The authors would like to acknowledge the "Ligue Nationale contre le Cancer" (Comit&#233; du Septentrion), the SIRIC ONCOLille (Grant INCa-DGOS-INSERM 6041) and the Institut pour la Recherche sur le Cancer de Lille (IRCL) for supporting this work. They would like to thank Delphine Taillieu and the animal facility staff for housing the mice and maintaining their welfare. We also thank Rapha&#235;lle Caillerez and Nathalie Jouy for their respective help in microscopy and flow cytometry.

动物住房和所有的实验过程由当地动物保护伦理委员会批准，CEEA.NPDC（协议no.512012），并根据实验动物的护理和使用的法国和欧洲的准则进行所有的实验。 1. 在 C1498细胞系的体外表征<ol><li> 在 C1498细胞的体外培养<ol><li>制备完全RPMI（罗斯韦尔园区纪念研究所）加入50毫升胎牛血清（FBS），5 ml青霉素1640培养基（100单位/毫升）-streptomycin（100微克/毫升）中，加入500μl的50mMβ巯基乙醇的，5毫升N-2-羟乙基-N-2-乙烷磺酸（HEPES），5毫升的非必需氨基酸和5毫升丙酮酸钠至500ml RPMI培养基。 </li><li>成长C1498细胞系完全RPMI。收获在悬浮液中的细胞，通过移液，将细胞转移到50毫升管中。离心机350 XG 10分钟，然后取出superna重要的。 </li><li>在350 XG加入20毫升磷酸盐缓冲盐水（PBS）（1×）溶液，离心10分钟，并除去上清液。 </li><li>悬浮细胞在10ml荧光激活细胞分选（FACS）缓冲液（2.5克牛血清白蛋白（BSA）的粉末和2毫升在500毫升PBS溶液的0.5M乙二胺四乙酸（EDTA）溶液）。计数使用托马细胞与台盼蓝染色细胞计数后室中的细胞。 </li></ol></li><li>在C1498细胞的表型鉴定采用免疫组化和流式细胞仪分析<ol><li>细胞表面染色<ol><li>准备流式细胞仪缓冲。 </li><li>调整在FACS缓冲液中收获的细胞以10 7个细胞/ ml和分配10 6个细胞（在100μl）对每个染色实验入流式细胞管中。 </li><li>标记用100μlFACS缓冲液稀释下列抗体或它们的相关同种型对照的细胞: <ol><li>对于M前体细胞和分化细胞的arkers，标号与抗CD11b /抗CD18（1），抗-LY-6G（1），抗CD19，抗B220（2），抗NK1.1，抗细胞CD49b，抗CD4（1），抗CD8（2），抗CD3（3），抗CD21 / 35，抗CD115，抗TCRVβ抗体。 </li><li>造血干/祖细胞标记物，使用抗CD34的结合/抗CD117 /抗的Sca-1，抗CD150 /抗CD117 /抗的Sca-1，抗CD117 /抗CD127或抗独自CD16 / 32 - 生物素的抗体。 </li><li>对的细胞功能（ 例如 ，粘附，抗原呈递，共刺激分子和受体）标记物，细胞染色用抗CD18（2）/抗CD11a，抗MHC I类，抗MHC II类，抗CD31，抗CD44，抗CD80-生物素，抗CD86和抗CD274抗体。 </li></ol></li><li>孵育所有流式细胞管中流动的，在4℃下30分钟。 </li><li>洗细胞通过加入2ml FACS缓冲液中向每个管中，离心分离，在350×g离心5分钟，两次，并去除上清。 </LI&gt; <li>加入100微升的FAC缓冲液中，以每管并通过（在FACS缓冲液1/100 1/200最终稀释度）加入100微升荧光链霉亲到生物素化的缀合的抗体进行二次染色。孵育管在4℃下30分钟。 </li><li>洗细胞两次如下:在350 xg离心加2ml FACS缓冲液中向每个管中，离心管5分钟，并通过移液除去上清液。 </li><li>重悬500微升冷PBS的细胞置于冰上的细胞，用铝箔覆盖管保持他们在黑暗中。分析使用流式细胞仪10个结果。 </li></ol></li><li>细胞内染色<ol><li>通过加入125毫升4％多聚甲醛（PFA）溶液至375毫升PBS溶液制备的固定缓冲液中。 注意:要在通风橱制成500毫升之4％PFA的，热400毫升PBS溶液至约60℃的搅拌板上。添加20克PFA粉末，并提高pH值直到PFA溶解。允许溶液冷却，调节pH至6.9，并补足体积至500毫升PBS中。 </li><li>通过加入0.5克皂苷和0.5g的BSA至500ml的PBS溶液制备透缓冲器。 </li><li>调整在FACS缓冲液中收获的细胞以10 7个细胞/ ml和分发10 6个细胞（100微升）为每个染色实验入流式细胞管中。离心细胞在350 XG 5分钟，去除上清。 </li><li>固定细胞在200μl的1％PFA溶液中并在4℃孵育10分钟。 </li><li>在350 XG2毫升透缓冲液添加至每个管，离心管5分钟，并通过移液除去上清液。添加100μl的透化缓冲液至每个管。 </li><li>标记用100μl下列抗体或它们相应的同种型对照的细胞透化缓冲液稀释它们后:抗CD3（2）/抗CD8（1），抗CD3（3）/一NTI-CD4（2），抗CD107b和抗CD3（3）/抗TCRVβ。 </li><li>孵育细胞在4℃下进行30分钟。 </li><li>通过加入2ml透化缓冲到每个管的两次洗细胞。离心管350 XG 5分钟，去除上清。 </li><li>添加100μl的透缓冲器向细胞。通过加入100微升荧光链霉在透化缓冲液至生物素标记的抗体稀释进行到二次染色。孵育管在4℃下30分钟。 </li><li>加2ml透化缓冲各管，在350 XG 5分钟离心管，去除上清。重复此步骤一次。 </li><li>重悬500微升冷PBS的细胞，然后将细胞在黑暗中的冰。分析用流式细胞仪10的流程的结果。 </li></ol></li></ol></li><li>细胞悬浮液在载玻片上为显微镜制备<ol><li>医管局洗10 6rvested C1498细胞（在步骤1.1.4获得）用5毫升冷的FAC缓冲液两次，稀释细胞在1ml冷的FACS缓冲液中。放置管在冰上。 </li><li>将滑入一次性室预过滤器附加卡和将这些成cytocentrifuge。 </li><li>加入100微升的FAC缓冲液中向每个腔室和过滤卡，并在4.52×g下旋转他们2分钟。 </li><li>加入100微升细胞对各室和过滤卡，和自旋细胞在4.52 xg离心2分钟。 </li><li>小心地从腔室取出幻灯片和髓过氧化物酶（步骤1.4），酯酶（步骤1.5）或月-Grünwald的吉姆萨（步骤1.6）染色之前风干的幻灯片。 </li></ol></li><li>髓过氧化物酶染色免疫<ol><li>通过在冷的甲醇中浸渍载玻片固定在载玻片细胞:丙酮（1:1）溶液，2分钟，然后空气干燥的幻灯片。 </li><li>到液体限制到含有细胞的滑动的面积，画一个词rcle周围使用防水笔细胞。 </li><li>冲洗在200μl冷PBS溶液的细胞10分钟。 </li><li>阻断细胞在200μl含有正常驴血清的10微升和10微克/毫升的纯化的抗CD16 / 32的抗体的3％BSA / PBS缓冲液中。 </li><li>应用200微升抗小鼠髓过氧化物酶（在3％BSA / PBS缓冲液稀释至20微克/毫升）的。孵育细胞O / N，在4℃的湿度室中。 </li><li>清洗细胞用200μl冷的0.1％BSA / PBS中。 </li><li>应用200微升在3％BSA / PBS缓冲液1/250稀释的抗山羊IgG抗体。孵育所述细胞在室温下在潮湿箱2小时。 </li><li>清洗细胞3次，用200μl0.1％BSA / PBS缓冲液中并两次冷PBS。 </li><li>用200μl的Hoechst的（在1微克/毫升的最终浓度）在室温2分钟在PBS中的1/1000稀释染色细胞核。 </li><li>用清水洗净，幻灯片和让他们mountin前风干G。应用安装介质1的小区中的一个下降，放置一个盖玻璃的一个边缘到幻灯片，并小心地将其放下到使用镊子将细胞。轻轻按压外盖玻璃，以消除任何气泡。 </li></ol></li><li>酯酶细胞化学 注:预暖的所有试剂室温。 <ol><li>固色剂的制备<ol><li>制备柠檬酸盐丙酮 - 甲醛（CAF）溶液中，添加2.5毫升柠檬酸盐溶液，6.5毫升丙酮和0.8ml 37％的甲醛至玻璃瓶中。轻轻混匀并在4℃下保存。 </li></ol></li><li>色酚AS-D氯乙酸酯酶（CAE）活性测定<ol><li>温的去离子水至37℃。 </li><li>在50ml管中，加入1毫升亚硝酸钠溶液到1ml的染料溶液。轻轻混匀，静置2分钟。加40毫升预热的去离子水，5毫升的pH 6.3缓冲液浓缩物和1的萘酚AS-D氯乙酸溶液。混合并转移到一个罐子科普林。 </li><li>固定细胞到幻灯片（见1.3节），用于与CAF溶液（见步骤1.5.1.1）30秒，并洗玻片45秒，用去离子水。 </li><li>转移滑入这是在步骤1.5.2.2中制备的溶液，并在37°C孵育载玻片在避光湿度室30分钟。 </li><li>干燥载玻片，然后通过浸入冲洗他们在去离子水中2分钟。 </li><li>通过加入苏木溶液几滴孵育他们1分钟染液细胞。 </li><li>与中性水（pH 7）洗涤载玻片并允许风干。应用安装介质2向小区中的一个下降，放置一个盖玻璃的一个边缘到滑动并小心地将其放下到使用镊子将细胞。轻轻按压外盖玻璃，以消除任何气泡。 </li></ol></li><li>阿尔法萘丁酯酶（NBE）活性测定<ol><li>使用前暖α萘丁解到37℃。 </li><li> NIT稀释钠一片RITE 6.25毫升去离子水。 </li><li>在50ml管中，加入为1.5ml亚硝酸钠片剂溶液和1.5毫升副玫瑰苯胺溶液。轻轻混合，并允许溶液静置5分钟。补充用40ml磷酸盐缓冲溶液中的溶液。通过仔细加入10 N滴加氢氧化钠带给pH值为6。加入5毫升α萘丁解决方案，混合整个解决方案，并将其转移到一个罐子科普林。 </li><li>用在RT下CAF溶液固定细胞在载片上，持续10秒，冲洗45秒，用去离子水。 </li><li>转印滑入包含在步骤1.5.3.2制备并一起1小时，在37℃的湿度室中孵育，同时避光溶液中的科普林缸。 </li><li>冲洗的幻灯片在中性水中2分钟（pH值为7）和空气干燥。 </li><li>染液与亚甲基蓝溶液中的细胞在幻灯片上加入几滴孵育4分钟。 </li><li>浸入deio幻灯片的发布水2分钟并允许风干。挂载幻灯片，应用安装介质2向小区中的一个下降，放置在盖玻璃的一个边缘到幻灯片，并小心地将其放下到使用镊子将细胞。轻轻按压外盖玻璃，以消除任何气泡。 </li></ol></li></ol></li><li>五月 - 格伦沃尔德姬姆萨（MGG）染色<ol><li>通过幻灯片浸入含五月 - 格伦沃尔德溶液中3分钟的科普林氏缸染色细胞（以1.3节准备）。 </li><li>转移的幻灯片到含有1分钟的pH 6.8的缓冲溶液中的科普林缸。 </li><li>将它们放置在含有10分钟的吉姆萨R染料溶液（在pH 6.8的缓冲液稀释到1/20）一个科普林缸染色的幻灯片。洗与中性水（pH 7）的幻灯片，持续10秒。 </li><li>漏和风干的幻灯片。通过施加固定介质2到细胞上的一滴装入滑动。将盖板玻璃的一个边缘到幻灯片，并小心地放下它到细胞中使用镊子。轻轻按压外盖玻璃，以消除任何气泡。 </li></ol></li></ol> 2. 在体内发育和急性白血病的表征注:4周大的雌性同类系的C57BL / 6J-Ly5.1小鼠无特定病原体的条件下维持（ 即，在无菌环境中）。当他们周5和6岁之间的小鼠注射。 <ol><li>静脉注射C1498细胞<ol><li>吹打收获悬浮培养细胞C1498。转移细胞到一个50毫升管中并离心，在350×g离心10分钟。清洗细胞在10ml冷PBS两次，并准备在PBS中的10 7个细胞/ ml的细胞悬浮液。广场上冰的细胞悬浮液进行注射前。 </li><li>鼠标放置在一个限制器和一个层流罩进行无菌条件下注入。 </li><li>使用29G针，用注射器注入细胞进入尾静脉。把握tail在远端，并用70％的乙醇中浸泡过的纱布海绵消毒它。检查，以确保有在注射器没有气泡，然后缓缓注入100微升C1498细胞悬浮液（10 6个细胞）至尾静脉。 </li><li>注射后，从尾部取出针，并通过施加压力以在注射部位中的无菌纱布海绵控制任何出血。返回动物笼子里，并仔细检查其健康在接下来的几个小时和几天。 </li></ol></li><li>眼眶采血<ol><li>监视PBS-和C1498-注射的小鼠的为白血病病（ 例如 ，立毛，从组隔离，并减少或在笼不运动）的迹象的行为。 注意:这通常发生17至19天之间的细胞注射后。 </li><li>只是安乐死之前执行眼眶采血（见步骤2.2.7）在无菌条件下在层流引擎盖下加热灯，以防止体温过低。 </li><li>麻醉，用氯胺酮150 mg / kg和赛拉嗪在10毫克/公斤。通过稀释1.5毫升氯胺酮和0.5ml甲苯噻嗪的18毫升PBS溶液制备麻醉剂溶液。 </li><li>麻醉控制和白血病的小鼠。用200微升每10g使用26G针小鼠的麻醉剂溶液的腹膜内注射和1ml注射器进行。检查踏板反射消失，确认麻醉。 </li><li>插入毛细管进入眼睛的内眦。血液将上升从眼窝窦入毛细管。通过轻轻地用无菌纱布海绵施加压力到眼睛控制出血。 注:100〜200微升血液的体积可以使用这种技术来收集。 </li><li>收集血液到EDTA管中，并分离单核细胞前在冰上样品存储。 </li><li>使用颈椎脱位安乐死鼠标和进行隔离器官（第2.3节）。 </li></ol></li><li>器官和细胞分离<ol><li>机关隔离<ol><li>放置安乐死鼠标到其上的塑料板回用针引脚动物的脚，以促进器官隔离。消毒用70％的乙醇进行的切口之前鼠标。 </li><li>使用无菌剪刀，从腹部皮肤到颈部执行腹侧切开。切断通过腹壁来访问肝脏。通过胸廓和访问肺部隔膜切。移动至肠的侧面和除去使用无菌剪刀和镊子脾脏。 </li><li>以分离骨髓，切腿在使用无菌剪刀接头上方的股骨的顶部。通过轻轻拉动断开股骨胫骨，然后取出使用镊子和剪刀骨头的皮肤和肌肉。 </li><li>放置在含有冷PBS 50ml管各器官和骨骼，并将它们放置在冰上。 </li></ol></li><li>从器官细胞的分离<ol><li>破坏细胞前称量脾脏。通过在一个50ml管中使用的针筒柱塞按压它们通过一个70微米的过滤器机械地扰乱脾，肺和肝脏，并收集细胞为30个毫升冷PBS中。 </li><li>收集的骨髓细胞，将股骨和胫骨在培养皿中在冰上，使用无菌剪刀剪四肢，并通过插入附连到含有5毫升冷的PBS 10ml的注射器26G针冲出骨髓。 <ol><li>通过将细胞悬浮液通过针/注射器破坏骨髓细胞，并通过一个70微米的过滤器，在50毫升管过滤细胞悬浮液。 </li></ol></li><li>离心机所有含每种器官和骨髓细胞在350×g离心10分钟的管子。弃去上清液，并重新悬浮在2毫升的裂解缓冲液（1x）和细胞从肺和骨髓收集细胞轻轻上下吹打该混合物从肝脏和脾脏s隔离在5ml裂解缓冲液（1×）的。管填充50毫升冷PBS。 </li><li>离心细胞，在350×g离心10分钟。重悬在FACS缓冲液将细胞进行流式细胞术分析或制备细胞显微术。算使用托马细胞计数室中的细胞用台盼蓝染色它们之后。 </li></ol></li></ol></li><li>从机关分离出来流式细胞仪分析细胞的细胞表面染色<ol><li>在流式细胞术管的流动，标签从器官中分离用10μg/ ml，在100微升的FAC缓冲液中纯化的抗CD16 / 32抗体的有10 6个细胞。 </li><li>至10 6个骨髓细胞，添加在FACS缓冲液稀释抗体的下列抗体或组合以及它们相应的同种型对照的100微升:抗CD11b /抗CD3（1）/抗的Ly6C /抗Ly6G（2），抗B220（1）/anti-CD45.2/anti-CD19，抗CD115 /抗CD3（1）/一个TI-的Ly6C /抗Ly6G（2），抗CD45.2，抗Ly6G（2），抗CD11b，抗CD115或单独为补偿设定抗CD19。 </li><li>到的脾细胞中，添加以下抗体的100μl的和其相应的同种型对照在FACS缓冲液稀释:（1抗CD11b /抗CD3（1）/抗的Ly6C /抗Ly6G（2），抗B220的组合）/anti-CD45.2/anti-CD19，抗CD45.2，抗Ly6G（2），抗CD11b或抗CD19补偿设置。 </li><li>肺和肝细胞，添加抗CD45.2抗体的100微升，并在FACS缓冲液1/100稀释其相应的同型对照。 </li><li>孵育所有30分钟的细胞溶液在4℃下。 </li><li>通过加入2ml FACS缓冲液中向每个管洗涤细胞。离心管在350 xg离心5分钟，并通过移液弃去上清。重复此步骤一次。 </li><li>重悬标记细胞在500微升冷PBS。置于冰上的电池和交流电进行术前流避光quisition和分析10。 </li></ol></li><li>外周血单个核细胞和免疫荧光染色的隔离流式细胞分析<ol><li>启动协议之前，预温分离溶液至室温。 </li><li>转移血样（100至200微升来自步骤2.2获得）到微量离心管中，并加入PBS / 1mM EDTA的溶液，直至溶液体积为500微升。仔细层500微升含有使用30G针和1ml注射器血液中的溶液下分离溶液。不要混合血液和分离解决方案。 </li><li>在800 XG（无制动器），在室温20 min离心管。离心后，收集用吸管蜂窝环（不透明的白色层）。转移细胞到一个离心管中。 注:不透明白色层含有淋巴细胞以及单核细胞和下层之间出现 - 分离溶液 - 和上层。 </li><li>加入1ml的PBS溶液，并离心管中，在350×g离心10分钟。悬浮细胞在600微升FACS缓冲液中。 </li><li>添加10μg/ ml的纯化的抗CD16 / 32的抗体与100微升的细胞悬液分配到六个独立的管（每100微升）。 </li><li>标记的细胞用100μlFACS缓冲液稀释下列抗体或它们的相关同种型对照的:抗CD3（1）/抗B220（1）/anti-CD45.2和抗的Ly6C /抗CD115的结合/anti-CD45.2或抗CD45.2，独自补偿设置抗CD115。 </li><li>孵育所有试管在4℃下30分钟。 </li><li>通过加入2ml FACS缓冲液中向每个管洗涤细胞，然后在350 xg离心离心管10分钟，并使用移液管弃上清。 </li><li>重悬标记细胞在500微升冷PBS。置于冰上的细胞和流式细胞仪采集和分析10执行流之前，避光。 </li></ol></li><li>骨髓细胞悬液在玻片上显微镜准备<ol><li>按照1.3节所述的步骤，但在步骤1.3.1，使用10 5骨髓细胞，在步骤1.3.4，旋细胞到每个室在72.26 XG 10分钟。 </li></ol></li><li>利用骨髓细胞酯酶活性测定<ol><li>要进行骨髓细胞化学酯酶试验，从出发步骤1.5至1.5.3.7。 </li></ol></li><li>骨髓细胞的五月-Grünwald的吉姆萨染色<ol><li>染色骨髓细胞，按照1.6节所述的协议，但在步骤1.6.1，孵化在5 - 格伦瓦尔德解决方案的幻灯片5分钟。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

在以前的研究中，C1498细胞系被描述为急性粒5，6粒单核细胞或NKT 7细胞白血病的诱导。然而，在文献中示范的数据是不存在或不完整的。这里介绍的协议使用不同的技术，如流式细胞术，免疫荧光，MGG染色和细胞化学测定法，以表征培养的C1498细胞，并确定在小鼠中诱导它们被注射后的白血病的性质。 当我们在体外培养的C1498细胞表型免疫染色后和流式细胞术进行分析，我们观察到一些限制，因为这些细胞中表达已在文献6,7之前已经描述几个细胞表面造血标记。与我们的结果一致，拉贝勒等人没有使用流动cytomet观察C1498细胞成熟TCR的细胞表面表达RY染色。然而，他们认为他们是NKT细胞系，他们发现CD3ε和TCRVβ8.2的mRNA 7之后。我们还观察到TCRVβ链和CD3ε分子的细胞内表达在大多数细胞（&gt; 70％），但无法确定他们的造血谱系因为也有在Mac-3分子的伴随的细胞内表达。 髓过氧化物酶，MGG染色和评估分析使用细胞化学功能酯酶表明，C1498细胞系具有髓系起源和组成monoblasts和原粒细胞。这些结果是一致的，在流量获得染色流式细胞仪分析认为Mac-3 +细胞的百分比。虽然不是定量的，这些步骤代表键实验中进行。事实上，它们仍然存在，到目前为止，用于表征表达没有或f造血细胞的谱系和分化阶段最好现有的方法EW特定表型标记。 流式细胞仪染色用于演示急性白血病的小鼠同类后发展C1498细胞静脉注射有帮助的。的CD45.2 + C1498细胞渗入外周血和各种器官进行分离，并测定它们的频率。还进行免疫定量分析后，分析内在髓质和脾细胞。当器官被检查的C1498细胞表型，因为它们表达了几个造血标记中遇到的限制（只有少数为B220 +）。来定义观察急性白血病的性质，可能 - Grünwald的Giemsa染色和单核细胞的活动进行分析并使用骨髓进行粒酯酶。结果表明，C1498细胞保留其髓和单核形态和功能，揭示单核细胞白血病的发病。 <p clasS ="jove_content"&gt;在考虑在本协议中所描述的关键步骤，应特别注意进行细胞化学反应和MGG染色时，由于pH值的错误可能会导致结果不正确的解释给予pH值。例如，α萘丁酸酯酶活性只在pH 6.0是特定于单核细胞，因为粒细胞和淋巴细胞也可以染色阳性此测试在较高的pH值。凝视细胞进行染色MGG之前不建议的，我们发现只有CAF固定使用C1498细胞进行酯酶当细胞化学反应提供了令人满意的结果。为了保持CD115分子的表达，并通过流式细胞其检测仪中，所有的样品（ 例如 ，血液，骨髓和脾细胞）应在手术过程中保持在冰上。如果没有染色流式细胞仪或/和免疫荧光实验，抗体的参考，其存储重新观察表彰和其稀释液应进行检查。在材料/设备表中指定的引用已经被选择用于流式细胞术或免疫荧光的应用程序。初级/次级抗体或它们的共轭荧光团可能已经失去了他们的活动，由于不恰当的存储（ 例如 ，暴露于光或热），稀释不当，大量的冷冻/解冻或使用受污染的缓冲区。运行阳性对照，以确保它们正常工作。使用小鼠骨髓或已知为表达目的蛋白脾来源的细胞。为了避免高的背景和非特异性染色，确保将细胞洗净，并保持在高湿度（对于免疫荧光），并且作为指示抗体稀释。使用相同的浓缩和稀释的同种型对照抗体和第一抗体精确地确定样品中的背景水平。酯酶细胞化学实验，试剂可通过使用含有纯化的小鼠脾粒（Ly6G +）和单核细胞（CD115 +）细胞阳性和阴性对照载玻片进行测试。 在这项研究中所描述的过程表明，许多的在小鼠中观察到C1498细胞注射后的白血病功能共享与人急性单核细胞白血病11,12共同特点。侵入白血病细胞导致减少成熟和未成熟（祖细胞和前体）骨髓造血细胞。 C1498细胞存在于外周血中的高频率（&gt; 20％），因为是单核细胞。观察肝脾肿大从白血病细胞的浸润导致，并在B淋巴细胞和骨髓细胞显著上升也观察陪脾肿大。当血液中的血小板数均使用血液分析仪血小板减少，估计也观察到。 这是秀N， 使用体外实验，即C1498细胞通过分泌可溶性因子13抑制正常小鼠造血功能。在几个肿瘤小鼠模型中，未成熟的骨髓细胞（包括单核细胞和粒细胞）也已经显示，从骨髓迁移到脾，在那里它们抑制抗肿瘤特异性T细胞活化和增殖14。因而，在骨髓中观察到，在造血细胞中的减少可能导致从任造血和/或从他们的移民缺乏。这后一种机理可以解释单核细胞增多的外周血中存在或放大的骨髓级分在脾脏的观察。还可以想到这些细胞可能已被从改进的脾造血而得。事实上，稳态条件下，脾B细胞的部分子集被确定为成熟的B淋巴细胞前体15。此外，炎症的条件下，配有ullary干细胞和祖细胞已被证明搬迁到脾诱导产生成熟单核细胞16。这个协议不允许我们得出关于所涉及的白血病的发展，和附加的功能以及分子检测应采用这样做机制结论。然而，这些数据包括有关急性单核细胞白血病的临床特点的详细信息，并帮助研究人员评估和了解新的治疗药物的影响。

急性髓性白血病（AML）的特征在于被阻止在成熟的不同阶段的造血骨髓细胞的不受控制的增殖。这种失调可影响粒，单核细胞，红细胞或megaryocytic分化途径1。 AML细胞积聚在骨髓，导致受损的造血，这导致血小板减少，淋巴细胞和贫血。白血病细胞也侵入血液和非淋巴器官。 的C1498小鼠模型已使用了几十年作为急性白血病，因为癌细胞从白血病10月龄的C57BL / 6（H-2 B）雌性小鼠在1941年分离出一个模型的文献描述了侵入血液，造血器官（ 例如 ，脾和淋巴结）和非造血器官（ 例如 ，肝，肺，卵巢，和肾）由高度增殖C1498细胞后，他们通过在注入travenous，皮下或腹膜内途径进入易感小鼠2-4。然而，这种小鼠模型报道诱发要么粒2,5或6粒单核细胞白血病。最近，发表在2002年的一项研究说明这种类型的癌症如鼠NKT细胞白血病7。因此，该文献的不同对本C1498细胞系的性质和它在小鼠中诱导的相关性白血病。这些差异主要是由于缺乏详细的和有关的细胞更新的公布的信息，并在一般的白血病的疾病，因为许多研究是在1950年进行的 - 70年代。 在这里，我们提供了一个详细的协议来描述如何表征C1498细胞，并分析该被诱导可以通过静脉注射到小鼠白血病病的性质。这个协议的第一部分是专门为那些在体外培养的细胞C1498的描述。荧光抗针对表面和细胞内的造血标记的尸体被用于使用流式细胞仪，以确定其表型。髓过氧化物酶的存在下，用免疫荧光显微镜进行评估，其造血谱系和分化阶段用细胞化学以评估酯酶的活性进行评价，并进行可能 - Grünwald的吉姆萨染色。然后C1498细胞注射到小鼠中，而且诱导的急性白血病病在本手稿的第二部分进行说明。相同的技术被用于确定频率和白血病和固有细胞在骨髓，外周血，脾和非造血器官（肝和肺）的表型。 此协议是高度可重复的，这里提供的数据将有助于科研人员评估新的治疗策略的影响。此白血病小鼠模型已被用于测试免疫接近ND不同肿瘤化疗药物8,9。其功效通过测定肿瘤负荷和存活率的演变进行评价。该协议可以被用来提供关于白血病和其它造血细胞群的治疗过程中的分布和生活的其他信息。

a detailed protocol for characterizing the murine c1498 cell line and its associated leukemia mouse model

已经执行了静脉注射C1498细胞进同源或同类系小鼠自1941年这些注射导致急性白血病的发展。然而，这种疾病的性质并没有得到很好的文献记载。这里，我们在体外表征C1498细胞和用于确定在体内诱发白血病的性质提供了技术的协议。这个过程的第一部分聚焦在确定所述造血谱系和培养C1498细胞分化的阶段。为了实现这一点，多参数流式细胞染色用于检测造血细胞标记。免疫荧光显微镜，细胞化学和五月-Grünwald的吉姆萨染色，然后进行评估髓过氧化物酶的表达，酯酶和细胞形态的活性，分别。该协议的第二部分专门描述了在诱导白血病病体内。后者可通过测定血液中的白血病和固有细胞的频率来实现的，使用特定的染色和流式细胞术造血器官（ 例如 ，骨髓和脾）和非淋巴组织（ 例如 ，肝，肺）分析。白血病的性质然后使用月-Grünwald的吉姆萨染色和染色为在骨髓中具体酯酶证实。这里，我们提出使用在年龄匹配C1498-和PBS注射的小鼠中这个协议得到的结果。

表征C1498小鼠模型中，我们进行了两个主要步骤。首先，将C1498细胞进行表征，以确定其体外造血谱系和成熟阶段（ 图1）。然后将这些细胞注射入同类小鼠和诱导白血病疾病的性质进行了评估，以确定不同的功能:白血病细胞浸润，它们的表型，所述造血细胞的骨髓一个量化（成熟和祖细胞/前体），该频率的C1498细胞和血液中的成熟的造血细胞和器官肿胀的评价（在脾，肝和肺）和细胞组合物。 为了表征体外 C1498细胞表型，细胞标记与针对分子的抗体是由造血前体和成熟细胞（ 表1）表示，并且使用流量cytomet结果进行分析RY。在C1498细胞呈阳性的Mac-1（CD11b的/ CD18）（〜7％），B220（&gt; 25％）的细胞表面表达，它们显示CD3ε，T细胞受体（TCR）Vβ链和Mac的胞内表达-3（ 图2A和B）。细胞为阴性的细胞表面标记物Ly6G，的Ly6C，CD115，CD21 / CD35，CD19，CD3，CD4，CD8，NK1.1，和泛NK细胞分子和对于CD4和CD8的胞内表达（数据未示出） 。然后，他们被检查的造血干细胞和祖细胞（ 表1）的标志物。他们也为阴性CD117，CD34，的Sca-1，CD150和CD16 / 32的细胞表面表达（数据未显示）。然后将这些白血病细胞进行试验，以确定粘附，抗原呈递和协同刺激分子的表达。细胞中表达的表面标记的LFA-1（CD11a / CD18），CD44，CD31（PECAM-1），和H-2D b和为阴性MHC II类，CD80，CD86和CD274（数据未显示）。 C1498细胞ŧherefore表示既髓（MAC-1，Mac的-3）和淋巴标记（B220，CD3，TCR）。 为了更好地表征自己的造血系统，髓过氧化物酶的表达，用免疫荧光显微镜进行评估。所有的细胞呈阳性的髓过氧化物酶，验证他们的骨髓来源（ 图3A）。大多数细胞还沾着积极为α萘丁酯酶（ 图3B，左图 ），其中一些染色的色酚AS-D氯乙酸酯酶（黑色箭头）（ 图3B，右图 ）。结果表明，该细胞含有单核细胞和粒细胞的混合物。进行五月-Grünwald的吉姆萨染色后，观察到C1498细胞具有高核质比，以显示一个爆炸样形态，在细胞核中3到5的核仁，核周卤素，大量液泡和嗜碱性细胞质（ 图3C ）。钍美国，C1498细胞系是由monoblasts和原粒细胞。 的C1498细胞（CD45.2 +）然后静脉注射到CD45.1 +小鼠。小鼠屈服于所述细胞注射后17至19天。这些小鼠处死，使他们死于这种疾病在他们的白血病类型可以进行分析。对照小鼠，该注射用PBS，在用于比较的相同的时间点进行分析。在C1498细胞注射的小鼠显示C1498细胞大量浸润到他们的骨髓，这表现在细胞的爆炸状外观后五月-Grünwald的吉姆萨染色进行（ 图4A）。他们还保存他们的单核细胞和粒表型（ 图4B和C），显示出单核和髓细胞的积累是急性单核细胞白血病的特征。 到Determine以下白血病细胞浸润，CD45.2 + C1498细胞，B淋巴细胞，单核细胞和粒群体（包括祖细胞，前体和成熟细胞）髓造血干细胞数量是否降低，用免疫荧光染色和多参数流式细胞分析进行定量。白血病细胞代表16向造血细胞的36％（未显示数据）。在其他类型的细胞均存在于显著较低的数字中比在PBS中注射小鼠的C1498-注射的小鼠（通过5倍平均为B细胞亚群，4倍平均粒细胞和平均3倍对于单核细胞亚群）（ 图5A至C）。 白血病和对照小鼠血液样品中的单核细胞的频率的一项调查表明，它们含有淋巴细胞（ 图6A）的可比较的百分比，但是单核细胞的更高频率和白血病细胞。这些特征代表急性单核细胞白血病11（ 图6B）的。 间急性单核细胞白血病12的另一特点，C1498注射小鼠带有肿肝脏（肝），肺和脾（脾肿大）（ 图7A）。使用免疫荧光染色在这些器官进行检测和流式细胞术分析（ 图7B）CD45.2 + C1498细胞的不同频率。脾肿大从高数量的单核细胞浸润导致的，我们也估计脾人口的比例。在B淋巴细胞，单核细胞和粒细胞部分的数分别显著越大，由平均2倍，2.5倍和3倍，分别在白血病脾脏比对照脾脏（ 图7C）。 薄页="1"&gt; <img alt="图1" src="/files/ftp_upload/54270/54270fig1.jpg" /> 首先确定图1中的协议设置为在体外培养的C1498细胞系和在急性白血病的体内描述表征。造血谱系和组织培养C1498细胞的分化阶段的示意图 。然后C1498细胞注射入同类系小鼠以诱导急性白血病的发展。进行骨髓，外周血，脾，肝和肺组织的隔离，以确定的频率，表型和形态变化的C1498细胞浸润后。四:静脉MGG:五月-格伦沃尔德姬姆萨<a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html>广告/ 54270 / 54270fig2.jpg"/&gt; 图2. C1498细胞表型分析在体外培养。代表流式细胞仪散点图和细胞表面（A）和细胞内（B）的直方图后，C1498-表示，用成熟的造血细胞分化相关分子所示。 C1498细胞从培养物中收获，洗涤并标记使用分别特异于细胞表面的CD11b，CD18和B220标记或它们的同种型对照的荧光抗体。对于细胞内染色，将细胞固定，透和使用针对的Mac-3，CD3ε抗体和TCR（T细胞受体）Vβ链或它们的同种型对照的共同表位的标记。使用带活细胞设门进行分析。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> ntent"FO:保together.within页="1"&gt; <img alt="图3" src="/files/ftp_upload/54270/54270fig3.jpg" /> 图3.功能和培养C1498细胞的形态学特征 。从培养物收获并离心在玻片上进行显微镜C1498细胞。（A）中 ，使用免疫荧光进行用于髓过氧化物酶表达染色。（B）中的细胞化学反应用于分析α萘丁酸酯酶（NBE）和萘酚AS-D氯乙酸酯酶（CAE）的C1498细胞活动。细胞被认为是阳性时褐色和红色紫色，大细胞质颗粒，分别观察每个标签。（c）可以-Grünwald的吉姆萨（MGG）C1498细胞染色。对于每个染色实验中，显微镜物镜放大倍率被表明。每个图像是代表三个独立的实验。large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54270/54270fig4.jpg" /> 图4.骨髓形貌在PBS-和C1498注射小鼠骨髓细胞从PBS-和C1498细胞注射的小鼠中分离，离心到幻灯片显微镜（A）可以-Grünwald的吉姆萨（MGG）染色。（B ）α萘丁酸酯酶（NBE）和AS-D氯乙酸酯酶（CAE）的功能是使用细胞化学评价（C）的萘酚。在面板A中，带（未成熟）或分段（成熟）嗜中性粒细胞是在比PBS注射的小鼠的C1498-注射的小鼠的骨髓中不可见。面板B和C表明，有相对于在对照骨髓中观察到的数字单核和粒细胞在白血病骨髓的积累。所有用放大100倍的目标进行微观分析。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54270/54270fig5.jpg" /> 图5.髓质种群PBS-和定量分析C1498注射小鼠骨髓细胞从PBS-和C1498细胞注射的小鼠中分离，并进行细胞计数后估计。免疫染色和流式细胞仪分析活细胞门控流之后，确定了不同的细胞群的频率。（A）中的B细胞亚群包括CD19 + B220 +细胞从原B细胞阶段到成熟B淋巴细胞（B）的粒细胞中的CD3 -和CD11b + Ly6G +谱系，包括前体的。第二未成熟和成熟粒细胞（C）中的单核细胞亚群被定义为CD3 - CD115 +和包含在祖细胞到成熟单核细胞的阶段。 N = 7只小鼠/组，并且数据表示为直方图示出了平均值±SEM表示。 ***，P &lt;0.0001和**，P &lt;0.01，非配对t检验比较PBS-C1498和注射小鼠。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54270/54270fig6.jpg" /> 在PBS-和单核细胞亚群图6.血液分析C1498注射小鼠 。代表流式细胞仪已分别定义为CD3 +和B220 +细胞在PBS-C1498和（一）T，B淋巴细胞百分比，散点图细胞注入以及CD115 +的Ly6C 高细胞-小鼠（B）以C1498白血病和控制单核细胞的细胞的频率（PBS）的小鼠通过分析CD115 +的Ly6C确定。通过门控活细胞进行分析。为了比较白血病和对照组，CD45.2 +细胞C1498排除。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54270/54270fig7.jpg" /> 在白血病和对照组小鼠脾种群图7.估计。 （A）的肝，肺和脾的代表性照片在白血病小鼠肿胀相比对照小鼠。收集脾，称重，脾细胞计数以下组织破坏。（B）柱状图表示差异白血病细胞频率erent器官（℃）后，对CD45.2 +细胞进行免疫染色，并使用流式细胞仪进行分析的结果。脾B，免疫染色后粒和单核细胞数的估计和流式细胞仪分析选通进行，以确定现场CD19 + B220 + ，CD3 -细胞CD11b + Ly6G +，CD3 -细胞CD11b +的Ly6C -和CD3 -细胞CD11b +的Ly6C 高细胞。对于肺，脾脏和肝脏中所示的比例条表示1厘米。 。每组5 - 8只小鼠/组，和该数据被表示在直方图为平均值±SEM * P &lt;0.05; **，p值= 0.0033，非配对t-检验比较PBS-和C1498注射小鼠<a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbodY&gt; <tr><td> 电池类型 </td><td> 膜或细胞内分子 </td></tr><tr><td></td><td></td></tr><tr><td> 前体和成熟细胞 </td><td></td></tr><tr><td> NK细胞</td><td> NK1.1 +，泛NK + </td></tr><tr><td> NKT细胞</td><td> NK1.1 +，泛NK +，TCR Vbeta +（8.2），CD3 + </td></tr><tr><td> T淋巴细胞</td><td> TCR Vbeta +，CD3 +，CD4 +，CD8 + </td></tr><tr><td> B细胞的前体，B淋巴细胞</td><td> B220 +，CD19 +，CD21 / 35 + </td></tr><tr><td>粒前体和粒细胞</td><td> Ly6G + MAC-1 +，+的CD11b </td></tr><tr><td>单核细胞前体和单核/巨噬细胞</td><td>的CD11b + MAC-1 +，苹果-3 +，CD21 / 35 +，CD115 +，Ly6Chi </td></tr><tr><td></td><td></td></tr><tr><td> 先觉 </td><td></td></tr><tr><td>多能祖细胞</td><td> CD117 +的Sca-1 + CD34 +（Lin-的CD150-） </td></tr><tr><td>淋巴引发的多潜能祖细胞</td><td> CD117hi的Sca-1hi CD127 +（Lin-的） </td></tr><tr><td>常见的淋巴祖</td><td> CD117lo的Sca-CD127 1LO +（Lin-的） </td></tr><tr><td>共同髓系祖细胞</td><td> CD16 / CD117 32lo + CD34int（Lin-的的Sca-1） </td></tr><tr><td>粒细胞 - 巨噬细胞祖细胞</td><td> CD16 / CD117 32hi + CD34hi（Lin-的的Sca-1） </td></tr><tr><td>巨核细胞祖红</td><td> CD16 / CD117 32lo + CD34lo（Lin-的的Sca-1） </td></tr><tr><td></td><td></td></tr><tr><td> 造血干细胞 </td><td> CD117 +的Sca-1 + CD150 +（Lin-的CD34-） </td></tr></tbody></table> 表1.造血细胞谱系和分化的标志物。 CD:分化簇;林:成熟细胞的标记;罗:低表达;喜:高表达; INT:中间表达; NK:自然杀伤细胞; TCR:T细胞受体。

Watch this Scientific Journal Video about A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model at JoVE.com

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

该原稿提供了可用于表征在体外和诱导小鼠的注射后急性白血病C1498细胞培养技术的过程。表型和功能分析使用流式细胞仪，免疫荧光显微镜，细胞化学和五月 - 格伦沃尔德姬姆萨染色进行。

详细的定性C1498小鼠细胞系及其关联白血病小鼠模型协议

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

a-detailed-protocol-for-characterizing-murine-c1498-cell-line-its

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

19.9K 次观看.  INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC). 该原稿提供了可用于表征在体外和诱导小鼠的注射后急性白血病C1498细胞培养技术的过程。表型和功能分析使用流式细胞仪，免疫荧光显微镜，细胞化学和五月 - 格伦沃尔德姬姆萨染色进行。 

视频: A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

临床前小鼠模型的外科手术和方法<em&gt; De Novo</em&gt;乳腺癌转移

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

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

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

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 ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

木尿造血干细胞和祖细胞和MLL-AF9驱动白血病线粒体反应氧物种的流细胞学分析

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

评估癌细胞3D球形培养中的细胞活力和死亡

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

毛尿胰腺腺腺腺癌超声引导正射植入

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

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 ...

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

使用膜结合IL-21修饰B细胞系的自然杀伤（NK）和CAR-NK细胞扩增方法

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...