Name: using crispr/cas9 to knock out gm-csf in car-t cells
Uploaded: 2019-07-22T12:30:00
Description: Watch this Scientific Journal Video about Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells at JoVE.com

这项工作得到了K12CA090628(SSK)、国家综合癌症网络(SSK)、梅奥个性化医学诊所中心(SSK)、普雷多林基金会(SSK)、梅奥诊所执业翻译办公室(SSK)的资助。梅奥诊所医学科学家培训计划罗伯特·豪厄尔医生-科学家奖学金(RMS)。

该协议遵循梅奥诊所机构审查委员会(IRB)和机构生物安全委员会(IBC)的指导方针。
1. CART19细胞生产
<ol><li>T细胞分离、刺激和前体培养<ol>
<li>利用适当的个人防护设备,在细胞培养罩中执行所有细胞培养工作。从在阿汇期间收集的去识别的正常供体血锥中采集外周血单核细胞(PBMC),因为这些已知是PBMC的一个可行来源。
</li>
		<li>要分离 PBMC,将 15 mL 的密度梯度介?...

<ol>
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在本报告中,我们描述了一种利用CRISPR/Cas9技术诱导CAR-T细胞二次修改的方法。具体来说,这证明使用慢病毒转导与病毒载体,含有gRNA靶向感兴趣的基因和Cas9生成GM-CSFk/o CART19细胞。我们之前已经表明,GM-CSF 中和在异种移植模型中可改善 CRS 和 NT。12如前所述,GM-CSFk/o CAR-T 电池允许在制造过程中抑制 GM-CSF,有效减少 GM-CSF 的生产,同时保持其他关键的 T 细胞功能,并增强体内...

嵌合抗原受体T(CAR-T)细胞治疗在癌症治疗中显示出巨大的前景。1,2两种针对CD19(CART19)的CAR-T细胞疗法最近在美国和英国批准用于B细胞恶性肿瘤,在多中心临床试验中取得了显著成果。3,4,5 CAR-T细胞更广泛地使用的障碍是实体肿瘤和相关毒性(包括细胞因子释放综合征(CRS)和神经毒性(NT)中的活性有限。3,5,6,7,8,9为了提高CAR-T细胞治疗的治疗指标,利用锌指核酸酶、TALEN和CRISPR等基因组工程工具进一步修改CAR-T细胞,以产生毒性较低或更有效的CAR-T细胞。10,11
在本文中,我们将介绍一种生成 CRISPR/Cas9 编辑的 CAR-T 单元格的方法。该方法的具体目标是通过CRISPR/Cas9在CAR-T细胞制造过程中对CAR-T细胞进行基因改造,以产生毒性较低或更有效的CAR-T细胞。开发该方法的原理基于从CAR-T细胞治疗的临床经验中吸取的经验教训,这表明迫切需要新的策略来增加CAR-T细胞治疗的治疗窗口,并将应用扩展到其他肿瘤,并支持合成生物学的最新进展,允许CAR-T细胞的多次修改,已经开始进入诊所。当几种基因组工程工具正在开发并应用于不同的环境,如锌指核酸酶、TALEN 和 CRISPR 时,我们的方法描述了 CRISPR/Cas9 对 CAR-T 细胞的修饰。10,11 CRISPR/Cas9 是一种基于RNA的细菌防御机制,旨在消除外来DNA。CRISPR依靠内切酶来切出通过导引RNA(gRNA)识别的目标序列。与其他基因组工程工具的CRISPR编辑CAR-T细胞具有若干优势。其中包括gRNA序列的精确性、针对感兴趣的基因设计gRNA的简单性、高基因编辑效率,以及同时使用多个gRNA系统靶向多个基因的能力。
具体来说,在本文描述的方法中,我们使用一种慢病毒编码CRISPR指南RNA和Cas9在CAR转导T细胞过程中破坏基因。在选择适当的技术来编辑CAR-T细胞时,我们建议这里描述的技术是一种产生研究级CAR-T细胞的有效机制,但由于Cas9永久集成到基因组的长期影响是未知的,我们提出这种方法,以开发概念证明研究级CAR-T细胞,但不用于生产良好的制造实践级CAR-T细胞。
特别是,在这里,我们描述了以人类CD19为目标的粒细胞巨噬细胞群落刺激因子(GM-CSF)敲除CAR-T细胞的产生。这些CAR-T细胞是通过转导与慢病毒颗粒编码的指南RNA特定于GM-CSF(基因名称CSF2)和Cas9产生的。我们之前发现,GM-CSF 中和改善 CRS 和 NT 在异种移植模型中.12个 GM-CSFk/o CAR-T 细胞允许在制造过程中抑制 GM-CSF,有效减少 GM-CSF 的生产,同时提高 CAR-T 细胞的抗肿瘤活性和在体内的存活率,与野生型 CAR-T 细胞相比。12因此,在这里,我们提供了一个生成 CRISPR/Cas9 编辑的 CAR-T 单元格的方法。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">CD3 Monoclonal Antibody (OKT3), PE, eBioscience</td>
			<td style="width:217px;">Invitrogen</td>
			<td style="width:123px;">12-0037-42</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD3 Monoclonal Antibody (UCHT1), APC, eBioscience</td>
			<td>Invitrogen</td>
			<td style="width:123px;">17-0038-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Choice Taq Blue Mastermix</td>
			<td>Denville Scientific</td>
			<td style="width:123px;">C775Y51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CTS (Cell Therapy Systems) Dynabeads CD3/CD28</td>
			<td>Gibco</td>
			<td style="width:123px;">40203D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">CytoFLEX System B4-R2-V2</td>
			<td style="width:217px;">Beckman Coulter</td>
			<td style="width:123px;">C10343</td>
			<td>flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">dimethyl sulfoxide</td>
			<td style="width:217px;">Millipore Sigma</td>
			<td style="width:123px;">D2650-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td style="width:217px;">Gibco</td>
			<td style="width:123px;">14190-144&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dynabeads MPC-S (Magnetic Particle Concentrator)</td>
			<td>Applied Biosystems</td>
			<td style="width:123px;">A13346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Easy 50 EasySep Magnet</td>
			<td>STEMCELL Technologies</td>
			<td style="width:123px;">18002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasySep Human T Cell Isolation Kit&#160;</td>
			<td>STEMCELL Technologies</td>
			<td style="width:123px;">17951</td>
			<td>negative selection magnetic beads; 17951RF includes tips and buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Fetal bovine serum</td>
			<td style="width:217px;">Millipore Sigma</td>
			<td style="width:123px;">F8067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC Mouse Anti-Human CD107a&#160;</td>
			<td>BD Pharmingen</td>
			<td style="width:123px;">555800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Fixation Medium (Medium A)</td>
			<td style="width:217px;">Invitrogen</td>
			<td style="width:123px;">GAS001S100</td>
			<td></td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:557px;">GenCRISPR gRNA Construct: Name: CSF2 
			CRISPR guide RNA 1; Species: Human, Vector: 
			pLentiCRISPR v2; Resistance: Ampicillin; Copy number: 
			High; Plasmid preparation: Standard delivery: 4 &#956;g (Free 
			of charge)</td>
			<td style="width:217px;">GenScript</td>
			<td style="width:123px;">N/A</td>
			<td>custom order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td style="width:123px;">A-21235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">https://tide.nki.nl.</td>
			<td style="width:217px;">Desktop Genetics</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human AB Serum; Male Donors; type AB; US</td>
			<td>Corning</td>
			<td style="width:123px;">35-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IFN gamma Monoclonal Antibody (4S.B3), APC-eFluor 780, eBioscience</td>
			<td>Invitrogen</td>
			<td style="width:123px;">47-7319-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 3000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td style="width:123px;">L3000075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation</td>
			<td>Invitrogen</td>
			<td style="width:123px;">L34966</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lymphoprep</td>
			<td>STEMCELL Technologies</td>
			<td style="width:123px;">07851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monensin Solution, 1000X</td>
			<td>BioLegend</td>
			<td style="width:123px;">420701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human CD28 Clone CD28.2</td>
			<td>BD Pharmingen</td>
			<td style="width:123px;">559770</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human CD49d Clone 9F10</td>
			<td>BD Pharmingen</td>
			<td style="width:123px;">561892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Human MIP-1&#946; PE-Cy7</td>
			<td>BD Pharmingen</td>
			<td style="width:123px;">560687</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Mr. Frosty Freezing Container</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:123px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NALM6, clone G5&#160;</td>
			<td>ATCC</td>
			<td style="width:123px;">CRL-3273</td>
			<td>acute lymphoblastic leukemia cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease Free Water</td>
			<td>Promega</td>
			<td style="width:123px;">P119C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.22uM, sterile</td>
			<td>Genesee Scientific</td>
			<td style="width:123px;">25-227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.45uM, sterile</td>
			<td>Genesee Scientific</td>
			<td style="width:123px;">25-228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Opti-MEM I Reduced-Serum Medium (1X), Liquid</td>
			<td style="width:217px;">Gibco</td>
			<td style="width:123px;">31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-CF594 Mouse Anti-Human IL-2</td>
			<td>BD Horizon</td>
			<td style="width:123px;">562384</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin-Glutamine (100X), Liquid</td>
			<td>Gibco</td>
			<td style="width:123px;">10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Permeabilization Medium (Medium B)</td>
			<td style="width:217px;">Invitrogen</td>
			<td style="width:123px;">GAS002S100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureLink Genomic DNA Mini Kit</td>
			<td>Invitrogen</td>
			<td style="width:123px;">K182001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin Dihydrochloride</td>
			<td>MP Biomedicals, Inc.</td>
			<td style="width:123px;">0210055210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td style="width:123px;">28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat Anti-Human GM-CSF BV421</td>
			<td>BD Horizon</td>
			<td style="width:123px;">562930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RoboSep-S</td>
			<td style="width:217px;">STEMCELL Technologies</td>
			<td style="width:123px;">21000</td>
			<td>Fully Automated Cell Separator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SepMate-50 (IVD)</td>
			<td>STEMCELL Technologies</td>
			<td style="width:123px;">85450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Sodium Azide, 5% (w/v)</td>
			<td style="width:217px;">Ricca Chemical</td>
			<td style="width:123px;">7144.8-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-VIVO 15 Serum-free Hematopoietic Cell Medium</td>
			<td>Lonza</td>
			<td style="width:123px;">04-418Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

using crispr/cas9 to knock out gm-csf in car-t cells

奇美抗原受体T(CAR-T)细胞治疗是癌症的前沿和潜在的革命性新治疗选择。然而,在癌症治疗中广泛使用它存在重大限制。这些限制包括发展独特的毒性,如细胞因子释放综合征 (CRS) 和神经毒性 (NT) 和有限的扩张、 效应器功能和抗肿瘤活性在实体肿瘤.提高CAR-T效能和/或控制CAR-T细胞毒性的一个策略是在CAR-T细胞制造过程中编辑CAR-T细胞本身的基因组。在这里,我们描述了CRISPR/Cas9基因编辑在CAR-T细胞中通过转导与含有引导RNA的慢病毒结构,以颗粒细胞巨噬细胞菌群刺激因子(GM-CSF)和Cas9。例如,我们描述了CRISPR/Cas9对GM-CSF的介导淘汰。已经证明,与野生型CAR-T细胞相比,这些GM-CSFk/o CAR-T细胞在维持关键T细胞功能的同时,有效产生较少的GM-CSF,并增强体内抗肿瘤活性。

图1显示了GM-CSFk/o CART19单元中GM-CSF的减少。为了验证T细胞的基因组被改变为敲除GM-CSF,TIDE测序用于GM-CSFk/o CART19细胞(图1A)。CAR-T细胞表面染色验证T细胞是否通过在活CD3+细胞上浇注在体外成功表达CAR表面受体(图1B)。通过流动细胞测定对GM-CSF的细胞内染色,通过对活CD3+细胞的注控,证明GM-CSF在GM-...

Watch this Scientific Journal Video about Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells at JoVE.com

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

在这里,我们提出一个协议,通过CRISPR/Cas9系统对CAR-T细胞进行基因编辑。

使用 CRISPR/Cas9 在 CAR-T 电池中敲除 GM-CSF

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

我们的协议描述了 CAR-T细胞在通过CRISPR技术制造过程中的基因改造，以产生更有效、毒性更小的产品。该技术的优点是，CAR-T细胞的产生和基因操作都可以在一个细胞中发生，效率高。展示这个程序的将是罗莎莉·斯特纳，一个医学博士，米歇尔·考克斯，一个技术专家和研究生，和雷奥娜·萨克穆拉，一个博士后研究员，从我的实验室。将T细胞从收获的外周血单核细胞中隔离后，首先制备T细胞介质，然后通过0.45微米无菌真空过滤器过滤，然后用0.22微米无菌真空过滤器进行消毒。从采集的外周血单核细胞开始培养T细胞。在T细胞刺激日或零日，在刺激前，将所需的珠子放入无菌1.5毫升微离心管中，并在一毫升的中药中重新保存，清洗CD3/CD28珠子。将管子与磁铁接触一分钟。然后吸进中医，再用一毫升新鲜中医重新洗珠子，再重复三次洗涤。最后，在一毫升的中医中重新暂停珠子。计算T细胞后，以三比一珠与细胞的比例将珠子转移到T细胞。然后稀释细胞，最终浓度为每毫升100万个细胞，并在37摄氏度、5%CO2下孵育24小时。在扁病毒载体中获取 CART19 结构后，首先在室温下孵育扁病毒质粒、包装载体、包络载体、预复杂试剂、转染试剂和转染介质进行 30 分钟的转染培养。使用 BSL2® 预防措施进行扁病毒工作，包括细胞培养罩、个人防护设备以及处理前用漂白剂对用过的材料进行消毒。孵育后，将这些转染试剂加入293个T细胞中，这些T细胞达到70至90%的汇合率，并在37摄氏度和5%CO2下培养转染细胞。在转染后24小时和48小时，收获、离心机、过滤和浓缩含有病毒的上流物质，通过超离心和冷冻在零下80摄氏度，供将来使用。在第一天，轻轻地重新暂停T细胞，在零日以每毫升100万细胞被刺激，以分解细胞的玫瑰花环。在适当的 BSL2® 预防措施下，在 3.0 的多重感染下，将新鲜或冷冻收获的病毒添加到模拟 T 细胞中。继续在37摄氏度，5%的CO2下孵育转导T细胞。第三天和第五天，计算 CAR-T 细胞，并将新鲜预热的 TCM 添加到培养物中，以保持 CAR-T 细胞浓度为每毫升一百万个细胞。模拟六天后，通过首先收获和重新暂停细胞，从转导的T细胞中去除珠子。将细胞在50毫升锥形管中转移，将管子放在磁铁中一分钟。在收集含有 CAR-T 细胞的上经剂并丢弃珠子后，将细胞以每毫升一百万个细胞的浓度放回培养瓶中。使用流式细胞学的细胞样本来评估 CAR 的表面表达式。孵育其余的恢复扩张，然后收获和冷冻保存细胞在第8天，如手稿中所述。要破坏粒细胞巨噬细胞群刺激因子，请使用手稿中描述的引导RNA。在室温下孵育转染试剂30分钟。孵育后，将它们添加到达到70-90%汇合的293个T细胞中，并在37摄氏度、5%CO2下培养转染细胞，产生扁病毒。在24和48小时，收获和浓缩含有病毒的上流水，通过超离心在50毫升超离心管和冻结在零下80摄氏度供将来使用。然后第一天，轻轻地重新暂停T细胞，以打破玫瑰花环。在 BSL2+ 批准的实验室中，为了生成 CAR-T 细胞，将 CAR19 扁家病毒和 GMCSF 靶向CRISPR扁病毒添加到模拟T细胞中。在第三天和第五天成功转导含有紫霉素抗药性克伦霉素的百联化T细胞，以每毫升1微克的紫丙胺素浓度治疗具有紫霉素的细胞。潮汐测序用于确认GM-CSF敲除C CART19细胞的GM-CSF减少，在活CD3阳性细胞上封闭的 CAR-T细胞表面染色的中断效率约为71%，表明野生型C前19和GM-CSF敲除C CART19均成功，以类似方式在体外表达 CAR表面受体。另一方面，通过流式细胞学对GM-CSF的细胞内染色也封闭在活的CD3阳性细胞上，表明与野生型C前19细胞确认敲解功能成功的野生T前19细胞相比，GM-CSF在敲解细胞中的表达减少。在这个程序的转导步骤中应特别注意，因为它们对转基因 CAR-T 细胞的发展至关重要。所述方法可能应用于各种基因，通过CRISPR/CAS9修改 CAR-T 细胞，以帮助生成毒性更低、更有效的产品。CRISPR/CAS9 技术提供了直接针对 CAR-T 细胞基因组的策略，以设计解决当前临床缺陷的解决方案。

Research

JoVE Journal

Cancer Research

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

寻找获得性抗靶治疗的驱动途径: 耐药亚克隆生成与基因击倒敏感性恢复

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

科研

癌症研究

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

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

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 ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

基于 crispr-cas9 的竞争检测对遗传依赖性的调查

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

A Spheroid Killing Assay by CAR T Cells

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

制造用于采用免疫疗法的奇美抗原受体（CAR）T细胞

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

使用单链DNA免疫荧光量化卵巢癌细胞中的复制应激

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

颅内插管植入小鼠连续局部区域嵌合抗原受体（CAR）T细胞输注

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...