotherapy. Slides were IF co-stained for ALP + + ^(+){ }^{+}osteoblasts (red) and Jagged1 (green). Scale bar: 40 μ m 40 μ m 40 mum40 \mu \mathrm{~m}. (B) Normalized signal intensity of Jagged1 staining to ALP staining. Signal intensity was quantified using ImageJ software (NIH). n = 14 samples per group; data presented as mean ± ± +-\pm SEM. < 0.05 < 0.05 ^(**) < 0.05{ }^{*}<0.05 by Student’s t-test. © Schematic representation of experimental procedures. 4T1.2 mouse mammary tumor cells were injected in the mammary fat pad of 6 8 6 8 6-86-8 week old female Balb/c mice. Two weeks later when the primary tumors progressed to about 5-8 mm in diameter, mice were treated with IgG, 15D11 (10 mg/kg), paclitaxel ( 20 mg / kg 20 mg / kg 20mg//kg20 \mathrm{mg} / \mathrm{kg} ) or both 15D11 and paclitaxel twice a week. Spontaneous bone metastasis progression was monitored by weekly X-ray images and by histological H&E staining and TRAP staining. n = 10 n = 10 n=10\mathrm{n}=10 for each experimental group. (D) X-ray images of these experimental mice at Week 5. (E) Quantification of the percentage of mice that developed spontaneous bone metastasis based on X-ray and H&E staining at the end point. (F) Quantification of osteolytic lesions based on X-ray images. AU: arbitrary unit. n = n = n=\mathrm{n}= 8; data presented as mean ± ± +-\pm SEM. p < 0.05 , p < 0.01 p < 0.05 , p < 0.01 ^(****)p < 0.05,^(****)p < 0.01{ }^{* *} \mathrm{p}<0.05,{ }^{* *} \mathrm{p}<0.01 by Student’s t-test. (G) Schematic model for Jagged1-Notch signaling in bone metastasis progression and chemoresistance.
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.

  1. 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 ^(10){ }^{10} Co-first author
    11 11 ^(11){ }^{11} Co-corresponding author
    12 12 ^(12){ }^{12} H.Z. current address: Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China Lead contact: Yibin Kang
    SUPPLEMENTAL INFORMATION
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    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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