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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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?...

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

Research

JoVE Journal

Cancer Research

5.8K 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

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

科研

癌症研究

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet exists. Progressive Resistance Exercise (PRE) is expected to improve lymphedema without additional swelling. This study showed the therapeutic effects of PRE on lymphedema by using ultrasonography to measure the change in thickness of the muscle and subcutaneous tissue. The thickness of subcutaneous tissue decreased more in the PRE group than in the non-PRE group. Ultrasonography is widely used in many clinics because of its easy accessibility, safety, and inexpensiveness. Ultrasound is one of the best tools for diagnosing and determining treatment efficacy on breast cancer-related lymphedema (BCRL).

This study utilized ultrasonography for diagnosis and follow-up tests to confirm treatment efficacy of Progressive Resistance Exercise (PRE) in breast cancer-related lymphedema (BCRL). Ultrasonography can be applied effectively to ensure diagnosis and treatment of lymphedema.

乳腺癌相关的淋巴水肿的超声评价

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs).

1%亚甲蓝染料作为单一技术在乳腺癌哨点节点活检中的应用

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery ...

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, ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

从妇科和乳腺癌肿瘤获得癌症干细胞球

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

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 ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

使用微生理系统模拟人体乳房组织中的乳腺癌

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

通过炎症性乳腺癌细胞的尾静脉注射模拟脑转移

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...