Name: tumor engraftment in a xenograft mouse model of human mantle cell lymphoma
Uploaded: 2018-03-30T12:30:00.000+00:00
Duration: 10 min 52 s
Description: Watch this Scientific Journal Video about Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma at JoVE.com

这项工作得到了 Genevoise 反癌症, Ferriere Dinu 李帕蒂, Oncosuisse 科索沃警察组织法团, OCS-02260-08-2008 和2914-02-2012 的支持, 瑞士国家科学基金会赠款31003A_156760 和310030-153456。

根据日内瓦大学医院当地伦理学和人类实验委员会批准的程序, 人体血液样本被使用。
动物程序是按照日内瓦、瑞士和州兽医局动物保育机构伦理委员会 (授权号: GE/26/15) 进行的。
1. 利用密度梯度分离制备原发外周血单个核细胞 (PBMCs)
注: 3-5 毫升的外周血是在白血病阶段 MCL 的患者身上获得的。诊断是根据标准诊断标准的流式细胞术 (CD5+, CD23−, CD200+, 单克隆 B 细胞人口), 并随后确认的存在染色体易位 t (11; 14) 和过度表达的细胞周期蛋白 D1。在所有情况下, 单克隆 b 细胞构成的总 b 细胞总数的 > 90%。其余细胞主要为单核和某些 T 细胞。
<ol><li>稀释 (1:1) 5 毫升的 MCL 血液样本5毫升的罗斯威尔公园纪念研究所中 1640 (RPMI) (图 1A)。</li>
	<li>在使用前多次反转密度梯度介质 (材料表) 以确保彻底混合。</li>
	<li>使用吸管吸引器将5毫升密度梯度介质添加到15毫升离心管中。</li>
	<li>用吸 (血密度梯度介质比应为 2:1,例如, 10 毫升血液到5毫升密度梯度介质) 轻轻地将稀释后的血液样本在密度梯度介质上的15毫升离心管上。应注意不要混合两个层 (图 1B, C)。</li>
	<li>离心机的样品在 400 x g 为40分钟在室温下与离心式刹车关闭了。</li>
	<li>使用无菌的巴斯德吸管, 轻轻地吸入中间层 (带白色的, 用箭头标记的薄环 (图 1D, E)), 其中包含单个核细胞并将其转移到干净的管中。</li>
	<li>单个核细胞的洗涤<ol>
<li>估计被转移的细胞悬浮的容量并且增加它3容量无菌1x 磷酸盐缓冲盐水 (PBS) 与1% 牛血清白蛋白 (BSA)。</li>
		<li>将悬浮液混合在一起, 吹打几次。</li>
		<li>离心机悬浮在 400-500 x g 在室温下 10-15 分钟。</li>
		<li>丢弃上清液以获得细胞颗粒并再次重复洗涤步骤。</li>
		<li>移除上清并并用重悬所需卷中的单元格,例如, 1 或2毫升 PBS。</li>
		<li>通过自动单元格计数器或手动使用 hemocytometer (Neubauer 室) 计算单元格数。</li>
	</ol>
</li>
</ol>2. b 细胞阴性选择对 b 淋巴细胞的富集
<ol><li>将 PBS 中的细胞稀释为 50 x 106细胞/毫升的最终浓度。</li>
	<li>将细胞转移到5毫升圆底聚苯乙烯管。添加100µL 抗体鸡尾酒从 B 细胞浓缩试剂盒到2毫升细胞悬浮, 并轻轻地混合吹打向上和向下 (图 2-1)。</li>
	<li>室温下孵化细胞10分钟。</li>
	<li>简要地涡流三十年代 B 细胞浓缩套件中提供的磁性微粒 (图 2-2)。添加150µL 的磁性粒子到2毫升的样品。</li>
	<li>在室温下孵化样品5分钟。</li>
	<li>将样品体积带到2.5 毫升, 1x PBS 含有2% 胎小牛血清 (FCS) 和1毫米乙二胺四乙酸 (EDTA)。将管放在磁铁中, 再孵化管 3-5 分钟 (图 2-3)。</li>
	<li>拿起管到位的磁铁, 并在一个连续的运动, 醒酒管, 使丰富的细胞悬浮收集在一个新鲜的管 (图 2-4)。通过自动单元格计数器或手动使用 hemocytometer 来计算单元格, 以获得 B 细胞的总产量。 注: 通常情况下, MCL 的正常 b 细胞和 5-50 x 106细胞/毫升的产量为 105 B 细胞/毫升。</li>
	<li>确认 B 细胞富集纯度<ol>
<li>取 ~ 5万细胞, 用 anti-CD19/CD20 抗体 (1:20 稀释) 和 anti-CD45 抗体与不同的荧光 (1:10 稀释) 结合在室温下进行 15-30 分钟的细胞孵化。</li>
		<li>用1毫升 1x pbs + 1% BSA, 离心细胞在 400-500 x g 为5分钟, 并并用重悬细胞在 ~ 200 µL 的 pbs。</li>
		<li>采用流式细胞仪采集细胞样本, 并对 B 细胞纯度进行分析。此方法的纯度主要 > 90% (图 3)。</li>
	</ol>
</li>
</ol>3. 移植小鼠模型的研制
<ol><li>挂起〜 40-60 x 106单元格, 在 150-200 µL 1x PBS (每个鼠标需要注入的卷)。</li>
	<li>将150µL 的细胞悬浮液静脉注射 (静脉注射) 到 6-7 周老 immunodeficient 点头免疫球蛋白 (核供应) 小鼠的每一个尾静脉, 不论性别 (图 4A)。</li>
	<li>允许老鼠发展人类淋巴瘤10周。 注: 与人类淋巴瘤细胞株相比, 原发性人类淋巴瘤细胞的发育要长得多 (约3周)。</li>
	<li>当动物开始表现出诸如体重减轻、头发竖起、活动减少、后肢瘫痪等致命疾病的症状时,等等, 用 CO2窒息后心脏穿刺获得血液来牺牲小鼠。</li>
	<li>收集不同的器官, 如骨髓, 脾脏, 肝脏通过解剖小鼠和收集外周血的血液从心脏在牺牲时, 为肿瘤植入分析 (图 4B -f)。</li>
</ol>4. 不同器官肿瘤细胞嫁接的嵌合体分析
<ol><li>收集不同的器官后, 用机械干扰处理它们, 用200µL 的1x 氯化铵钾缓冲液在血和骨髓、500µL 脾和2毫升的肝脏中生成单细胞悬浮液。.</li>
	<li>使用人体 B 细胞特异抗体 (即、anti-CD19、anti-CD20 和 anti-CD45) 对细胞进行计数和染色。 注: 核供应的小鼠缺乏 b 细胞, 因此进行分析, 细胞被染色的人特定的 B 细胞标志物。</li>
	<li>通过对 B 细胞的门控 (CD19+、CD20+、CD45+), 对各器官的细胞进行流式细胞术分析。</li>
	<li>根据步骤 4.3 (图 4G) 中所示的门控单元, 量化 MCL 注射小鼠的不同器官中的嫁接肿瘤细胞。 注: 有关详细信息, 请参阅材料表。</li>
</ol>

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作者声明他们没有竞争的财政利益。

临床试验是可能的药物, 处于晚期的发展阶段, 但不能用于药物发现。为了测试早期药物, 人们一直在努力为人类细胞移植开发动物受体。在这里, 我们提出了一个动物模型, 避免人移植排斥, 并可以建立一个模型, 人类疾病, 如 MCL。这是目前的一种状态的艺术移植模型研究的机制, 人类肿瘤植入和肿瘤的生长。在这里, 我们使用核供应的小鼠, 一个最免疫缺陷的老鼠模型迄今, 以取得更大的成功淋巴瘤...

淋巴细胞的性质在免疫监测中起着重要作用, 淋巴细胞贩运是增强抗原特异免疫的关键步骤1,2。这一过程包括从胸腺到血流的天真 T 淋巴细胞的迁移, 从那里到次生淋巴器官, 包括淋巴结, Peyer 的补丁, 或脾脏, 在那里他们满足同源抗原。B 淋巴细胞在骨髓中分化, 并作为天真细胞迁移到次级淋巴器官的卵泡中3。其中一些 B 细胞将抗原与受体结合, 并由特定的 T 细胞激活。这些 b 细胞的增殖和分化将非活化的、天真的 b 细胞推入卵泡的地幔区。激活细胞然后可以区分为记忆 B 细胞, 它巡逻的身体, 或成熟的免疫球蛋白分泌血浆细胞迁移到骨髓4。
当地幔区的天真 B 淋巴细胞转化为肿瘤时, MCL 发生。这些淋巴瘤细胞驻留在淋巴器官的微环境中, 并独立于特定的 T 淋巴细胞控制而增殖。然而, 在一定的密度阶段, 他们从这个利基和超量的血液中逃脱, 寻找其他器官的利基。考虑到粘附分子的复杂性和趋化因子及其受体的混杂性, 这种细胞贩运的机制在体内的作用不太清楚, 因此阻碍了治疗。为了防止淋巴瘤 B 细胞接触到新的微环境, 需要采用新的方法来有效阻断这一迁移过程。
MCL 是治疗 B 细胞恶性肿瘤最难的一种。MCL 肿瘤表型的发展是多步级联的结果, 其特点是获得独特的生物特性。在诊断时, 大多数患者 (70%) 已经出现了传播疾病, 大多数病例显示淋巴结外参与脾脏, 骨髓和/或胃肠道 5, 6.在治疗的患者中, 几年内抗肿瘤复发是常见的, 尽管常规化疗能在短期内诱发高缓解率7,8。在这里, 我们提出了一个新的疾病模型, 可以帮助了解 MCL 传播及其基础生物学: 我们建立了一个人 MCL 移植鼠模型, 起源于原发性肿瘤细胞的病人。我们希望这一模式将有助于制定抗 MCL 传播的治疗策略, 并可能为复发患者的最佳诊断和治疗提供新的临床前景。

<table><tbody><tr><td>Ficoll-paque media</td><td>GE Healthcare</td><td>17-1440-02</td><td>for separation of mononuclear cells</td></tr><tr><td>RPMI Medium 1640</td><td>Gibco-Life technologies</td><td>61870-010</td><td>for dilution of blood sample</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td>washing of cells</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A8412</td><td>for preparation of PBS with 1% BSA used in washing cells during isolation</td></tr><tr><td>CD19-APC700</td><td>Beckman Coulter</td><td>B49212</td><td>human pan-B cell marker</td></tr><tr><td>CD20-APC</td><td>Beckman Coulter</td><td>A21693</td><td>human pan-B cell marker</td></tr><tr><td>CD20-ECD</td><td>Beckman Coulter</td><td>IM3607</td><td>human pan-B cell marker</td></tr><tr><td>CD5-PC 5.5</td><td>Beckman Coulter</td><td>PN A70203</td><td>human T cell marker</td></tr><tr><td>CD23-PE</td><td>Pharmingen</td><td>555711</td><td>Cell surface protein typically absent in MCL</td></tr><tr><td>CD45 KO</td><td>Beckman Coulter</td><td>B36294</td><td>Pan-leucocyte marker</td></tr><tr><td>CD200-PE</td><td>Pharmingen</td><td>552475</td><td>Cell surface protein typically absent in MCL</td></tr><tr><td>NOD scid gamma (NSG) mice</td><td>Charles River Laboratories</td><td>5557</td><td>used to develop MCL xenografts in this study</td></tr><tr><td>Easy sep Human B cell enrichment kit</td><td>Stem cell technologies</td><td>19054</td><td>used to enrich B cells to obtain pure cells for injecting into mice</td></tr><tr><td>FACS</td><td>Beckman Coulter</td><td>Navios</td><td>used to characterize MCL sample and to study the organs for MCL engraftment</td></tr><tr><td>1X ammonium chloride potassium buffer</td><td></td><td></td><td>red blood lysis buffer (NH4Cl 8,024 mg/l; KHCO3 1,001 mg/l; EDTA.Na2&amp;middot;2H2O 3.722 mg/l )</td></tr></tbody></table>

tumor engraftment in a xenograft mouse model of human mantle cell lymphoma

B 淋巴细胞是免疫细胞循环的重要参与者, 它们主要栖息在淋巴器官中。当正常 b 细胞只在 T 淋巴细胞刺激下增殖时, 致癌 b 细胞在未定义的器官龛位中存活并自主扩张。地幔细胞淋巴瘤 (MCL) 是一种 B 细胞紊乱, 其中中位存活率为 4-5 岁。这就需要建立有效的机制, 使这些细胞的归巢和植入被阻断, 以增加患者的生存和长寿。因此, 开发一种异种移植小鼠模型来研究 MCL 疗法的效果, 通过阻断自导机制在体内是至关重要的。人类细胞异种移植动物受体在早期药物检测中的发展一直以来都在进行, 因为相关的前体小鼠模型对于筛查新的治疗剂至关重要。这种动物模型是为了避免人的移植排斥和建立人类疾病模型, 它可能是一个非常有用的工具来研究不同类型淋巴瘤的疾病进展, 并进行候选药物的临床前检查恶性血液病像 MCL 一样我们建立了一种移植小鼠模型, 作为研究和开发新的治疗方法的 MCL 的一个优秀的资源。

该手稿描述了一个优化的协议, 成功地开发了移植小鼠模型植入的 MCL 细胞。制备纯细胞种群 (在这种情况下 MCL 细胞), 是非常关键的发展成功的 MCL 移植。图 1表示通过密度梯度分离从 MCL 患者的血液中分离出单个核细胞的制备步骤。对单个核细胞进行进一步处理, 以获得纯 b 细胞, 使用阴性 b 细胞浓缩试剂盒获得纯细胞种群, 用于移植小鼠。为了获得?...

Watch this Scientific Journal Video about Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma at JoVE.com

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

地幔细胞淋巴瘤 (MCL) 是一种难以治疗 B 细胞紊乱的方法, 建立一只移植小鼠模型 MCL 的研究和开发治疗同样困难。在这里, 我们描述成功地建立了 MCL 移植在小鼠, 以帮助了解其基础生物学。

人地幔细胞淋巴瘤移植小鼠模型中肿瘤植入的研究

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

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JoVE Journal

Cancer Research

11.1K 次观看.  University of Geneva. 地幔细胞淋巴瘤 (MCL) 是一种难以治疗 B 细胞紊乱的方法, 建立一只移植小鼠模型 MCL 的研究和开发治疗同样困难。在这里, 我们描述成功地建立了 MCL 移植在小鼠, 以帮助了解其基础生物学。

视频: Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

原位Xenografting人类荧光素酶标记的恶性外周神经鞘瘤细胞在体内测试候选治疗药物

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

科研

医学

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

癌症研究

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

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

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

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

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

用于转化免疫肿瘤学(I-O)研究的人类外周血单核细胞(PBMC)的人工化异种移植模型

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

在免疫缺陷小鼠中生成肝脏正交人子宫黑色素瘤异种移植平台

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

在异种性鼠模型中的移植-无源疾病的诱导和评分,以及使用数字PCR对小鼠组织中人类T细胞的定量

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

免疫与感染

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

异种移植皮肤模型在体内操纵人类免疫反应

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

Patient-derived Xenograft Modeling: A Technique to Generate Melanoma Mouse Models

Source: Min Xiao et al. <a href="https://www.jove.com/v/59508"> A Melanoma Patient-Derived Xenograft Model</a>. J. Vis. Exp. (2018)
In this video, human-derived melanoma cells are implanted subcutaneously into the flank region of an immunocompromised mouse. The PDX model allows preclinical investigation of melanoma cells that better recapitulate the tumor heterogeneity and melanoma aggressiveness observed in in vivo conditions.