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植入。核供应的小鼠缺乏成熟的 T 细胞、B 细胞和 NK 细胞。他们也有损害细胞因子信号, 并有缺陷的先天免疫应答。
用密度梯度法从原 MCL 样品中制备淋巴细胞是去除其他细胞类型如红细胞和血小板的重要步骤。传统上, 密度梯度离心允许成功地分离淋巴细胞。进一步处理这些淋巴细胞, 以丰富的 b 细胞的人口是通过使用 B 细胞浓缩试剂盒后, 本手稿中所述的协议。应注意达到高纯度的 B 淋巴细胞种群达到最佳植入淋巴瘤细胞。MCL 细胞通过侧尾静脉静脉注射 (至少 40-60 x 106细胞/小鼠), 并允许小鼠发展为10周的淋巴瘤。这些小鼠每天都被严密检查, 因为疾病症状包括体重减轻、发皱、活动减少、后肢瘫痪、等.流式细胞仪对脾、肝、骨髓、血等不同器官的分析显示 MCL植入.最近, 已发现在20周的注射剂9的照射下, MCL 细胞嫁接在骨髓和脾脏中被辐照的核供应的小鼠。独立于这一发现, 我们成功地开发了我们自己的移植模型的原发性 MCL 与更快速的肿瘤形成。
这种异种移植小鼠模型可能成为研究不同淋巴瘤类型疾病进展的非常有用的工具。异种移植模型还提供了强有力的工具, 以执行候选药物的临床前测试如 MCL, 但是, 有一定的局限性, 例如, 复发的主要克隆在患者中不一定是克隆在异种移植10中出现。这可能是由于异种移植在不同宿主系统中的选择性压力不同。这种菌株的造血成分并不能完全概括人类造血, 从而限制了人类细胞的长期维持。使用人体肿瘤移植的优点覆盖了这些限制, 因为在几周后, 人类肿瘤活检就能得到治疗的结果。药物反应并不经常与患者的临床活动相关, 当人类细胞系取代原发肿瘤细胞时, 它们就会成为可能的治疗反应的基础, 而11 。将原发肿瘤作为正交异性异种异体移植具有更强的预测反应值, 特别是当使用临床相关药物剂量时11,12,13。淋巴瘤细胞归巢是生存和建立肿瘤植入的重要先决条件。这一过程通过淋巴系统得到严格的调节, 通过高内皮静脉循环淋巴细胞的进入维持了淋巴结的淋巴细胞稳态。
B 淋巴细胞通过淋巴结循环, 需要通过与不同黏附分子的协调相互作用, 跨越内皮屏障和趋化因子触发细胞迁移。因此, 靶向控制淋巴细胞的归巢的分子, 以其生存的利基可能构成一种新的治疗策略的 B 细胞淋巴瘤, 因为它们的行为类似正常淋巴细胞。我们的目的是针对分子果酱-C, 一个交界黏附分子, 这是存在于 MCL。该移植模型将用于研究果酱 C 抗体对原发淋巴瘤细胞的归巢和植入的治疗作用。最近, 我们已经显示了抗果酱 C 抗体在小鼠收到的 MCL 细胞线, Jeko-114的效果。该抗体对 Jeko-1 细胞的归巢有阻断作用, 并且在定期进行管理时, 有效地防止了 Jeko-1 细胞的植入。除了对归巢的影响, 抗体治疗根除了淋巴瘤嫁接在这只老鼠的大多数器官中测试14。
鉴于目前正在讨论的科学和临床问题, 这些移植肿瘤代表了一个有趣的模型来研究 MCL 的治疗结果。

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&#183;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。为了获得成功的 MCL 植入, 应注意获取最大纯度。使用这种方法获得的纯度通常 > 90%。其余的细胞主要是单核和一些 T 细胞 (数据没有显示)。
图 2表示 B 单元纯化协议的不同步骤, 如 "方法" 部分所述。利用不同的标记物 (CD45、CD19、CD20、CD23、CD200、CD5、卡伯和 lambda), 通过流式细胞仪对其纯度进行进一步分析。按照图 3中所示的顺序门控, 可以选择 MCL 单元格的特征: CD45+、CD19+、CD20+、CD5+、CD23−和 CD200−。根据标准细胞仪的建立, 对每种荧光使用单一染色控制, 对多色染色进行补偿。
图 3E, G表示富集前后 B 细胞的典型点图。在这种情况下, > 95% 的丰富细胞使用此套件是 B 细胞。这些细胞在 pbs 中悬浮在 40-60 ×106细胞的浓度, 在 150-200 µL PBS 中, 如协议所述。他们立即注射静脉注射到核供应的小鼠。10周后, > 90% 的注射动物发育淋巴瘤显示末期疾病的迹象 (体重减轻, 头发竖起, 活动减少,等等)。在牺牲以后, 脾脏和肝脏被去除并且处理通过机械中断获得单细胞悬浮 (图 4D, F);血液是精心绘制心脏穿刺和骨髓的处理通过切割股骨两端和冲洗它使用2毫升注射器填充 RPMI 培养基或 PBS (图 4C)。细胞由流式细胞术进一步处理, 使用人 B 细胞特异标记如 CD19/CD20 和 CD45。我们使用人类 B 细胞特定标记, 因为受体小鼠应变 (核供应集团) 缺乏 B 细胞。图 4B-F表示牺牲后的器官集合和进一步处理的步骤。来自两个病人的 MCL 细胞的植入程度在图 4G中表示。植入模式在不同的器官和患者之间可以比较后, 这种技术。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56023/56023fig1.jpg"> 图 1: 从全血中分离 PBMCs.从 MCL 患者提取的血液稀释1:1 在 RPMI 介质 (a), 并小心放置在密度梯度介质层 (B) 的顶部, 而不将血液混合到这个密度梯度介质 (C)。离心在 400 x g 为45分钟分离单个核细胞, 出现白色圆环 (箭头表明单个核细胞层) (D)。此层在不与其他层混合的情况下轻轻 pipetted, 以进行进一步处理 (E)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56023/56023fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56023/56023fig2.jpg"> 图 2: 从 PBMCs 中富集 B 细胞.采用密度梯度离心分离的 PBMCs 与 PBS 清洗两次。使用负 B 细胞浓缩套件, 并按照制造商的协议, 获得 b 单元格的纯填充 (14)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56023/56023fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56023/56023fig3.jpg"> 图 3: MCL 细胞的流化分析和富集前后 B 细胞的纯度.以 CD45、CD19、CD20、CD23、CD200、CD5、卡伯和 lambda 等不同标记物为特征, 对单个核 MCL 细胞进行了流点分析。选择用于 CD23 和 CD200 的 CD45、CD19、CD20、CD5 和负值的单元格 (AF)。G表示在浓缩后使用否定选择工具包与E相比, 在富集之后的单元格填充。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56023/56023fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56023/56023fig4.jpg"> 图 4: 核供应的小鼠作为植入 MCL 细胞的移植模型.从 MCL 患者中提取的 B 细胞通过核核组织小鼠的侧尾静脉注入静脉注射 (A)。经过几天的肿瘤嫁接, 小鼠被牺牲, 并解剖 (B) 收集不同的器官, 如骨髓 (C), 脾脏 (D), 外周血 (E) (绘制心脏穿刺), 并肝脏 (F)。通过流式细胞仪对 B 细胞的存在进行了进一步的处理和分析。G表示 MCL 细胞在 MCL 注射的核型核组织小鼠的不同器官中的植入模式。结果由两名患者显示。数据显示为平均值, 即扫描电镜, n = 3。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56023/56023fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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 ...

tumor-engraftment-xenograft-mouse-model-human-mantle-cell

Nanyang Technological University

Research

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

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

开发和维护的临床前患者来源的肿瘤异种移植模型为新的抗癌疗法的调查

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

科研

癌症研究

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

同源小鼠 B 细胞淋巴瘤模型对 CD19 轿车 T 细胞的临床前评价

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

黑色素瘤患者衍生异种移植模型

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

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. ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

研究癌症相关成纤维细胞在肿瘤生长中的作用的小鼠模型

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

研究利普托梅宁格疾病直接靶向治疗的穆林·奥马亚异种格拉夫特模型

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...