Name: multimodal bioluminescent and positronic-emission tomography/computational tomography imaging of multiple myeloma bone marrow xenografts in nog mice
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice at JoVE.c

这项工作得到了美国退伍军人事务部生物医学实验室研究和发展处 (blrds) 向 p. f. 提供的 va merit 赠款 1i01 bx001532 的支持, 欧洲经济合作署感谢 va 临床科学 & d 服务 (优异奖 i01cx001388) 和 va 康复 r & d 服务 (优异奖 I01CX001388)。进一步的支持来自加州大学洛杉矶分校向 j. k. 提供的种子赠款。这些内容不一定代表美国退伍军人事务部或美国政府的观点。

下面描述的所有动物程序都得到了大洛杉矶 va 保健系统的动物护理和使用机构委员会 (iacuc) 的批准, 并在无菌和无病原体的条件下进行。
1. 荧光素表达8226个细胞 (8226-lu) 的制备
<ol><li>在含有95% 空气和5% 二氧化碳 (co 2) 的加湿大气中, 在37°c 条件下保持人类 mm 细胞系 8226, 在 rpmi-1640 培养基中补充10% 的胎儿牛血清 (fbs) 和1% 的青霉素链霉素.</li>
	<li>通过转染 1 x 10 6 8226 ?...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laglambda-1:+A+Clinically+Relevant+Drug+Resistant+Human+Multiple+Myeloma+Tumor+Murine+Model+That+Enables+Rapid+Evaluation+of+Treatments+for+Multiple+Myeloma.">Laglambda-1: A Clinically Relevant Drug Resistant Human Multiple Myeloma Tumor Murine Model That Enables Rapid Evaluation of Treatments for Multiple Myeloma.</a> International Journal of Oncology. 28 (6), 1409-1417 (2006).</li><li>Miyakawa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+New+Model+of+Human+Multiple+Myeloma+Using+Nod/Scid/Gammac(Null)+(Nog)+Mice.">Establishment of a New Model of Human Multiple Myeloma Using Nod/Scid/Gammac(Null) (Nog) Mice.</a> Biochemical and Biophysical Research Communications. 313 (2), 258-262 (2004).</li><li>Ito, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nod/Scid/Gamma(C)(Null)+Mouse:+An+Excellent+Recipient+Mouse+Model+for+Engraftment+of+Human+Cells.">Nod/Scid/Gamma(C)(Null) Mouse: An Excellent Recipient Mouse Model for Engraftment of Human Cells.</a> Blood. 100 (9), 3175-3182 (2002).</li><li>Frost, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Antitumor+Effects+of+the+Mtor+Inhibitor+Cci-779+against+Human+Multiple+Myeloma+Cells+in+a+Xenograft+Model.">In Vivo Antitumor Effects of the Mtor Inhibitor Cci-779 against Human Multiple Myeloma Cells in a Xenograft Model.</a> Blood. 104 (13), 4181-4187 (2004).</li><li>Asosingh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Hypoxic+Bone+Marrow+Microenvironment+in+5t2mm+Murine+Myeloma+Tumor+Progression.">Role of the Hypoxic Bone Marrow Microenvironment in 5t2mm Murine Myeloma Tumor Progression.</a> Haematologica. 90 (6), 810-817 (2005).</li><li>Storti, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-Inducible+Factor+(Hif)-1alpha+Suppression+in+Myeloma+Cells+Blocks+Tumoral+Growth+in+Vivo+Inhibiting+Angiogenesis+and+Bone+Destruction.">Hypoxia-Inducible Factor (Hif)-1alpha Suppression in Myeloma Cells Blocks Tumoral Growth in Vivo Inhibiting Angiogenesis and Bone Destruction.</a> Leukemia. 27 (8), 1697-1706 (2013).</li><li>Fryer, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Novel+Mouse+Model+of+Multiple+Myeloma+and+Its+Use+in+Preclinical+Therapeutic+Assessment.">Characterization of a Novel Mouse Model of Multiple Myeloma and Its Use in Preclinical Therapeutic Assessment.</a> PLoS One. 8 (2), e57641 (2013).</li><li>Gould, S. J., Subramani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firefly+Luciferase+as+a+Tool+in+Molecular+and+Cell+Biology.">Firefly Luciferase as a Tool in Molecular and Cell Biology.</a> Analytical Biochemistry. 175 (1), 5-13 (1988).</li><li>Czernin, J., Phelps, M. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+Vivo+Molecular+Imaging+Probe+(18)F-Annexin+B1+for+Apoptosis+Detection+by+Pet/Ct:+Preparation+and+Preliminary+Evaluation.">An in Vivo Molecular Imaging Probe (18)F-Annexin B1 for Apoptosis Detection by Pet/Ct: Preparation and Preliminary Evaluation.</a> Apoptosis. 18 (2), 238-247 (2013).</li></ol>

尽管 mm6、9、11、12、13的临床前异种移植模型多种多样, 但研究 bm 微环境中肿瘤/bm 相互作用的能力仍然困难14. 这里所述的技术允许在 nog 小鼠的骨骼中快速和可重复地雕刻8226-luc 肿瘤细胞。
该协议的关键步骤包括建立表达 lm 细胞系的?...

mm 是一种不治之症, 由恶性血浆 b 细胞组成, 浸润 bm, 导致骨破坏、贫血、肾功能损害和感染。mm 占所有血液恶性肿瘤的10%-15% 1, 是最常见的癌症涉及骨骼 2。mm 的发展源于长寿命浆细胞的致癌转化, 这些细胞在最终归属于 bm3之前建立在淋巴组织的萌发中心。bm 的特点是高度异质的利基;包括不同和关键的细胞成分, 低 po 2 (缺氧) 的区域, 广泛的血管, 复杂的细胞外基质, 细胞因子和生长因子网络, 所有这些都有助于 mm 肿瘤发生 4.因此, 一个传播 mm 异种移植模型的特点是肿瘤, 严格地在 bm 相关的工具, 研究 mm 病理在体内5,6。然而, 许多技术障碍可能会限制大多数异种移植模型的有效性, 使其成本高昂, 难以应用。这包括与 bm 生态位内一致和可重复的肿瘤雕刻相关的问题, 肿瘤发育时间长, 以及直接观察和测量肿瘤生长生存率变化的能力有限, 而无需牺牲老鼠在实验过程中7,8。
该协议使用了最初由宫川等人开发的修改后的异种移植模型.9. 其中与骨髓瘤细胞的静脉注射 (iv) 挑战导致 "传播" 肿瘤, 在 nod/scid/il-2γ (空) 小鼠 bm 中持续和可重复地移植 10.这些肿瘤的原位可视化是通过8226人 mm 细胞系的稳定转染和 luc 等位基因和依次测量这些移植肿瘤细胞产生的 bii 的变化 6.重要的是, 该模型可以扩展到利用各种其他表达 luc 的人 mm 细胞系 (如u266 和 opm2), 具有类似的倾向, 特别是在 nog 小鼠的骨骼中移植。在通过小鼠的生物发光成像来识别肿瘤之后, pet任何 ct 测量放射性药物探针 (如18f-fdg) 的吸收情况。肿瘤/bm 微环境中的途径 (即代谢改变、缺氧变化和诱导细胞凋亡)。该模型的主要优势可以通过提供广泛的放射性标记、生物发光和荧光探针和标记来突出, 这些探针和标记可用于研究 mm 的进展和体内病理。

<table>
	<tbody>
		<tr>
			<td>8226 human myeloma cell line</td>
			<td>ATCC</td>
			<td>CCL-155</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ Mice (NOG)</td>
			<td>Jackson Labs</td>
			<td>5557</td>
			<td></td>
		</tr>
		<tr>
			<td>VivoGlo Luciferin substrate</td>
			<td>Promega</td>
			<td>P1041</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxyprobe-1Kit</td>
			<td>HPL</td>
			<td>HP1-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CD45 (clone H130)</td>
			<td>BD Biosciences</td>
			<td>555483</td>
			<td>Used for flow cytometry to identify human CD45+ tumor cells in BM exudate</td>
		</tr>
		<tr>
			<td>rabbit anti-human CD45 (clone D3F8Q)</td>
			<td>Cell Signaling Technology</td>
			<td>70527</td>
			<td>Primary antibody used for Immunohistochemistry of excised bone</td>
		</tr>
		<tr>
			<td>Goat Anti-rabbit IgG (HRP conjugated)</td>
			<td>ABCAM</td>
			<td>ab205718</td>
			<td>Seconday antibody used for Immunohistochemistry of excised bone</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>pGL4.5 Luciferase Reporter Vector</td>
			<td>Promega</td>
			<td>E1310</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina XRMS In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sofie G8 PET/CT Imaging System</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

multimodal bioluminescent and positronic-emission tomography/computational tomography imaging of multiple myeloma bone marrow xenografts in nog mice

多发性骨髓瘤 (mm) 肿瘤在骨髓 (bm) 中移植, 其生存和进展取决于存在于这种微环境中的复杂分子和细胞相互作用。然而, bm 微环境不可能在体外轻易复制, 这可能会限制许多体外和体外实验模型的生理相关性。这些问题可以通过利用异种移植模型来克服, 在该模型中, 荧光素酶 (luc) 转染 8226 mm 细胞将专门移植到小鼠的骨骼中。当这些小鼠被给予适当的底物 d-荧光素时, 可以通过测量肿瘤在体内产生的生物发光图像 (bli) 的变化来分析治疗对肿瘤生长和存活的影响。该 bri 数据结合正电子发射断层扫描 (pet/ct), 使用代谢标记 2-deoxy-2--(18f) 氟 d-葡萄糖 (18f-fdg) 进行分析, 用于监测肿瘤代谢随时间的变化。这些成像平台允许在肿瘤/bm 微环境中进行多种无创测量。

在初步的试点研究中, 在 nodse:scid 小鼠体内注射8226-luc 细胞并没有发生 bm 移植的 mm 肿瘤, 尽管鳞状 mm 肿瘤很容易形成 (100% 成功率)。相反, iv 挑战8226个细胞进入 nog 小鼠产生的骨骼肿瘤 (很少在非骨骼组织中形成肿瘤, 如肝脏或脾脏)。由于骨骼中的肿瘤无法通过对动物进行物理检查来直观地证实, 因此必须考虑其他方法来确认成功的肿瘤雕刻和 bm 中肿瘤的位置 (<strong class="x...

Watch this Scientific Journal Video about Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice at JoVE.c

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

在这里, 我们使用生物发光, x 射线和正电子发射断层扫描断层扫描断层扫描成像, 以研究如何抑制 mtor 活性影响骨骨髓移植骨髓瘤肿瘤在异种移植模型。这使得生理相关, 非侵入性, 多模态分析的抗骨髓瘤的作用治疗针对骨髓移植骨髓瘤肿瘤在体内。

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

该方法可用于回答有关骨髓微环境作用和骨髓瘤肿瘤生存性等关键问题。该程序的主要优点是，它可以应用于纵向抗药物研究，并用于以非侵入性的方式检测多种生化途径。在实验当天，首先将8226个路西拉酶转成细胞在200 RCF的冰冷PBS中三次清洗，每200个RCF进行5分钟的离心，并在新鲜冰冷PBS中重新供应颗粒，每200微升冰冷PBS的5倍至第6个细胞。接下来确认在麻醉的四到六周大的NOG小鼠中对手趾捏缺乏反应，并在动物的眼睛上涂抹眼科软膏。将细胞装入装有26针的一毫升胰岛素注射器。将整个细胞体积注入动物的尾静脉，然后将鼠标返回到其笼子，进行监测，直到完全恢复。挑战后10至20天，在无菌盐水内注射200微升体内D-西西芬基质。注射后 5 至 10 分钟内将麻醉动物放在小型动物成像系统中的上角位置。测量相应成像软件中选定感兴趣区域的平均辐射。对于8226路西法酶肿瘤的靶向治疗，在测量了每个动物的基线生物发光后，随机将动物分为治疗组，并用一系列200微升IP注射Temsirolimus或盐水对照治疗每只小鼠。每周测量路西法酶活性两次，并绘制生物发光随时间的变化。要测量肿瘤代谢的变化，首先从小鼠的家笼中取出食物24小时，以避免过量的非特异性放射性标记的氟化物葡萄糖吸收。第二天，将无菌盐水中18个氟放射性标记氟化物葡萄糖探针的500至100微曲线稀释至每只小鼠100微升的最终体积，并记录探头的时间和活性，使用剂量校准器。当探头准备就绪时，将第一只麻醉动物放在加热垫上，头部朝向，并使用装有 26 量针的屏蔽一毫升胰岛素注射器将探针的 100 微升注射到尾静脉中。使用剂量校准器测量针头和注射器中的残余放射性，并记录活性和时间。然后测量与小型动物 PET CT 成像系统中的带辐射肿瘤的 Fludeoxyglucose 在选定感兴趣区域中的活性。如证明，通过生物发光成像可以证实一个成功的骨髓肿瘤移植。多个动物的串行成像可用于可视化骨髓移植多发性骨髓瘤肿瘤的分布。此外，光学成像 X 射线分析显示快速和非侵入性测定小鼠骨架内多发性骨髓瘤肿瘤的确切位置和分布。这些被感染的肿瘤细胞产生的生物发光可以连续和非侵入性测量，以评估肿瘤生长的变化。此外，使用mTOR抑制剂Temsirolimus治疗的小鼠的生存情况可以监测，正电子发射和计算断层扫描分析的肿瘤放射性标记氟化物吸收可用于证明特姆西罗利莫斯调节葡萄糖代谢的变化。执行此过程时，注射细胞 IEV 非常重要。我们发现，如果不将细胞注射到静脉中，在注射部位附近形成非骨髓移植的肿瘤细胞。按照这个程序，其他方法，如免疫组织化学和微CT可用于解决肿瘤对骨骼结构的影响以及骨髓环境的非肿瘤细胞和分子成分的问题。

Research

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肿瘤细胞系基因表达的条件性击倒研究单核/巨噬细胞在微环境中的招募

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

科研

癌症研究

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

测定急性髓系白血病可测残留病和白血病干细胞的骨髓标本和过程综合协议

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

黑色素瘤细胞衍生因子对骨髓脂肪分化的双重影响

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

胶质瘤移植中体内成像的荧光分子层析成像研究

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube SWCNT-delivered MALAT1 Antisense Oligos

单壁碳纳米管 (sscnt) (mallat1 反义寡 3号) 抑制多骨髓瘤细胞在体内的生长

The single-wall carbon nanotube (SWCNT) is a new type of nanoparticle, which has been used to deliver multiple kinds of drugs into cells, such as ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

在斑马鱼肿瘤异种移植物中宿主血管反应的Vivo成像和定量

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

使用[18F]四氟硼酸盐正电子发射断层扫描/计算机断层扫描对嵌合抗原受体T细胞进行动态成像

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...