这项工作得到了美国国家卫生研究院（R37CA228304和R01HL146642至西陈，CA148761至杰弗里·罗森），美国国防部（W81XWH-19-1-0524至西陈， W81XWH-19-1-0035 至徐向东、美国癌症协会（RSG-18-181-01-TBE 至西辰）和德克萨斯州癌症预防与研究所（RR150009 CPRIT 癌症研究学者奖授予陈希） 贝勒医学院的患者衍生异种移植和高级体内模型核心（来自RP170691 CPRIT核心设施奖和NCI-CA125123 P30癌症中心支持补助金）。

使用动物的所有协议都已得到机构动物护理和使用委员会（IACUC）的审查和批准。肿瘤片段，约1×2毫米3 的大小，是从贝勒医学院的患者衍生异种移植和高级在Viva模型核心获得的可冷冻库存。
1. 准备冷冻保存的乳腺肿瘤片段进行移植
<ol><li>将带有肿瘤片段的冷冻液从液氮转移到 37 °C 的水浴中。</li>
	<li>解冻时偶尔轻轻轻拂一下，搅拌低温。</li>
	<li>组织解冻后，将冷冻浴缸取出，通过温和的反转混合。</li>
	<li>干燥冷冻室外，用70%乙醇喷洒。将其转移到生物安全罩。</li>
	<li>将解冻的肿瘤组织转移到一个15mL的圆锥管中，管中充满10兆L的冷杜尔贝科改良鹰介质（DMEM）。倒管混合好。让组织碎片沉淀到管子底部。</li>
	<li>吸气超高音，并在10 mL的冷DMEM中重新吸气。倒管混合好。让组织碎片沉淀到管子底部。</li>
	<li>吸气超高音，并在10 mL的冷DMEM中重新吸气。把管子放在冰上。 注:组织已准备好移植。</li>
</ol>2. 收集和准备用于移植的新鲜乳腺肿瘤
<ol><li>对乳腺肿瘤携带小鼠实施安乐死。 注:PDX宿主可能是SCID/米色，NSG或NRG雌性小鼠，而在目前的研究中，使用3~5周雌性巴尔布/C小鼠。</li>
	<li>用70%的乙醇喷洒安乐死小鼠的肿瘤区域。 注意:尽量避免头发与肿瘤样本，这可能会导致肿瘤片段污染冷冻储存或移植。</li>
	<li>用锯齿状钳子捏合和抬起肿瘤周围的皮肤，用剪刀做一个短切口。</li>
	<li>用剪刀将肿瘤从皮肤中分离，从宿主小鼠身上解剖整个肿瘤。从肿瘤的外表面修剪掉任何剩余的老鼠脂肪垫组织。将肿瘤放在一个15mL的圆锥管中，里面装满了5mL的冷DMEM。 注意:使用直径最大为 1 厘米的肿瘤，因为较大的肿瘤可能含有坏死核。</li>
	<li>在生物安全罩中，将解剖的肿瘤转移到含有足够 DMEM 以防止干燥的无菌 10 厘米培养皿中。</li>
	<li>在无菌条件下用手术刀或刀片将肿瘤切成 1 mm3 片段。 注意:手术刀或刀片应在生物安全罩的紫外线下暴露至少 20 分钟才能使用。</li>
	<li>将肿瘤片段转移到充满冷DMEM的15mL锥形管中。把管子放在冰上。 注:组织已准备好移植。从第2.4步解剖的肿瘤片段可以，1）捕捉冷冻在液氮蛋白质和RNA/DNA提取， 2） 固定 4% 副甲醛 （PFA） 或 10% 中性缓冲甲醛 （NBF） 用于血氧林和欧辛 （H&E） 和免疫造血 （IHC） 分析， 或 3） 冷冻保存在 1.25 mL 冷冻介质 （10% 二甲基硫氧化物 [DMSO]， 40% DMEM 和 50% 胎儿牛血清 [FBS]）通过在 -80 °C 的冰柜中缓慢冷冻过夜，然后转移到液氮进行长期储存。</li>
</ol>3. 准备动物手术
<ol><li>对于动物疼痛管理，皮下注射丁丙诺啡持续释放手术前60分钟剂量1毫克/千克或遵循机构指南72小时的镇痛覆盖。</li>
	<li>根据无菌手术的机构指南设置手术套件。</li>
	<li>以11.25 mL/h的速度在异黄素麻醉机的感应室麻醉一名4周大的女性。将鼠标转移到手术区域，转移到无菌（硅橡胶）手术板上，通过小面罩接受麻醉。把鼠标放在它的背上，把腿贴在自然的位置。 注:SCID/米色、NSG 或 NRG 用于 PDX，Balb/c 用于 GEMM 移植模型。</li>
	<li>将眼科软膏涂在眼睛上，以防止干燥。</li>
	<li>通过脚趾捏确认适当的饱和度。 注:动物没有反应/移动表明动物已经麻醉充分，并准备手术。</li>
	<li>剃掉下腹部的老鼠，尤其是第四个周围进行手术的区域。</li>
	<li>使用圆形运动，从手术部位的中心开始，用孔隙碘手术磨砂向外边缘工作，然后用 70% 异丙基乙醇垫去除孔维多碘。重复两次。</li>
</ol>4. 将肿瘤片段移植到第四个（内在）乳腺脂肪垫中
<ol><li>使用无菌手术窗帘覆盖动物的身体，切口部位除外。 注:在切口前，通过脚趾捏确认适当的饱和度。</li>
	<li>使用锯齿状钳子捏和抬起皮肤在 # 4 。</li>
	<li>钝面向下，用剪刀做短（约 1 厘米），寄生精切口，从 # 4 朝头部。</li>
	<li>使用棉尖施用器，将皮肤与切口内侧的腹膜分离。</li>
	<li>握住切口的侧面时，使用相同的拭子轻轻剥去皮肤上的 #4 乳腺脂肪垫。</li>
	<li>一旦脂肪垫分离，用27G针固定皮肤靠近动物的身体。</li>
	<li>如果在实验中有必要清除内源性小鼠乳腺上皮的脂肪垫，则执行以下步骤。<ol>
<li>使用锯齿状钳子，轻轻地将脂肪垫从身体中伸出，并定位在腺体主要容器的交叉点（靠近钳子将持有腺体的位置）下方的静脉淋巴结。</li>
		<li>小心地将容器内化到淋巴结，将脂肪连接到形成三角形区域的第四和第五脂肪垫中。 注:暂时关闭此步骤的氧气源。</li>
		<li>使用微解剖剪刀，一次切开每个烧灼的容器（以确保每次切口后不会出血），直到切除含有淋巴结的脂肪垫部分。</li>
		<li>丢弃"清除"的脂肪垫组织。</li>
	</ol></li>
	<li>用钝分离钳握住第四个乳腺脂肪垫。另一方面，将倾斜的细钳的封闭尖端插入剩余容器上方的脂肪垫中间，并靠近腹膜壁。慢慢地打开钳子，做一个小口袋。从脂肪垫中取出倾斜的细钳。</li>
	<li>使用倾斜的细钳，取一块肿瘤片段并插入口袋。</li>
	<li>慢慢打开钳子尖，将肿瘤片段释放到脂肪垫口袋里。</li>
	<li>小心地提取角度细小的钳子。 注意:查看口袋，确认提取钳子时肿瘤片段仍保留在乳腺脂肪垫的口袋里。当实验或银行需要多余的肿瘤组织时，肿瘤可以植入两个反向脂肪垫中。由于已知反向肿瘤之间的相互作用，不建议进行双面移植的动物进行治疗研究。或者，可能用于植入过程的托车设备。</li>
	<li>从切口底部开始，使用爪子和锯齿状钳子收集两侧的皮肤。将两侧聚集在一起，稍微抬起，为伤口夹应用准备皮肤。用爪钳解开切口的边缘，将边缘捏在一起，在顶部形成连续的表面。</li>
	<li>将两侧与锯齿状钳子结合，使用伤口夹施用器将伤口夹放在切口的中心。如有必要，将组织粘合剂涂抹在切口的末端，以保持其闭合和安全。</li>
	<li>将动物放入一个干净的笼子里，笼子在变暖的表面上。监测手术后出血、脱血和疼痛的迹象。动物应该在术后几分钟内起床移动。</li>
	<li>手术后至少3天密切关注切口部位和动物的整体健康。遵循疼痛管理的机构准则。</li>
	<li>在继续下一次移植之前，用玻璃珠消毒器清洁10s的手术工具。等待工具在使用前冷却。</li>
	<li>重复步骤 4.1+4.15，直到所有小鼠都移植。 注:雌激素补充是需要ER+ 肿瘤，可以通过水或缓慢释放颗粒59提供。</li>
</ol>5. 监测肿瘤生长以响应药物治疗
注:根据内在肿瘤的生长速度，已建立的可移植PDX和p53-空肿瘤的可移植肿瘤在手术后需要2周至8周才能发育。
<ol><li>每周使用两次卡钳测量两个维度的肿瘤。使用公式计算体积: 肿瘤体积 （mm3） = W2 x L/2 其中 W 是宽度， L 是肿瘤的长度。</li>
	<li>当肿瘤体积达到150~300mm3时，开始药物治疗。 注:根据药物的特性，口服加瓦奇或腹内注射可用于提供药物。</li>
	<li>收集肿瘤样本，执行IHC染色60，蛋白质和RNA/DNA分离和免疫表型61 为各种目的。收集血液并进行免疫表型或将其用于药理动力学/药理动力学研究。</li>
</ol>

MTL 是斯特姆梅德有限公司有限合伙人的创始人，也是斯特姆梅德控股公司（其总合伙人）的创始人和经理。MTL 是特瓦迪治疗公司 LED 的创始人和股东，是 StemMed 有限公司的薪酬员工。

为了减少动物肿瘤生长的变化，将肿瘤组织切成1毫米3 的片段进行移植至关重要。生长软组织的模型更难处理，肿瘤片段需要稍微大一点（1~2毫米3）。当将组织放入乳腺脂肪垫口袋时，请注意不要将组织分割成多个块，因为这会导致多个小肿瘤或形状奇怪的肿瘤。
此外，使用新鲜肿瘤移植动物，将用于药物治疗研究。从冷冻保存中植入组织将产生更可变的接受率和稍慢的生长动力学。一旦肿瘤从冷冻保存材料中生长，第二代移植将产生该模型的典型接受率和生长动力学。此外，尝试使用肿瘤没有或轻度坏死因移植。对于大多数型号，这将是一个大小范围为400-600毫米3。如果观察到明显的坏死核心，则使用肿瘤外层的组织进行移植，不使用坏死区域。重要的是要保持肿瘤组织在冰上，并防止干燥。
为了减少可能来自周围或肿瘤核心的具有不同微环境的 GEMM 肿瘤块之间的变异性，另一种方法是准备原细胞悬浮，并根据肿瘤模型将大约 5，000 至 30，000 个细胞移植到乳腺脂肪垫中。有限的拼贴酶消化是在DMEM/F12中用1毫克/mL型I拼贴剂进行的，在125转/分时旋转37°C时，没有任何2小时的添加剂。乳腺肿瘤细胞可以通过3个短的离心步骤进行浓缩。简言之，将电池悬架以 450 x g 为 7s 将电池悬架转移到 15 mL 圆锥形管和离心机。吸气超高分子，并在10 mL的1x杜尔贝科磷酸盐缓冲盐水（DPBS）中重新吸气颗粒。重复脉冲离心两次。这将有助于随机化块之间的差异。
正常乳腺上皮的移植不会再生形态正常和功能导管树存在内源性小鼠上皮。有必要去除内源性小鼠上皮（清除），使正常的上皮移植生长62。然而，肿瘤组织能够生长，即使在完整的内源性小鼠上皮的存在。然而，这并不一定意味着不存在这种抑制信号。对于某些实验协议来说，乳腺脂肪垫清除是必要的，以禁止内源性小鼠乳腺上皮与所附材料的相互作用。此外，内源性上皮可能会使一些下游分析复杂化，如基因组、转录组和蛋白质组分析。
PDX模型和可移植的创业板可以忠实地概括临床亚型的异质性和人类乳腺癌药物治疗的反应。重要的是，这些模型很容易移植，并在有限的序列段落中保持稳定的表型。肿瘤生长可以很容易地用卡钳测量。PDX 模型和可移植 GEMM 的一个警告是，这些模型不能概括肿瘤启动的早期步骤。此外，PDX模型缺乏肿瘤与功能免疫系统的相互作用。这些临床前模型为研究乳腺癌生物学和评估药物反应提供了宝贵的系统。将药物反应与每个肿瘤模型的基因组和蛋白菌信息相结合，将有助于识别生物标志物，以建立反应预测和治疗耐药机制。这些类型的数据可能导致新的靶向疗法，可以单独使用，并结合化疗或免疫疗法，以改善患者的结果。

大多数乳腺癌死亡可归因于对常规疗法1，2具有抗药性的复发性疾病。乳腺癌的肿瘤间和肿瘤内异质性有助于治疗抵抗。此外，肿瘤异质性会影响准确的预后，并挑战疾病管理3，4。识别反应的预测生物标志物将显著改善乳腺癌患者的临床结果。尽管大多数乳腺癌类型是免疫学上"冷"的肿瘤，可能对免疫治疗没有反应，但免疫检查点抑制剂在临床试验2，5中已显示出希望。例如，第三阶段的试验显示，无病生存（DFS）和初步证据表明，atezolizumab（对PD-L1的单克隆抗体）与纳布-帕利塔塞尔相结合，可以提供整体生存效益相比，纳布-帕利塔塞尔单独在肿瘤中≥1%PD-L1染色6。开发使乳腺肿瘤对免疫疗法敏感的疗法将彻底改变治疗方案。
临床前模型，忠实地回顾人类乳腺癌异质性和药物反应是研究肿瘤生物学和确定潜在的生物标志物有针对性的治疗的关键。不朽的细胞系被广泛用于乳腺癌研究，因为这些细胞系很容易生长和基因改造，以研究分子机制。然而，由于来自体外长期细胞培养的选择性压力，遗传漂移可能会随着时间的推移而发生，乳腺癌细胞系可能携带不同于原发性乳腺肿瘤7、8、9畸变的细胞系特异性基因组改变。
患者衍生的异种移植（PDX）肿瘤块能够概括人类疾病的异质性，在组织学和免疫造血学上类似于原发肿瘤10、11、12、13、14、15、16、17、18、19、20、21、22 ，23，24，25，26，27，28，29。重要的是，PDX模型在多种移植中表型稳定，从组学、转录学、蛋白质组和基因组分析10、11、12、13、14、15、16、17、18、19、20、21、22中可以证明这一点，23，24，25，26，27，28，29。PDX 模型显示的治疗反应与临床观察到的 10、11、12、13、14、15、16、17、18、19、20、21、22、23、24 等治疗反应相当， 25，26，27，28，29。已建立了雌激素受体阳性（ER+）、黄体酮受体阳性（PR+）、表皮生长因子2阳性（ERBB2+、HER2+）和三阴性乳腺癌（TNBC）PDX模型的PDX模型，为检测内分泌、化疗和靶向治疗提供了一个极好的平台。然而，PDX模型目前的主要警告之一是小鼠缺乏功能免疫系统。
基因工程小鼠模型（GEMM），如Trp53同源空、cMyc、Wnt1、PyMT或Her2过度表达模型，允许在完整的免疫系统背景下研究自发肿瘤的启动、进展和转移。然而，肿瘤延迟时间长，这使得它很难进行临床前试验与多臂30，31。然而，GEMM可以移植到同步宿主，产生足够数量的肿瘤，密切回顾人类肿瘤32，33，34，35，36，37，38，39，40，41，42，43，44，45，46，47，48，49，50，51，52，53，54，55。例如，从p53-空BALB/c小鼠的乳腺上皮被移植到同步野生型受体小鼠的清除脂肪垫中，形成原发性肿瘤，可以进一步移植到同步宿主56，57。p53-空肿瘤回顾了人类肿瘤的不同亚型。
PDX模型和可移植GEM的结合为研究乳腺癌生物学、药物反应和抗肿瘤免疫提供了宝贵的临床前工具。在目前的协议中，描述了PDX和GEMM肿瘤片段在小鼠乳腺脂肪垫中的矫形移植方法。这些模型适合串行段落，通常保留稳定的表型。为了减轻遗传漂移或失去异质性的风险，随着时间的推移，多个组织片段被冷冻保存在每一个通道，以备随后移植的情况下，生物或形态的变化观察到时间29，58。

<table><tbody><tr><td>1 mg/mL Buprenorphine-SR</td><td>ZooPharm (via BCM veterinarians)</td><td></td><td>Sterile</td></tr><tr><td>26G syringe</td><td>BD</td><td>148232E</td><td>Sterile</td></tr><tr><td>Betadine Scrub</td><td>Fisher</td><td>19-027132</td><td></td></tr><tr><td>Cotton Swabs</td><td>VWR International Laboratory</td><td>89031-272</td><td>Sterile</td></tr><tr><td>DMEM</td><td>Fisher</td><td>MT 10-013-CM</td><td>Sterile</td></tr><tr><td>Electric shaver</td><td>Oster</td><td>78005-050</td><td></td></tr><tr><td>Glass beads sterilizer (Germinator)</td><td>Roboz Surgical Store</td><td>DS-401</td><td></td></tr><tr><td>Lubricant ophthalmic ointment</td><td>Akorn Animal Health</td><td>17478-062-35</td><td></td></tr><tr><td>Micro Dissecting Forceps; Serrated, Angular (regular forceps)</td><td>Roboz Surgical Store</td><td>RS-5139</td><td>Sterile</td></tr><tr><td>Micro Dissecting Spring Scissors (fat pad cutter)</td><td>Roboz Surgical Store</td><td>RS-5658BT</td><td>Sterile</td></tr><tr><td>Micro Forceps (tissue placing forceps)</td><td>Roboz Surgical Store</td><td>RS-5069</td><td>Sterile</td></tr><tr><td>Petri Dish</td><td>Fisher</td><td>08-757- 100D</td><td>Sterile</td></tr><tr><td>Sterile drape</td><td>Sai Infusion Technology</td><td>PSS-SD1</td><td>Sterile</td></tr><tr><td>Surgery scissors</td><td>Roboz Surgical Store</td><td>RS-5960</td><td>Sterile</td></tr><tr><td>Tissue Forceps (claw forceps)</td><td>Roboz Surgical Store</td><td>RS-5158</td><td>Sterile</td></tr><tr><td>Wound clip applier</td><td>BD Autoclip Wound System</td><td>01-804</td><td>Sterile</td></tr><tr><td>Wound clip remover</td><td>BD Autoclip Wound System</td><td>01-804-15</td><td>Sterile</td></tr><tr><td>Wound clips</td><td>BD Autoclip Wound System</td><td>01-804-5</td><td>Sterile</td></tr></tbody></table>

orthotopic transplantation of breast tumors as preclinical models for breast cancer

临床前模型，忠实地回顾肿瘤异质性和治疗反应是关键转化乳腺癌研究。不朽的细胞系很容易生长和基因改造，以研究分子机制，但来自细胞培养的选择性压力往往导致遗传和表观遗传的变化随着时间的推移。患者衍生异种移植（PDX）模型忠实地概括了人类乳腺肿瘤的异质性和药物反应。PDX模型在矫形移植后表现出相对较短的延迟，有助于乳腺肿瘤生物学和药物反应研究。可移植的基因工程小鼠模型允许研究乳腺肿瘤免疫力。目前的协议描述了将乳腺肿瘤片段矫止移植到乳腺脂肪垫中的方法，然后是药物治疗。这些临床前模型为研究乳腺肿瘤生物学、药物反应、生物标志物发现和耐药机制提供了有价值的方法。

图1显示了矫形移植的设备（图1A）和关键程序（图1B）。图2显示了移植PDX肿瘤（MC1）的特征。MC1模型的肿瘤片段（1毫米3）被移植到SCID/米色小鼠的#4脂肪垫中。一个月后，肿瘤平均大小达到350毫米3左右。肿瘤体积每周监测两次，为期一个月。通常，我们在大约2周到8周内获得各种PDX或创业板的明显肿瘤，移植1毫米3肿瘤片段（图2A）。肿瘤样本可以收集用于形态和信号分析（图2B+D）。进行了H&E染色来分析病理学（图2B）。IHC用于监测特定细胞谱系（角蛋白19（K19）、上皮细胞、图2C）、细胞状态（Ki67、增殖、图2D）或感兴趣的信号分子的标记。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61173/61173fig1.jpg"> 图1:示意图显示手术技术。 （A） 矫位移植所需的手术设备。（B） 代表图片显示肿瘤躯干移植乳腺脂肪垫的暴露。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61173/61173fig1large.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/61173/61173fig2.jpg"> 图2:移植肿瘤的特征。 （A） 以卡钳测量的肿瘤生长的代表性动力学。肿瘤体积 （mm3） = W2 x L/2 （W = 宽度和 L = 长度）。（B） 显示MC1PDX病理的H&E染色。IHC显示上皮标记角蛋白19（C） 和增殖制造商Ki67（D） 在MC1 PDX.比例栏 = 20 μm， 放大倍数 = 40 倍。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61173/61173fig2large.jpg" target="_blank">请点击这里查看此图的较大版本。</a>

Watch this Scientific Journal Video about Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer at JoVE.com

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

患者衍生异种移植（PDX）模型和可移植的基因工程小鼠模型忠实地概括了人类疾病，是基础和转化乳腺癌研究的首选模型。在这里，一种方法被描述为正位移植乳房肿瘤片段到乳腺脂肪垫研究肿瘤生物学和评估药物反应。

乳腺癌临床前模型乳腺肿瘤的矫形移植

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

orthotopic-transplantation-breast-tumors-as-preclinical-models-for

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

5.9K 次观看.  Baylor College of Medicine. 患者衍生异种移植（PDX）模型和可移植的基因工程小鼠模型忠实地概括了人类疾病，是基础和转化乳腺癌研究的首选模型。在这里，一种方法被描述为正位移植乳房肿瘤片段到乳腺脂肪垫研究肿瘤生物学和评估药物反应。

视频: Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

科研

癌症研究

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

乳腺癌细胞原位注射到小鼠乳房脂肪板

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>体内成像测量原位注射乳腺肿瘤细胞的自发性肺转移

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

医学

<meta charset="utf8"></head><body>乳腺癌患者来源的异种移植物的高通量解离和原位植入

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 &#181;L suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&amp;E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

乳腺癌患者来源的异种移植物的高通量解离和原位植入

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which ...

乳腺癌临床前模型乳腺肿瘤的矫形移植

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乳腺癌临床前模型乳腺肿瘤的矫形移植

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研究使用正交乳腺癌模型的三阴性乳腺癌

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体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

乳腺癌患者来源的异种移植物的高通量解离和原位植入

乳腺癌患者来源的异种移植物的高通量解离和原位植入

乳腺癌患者来源的异种移植物的高通量解离和原位植入

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移