作者承认Beth Chaffee博士，DVM，PhD，DACVP对该技术发展的重要贡献。

所有描述的动物实验均获得英国剑桥大学机构动物护理和使用委员会的批准。
1. 细胞系的制备
<ol><li>根据实验室的标准细胞培养方案培养细胞系，用于传统细胞培养或注射到小鼠体内。这里使用的标准方案是在Dulbecco的改良Eagle培养基中生长，其中含有10%胎牛血清（FBS），L-谷氨酰胺和青霉素/链霉素（以下简称完全生长培养基）。 注意:在本实验中，艾布拉姆斯骨肉瘤细胞用于Balb / c Foxn1 nu / nu 小鼠。对于乳腺癌研究，使用Balb / c小鼠中的4T1细胞和C57BL / 6小鼠中的EO771细胞。</li>
	<li>在37°C的5%CO2中在通风的组织培养瓶或6孔组织培养板中培养细胞。</li>
	<li>传代感兴趣的细胞系，并在细胞达到通常用于将这些细胞注射到小鼠中的汇合度时准备注射细胞。</li>
</ol>2. 动物
<ol><li>使用至少6-8周龄的Balb/c Foxn1 nu/nu 小鼠进行皮下肿瘤生成，以确保动物超越快速生长期并已达到成年和骨骼成熟。</li>
	<li>使用雄性或雌性小鼠。选择激素反应性细胞系时要例外（例如，雌性小鼠的乳腺癌细胞和雄性小鼠的前列腺癌细胞）。</li>
	<li>对于异种移植实验，使用基于细胞系在正常条件下与完整小鼠免疫系统不相容的免疫缺陷无胸腺裸鼠。</li>
	<li>对于使用鼠细胞系的同种异体移植实验，也建议基于不同的小鼠遗传和免疫背景进行同种异体移植实验。然而，对于同系实验，使用与感兴趣的细胞系相同菌株的动物。</li>
	<li>根据机构的饲养政策，以标准密度饲养动物。</li>
</ol>3.皮下肿瘤
<ol><li>通过胰蛋白酶消化从培养物中收获细胞系，并重悬于无菌磷酸盐缓冲盐水（PBS）中。</li>
	<li>评估细胞活力并通过台盼蓝排除法确定细胞密度。使用血细胞计数器或自动细胞计数器对细胞进行计数。最低细胞活力为90%用于注射到小鼠体内以产生皮下肿瘤。</li>
	<li>调整细胞密度以在 0.1 至 0.15 mL（100 至 150 μL）无菌 PBS 的最终体积中注入 1-2 x 105 个细胞。将细胞保持在冰上直到注射。</li>
	<li>或者，通过以800 ×g 离心5分钟来沉淀细胞。弃去上清液并将沉淀的细胞重新悬浮在未稀释的无菌基底膜基质培养基中，以获得 1-2 x 10 5个细胞， 最终体积为 0.1 至 0.15 mL（100 至 150 μL）。将细胞放在冰上直到进一步使用。</li>
	<li>麻醉小鼠，用于氧麻醉中异氟醚的皮下肿瘤生长。在2 L / min氧气中使用5%异氟烷的诱导剂量，在2 L / min氧气中使用2-3%异氟醚的维持剂量。在继续之前检查是否缺乏眨眼或踏板反射。 注意:异氟醚是一种吸入麻醉剂。在通风良好的区域使用异氟醚，并有适当的清除和自由气体收集系统。请咨询机构兽医人员，制定麻醉诱导、维护和监测计划，并确保实验室工作人员在麻醉监测和吸入麻醉剂处理方面接受过适当的培训。</li>
	<li>用脱毛溶液或电剪从麻醉小鼠的胸部或腹部的背部区域去除毛发。脱毛溶液是首选，以尽量减少对皮肤的潜在创伤。如果使用无胸腺裸鼠，请跳过此步骤。</li>
	<li>在注射细胞悬液之前，用70%乙醇拭子清洁制备的区域。</li>
	<li>使用带有 27 G 针头的 1 mL 结核菌素注射器在胸部或腹部的背侧区域皮下注射细胞，不受肩胛骨运动的影响。或者，将细胞皮下注射作为悬浮液在市售的细胞外基质中。 注意:注射市售的细胞外基质将限制细胞悬液在皮下空间中的迁移，因为这些基质在室温下固化。</li>
	<li>在单个笼子的加热垫上恢复小鼠，直到走动。然后可以将小鼠放在带有干净干燥的床上用品的正常笼子中。</li>
	<li>用卡尺监测覆盖在背胸或腹部的皮下肿瘤的大小，并每周测量体重，以确保皮下肿瘤不会溃疡或小鼠符合机构动物护理和使用委员会制定的早期切除标准。建议任何尺寸的最大肿瘤大小为 15 mm，以降低皮肤溃疡或中心性肿瘤坏死的风险。 注意:请查阅当地指南以确定最大允许的肿瘤大小/体积。</li>
	<li>通过吸入CO2 然后颈椎脱位在三到四周后对携带皮下肿瘤的小鼠实施安乐死。遵循机构可接受的小鼠安乐死政策。</li>
	<li>使用无菌手术技术收获皮下肿瘤。去除毛发后，像以前一样用70%乙醇对覆盖在肿瘤上的皮肤进行消毒（如果适用）。用#15手术刀刀片（有或没有手术刀刀片手柄）切开覆盖肿瘤的皮肤。用一把无菌手术剪刀从周围附着的软组织中尖锐地解剖肿瘤。</li>
	<li>将肿瘤置于含有完整生长培养基的6孔组织培养板中，并切成预先确定大小的多个小片段（~ 0.6 mm x 0.6 mm x 0.6 mm – 0.25 mm 3 至 1 mm x 1 mm x 1 mm – 1 mm3）与 #15 手术刀刀片（带或不带手术刀刀片手柄）。</li>
	<li>在室温下将肿瘤片段维持在无菌完全生长培养基中，直到胫内植入。对于携带荧光素酶或荧光报告基因的细胞系，在胫内植入小鼠之前，使用离体生物发光或荧光成像来确认肿瘤活力。</li>
	<li>为了冷冻保存，将多个片段放在补充有20%FBS和10%二甲基亚砜（DMSO）的完全生长培养基中的同一冷冻管中。使用商业冷冻保存系统在–80°C下逐渐冷冻，并在液氮中长期储存。通过使用液氮浸没快速冷冻，保留肿瘤碎片以供后续分析，但不用于将来的植入。将这些冷冻肿瘤片段长期储存在–80°C。 注意:先前有报道称，快照冷冻肿瘤不会在体内移植和生长8。</li>
</ol>4.手术植入皮下肿瘤碎片
<ol><li>在手术植入之前，将新鲜或冷冻保存的皮下肿瘤片段在完全生长培养基中置于室温。</li>
	<li>如第3节所述，在氧麻醉中使用异氟醚麻醉感兴趣的菌株的小鼠。在继续之前检查是否缺乏踏板反射。皮下注射丁丙诺啡，剂量为0.02-0.05mg/kg，以提供围手术期镇痛。如果需要，可以在术后每6-8小时重复一次。</li>
	<li>用脱毛溶液去除右膝关节和后肢胫骨近端的毛发，以尽量减少对皮肤的潜在创伤。</li>
	<li>用手术防腐剂擦洗准备好的区域。首先用70%乙醇棉签擦洗，然后用洗必泰和盐水交替擦洗。</li>
	<li>将胫骨近端想象为膝关节远端的区域，同时弯曲和伸展关节。</li>
	<li>用 #15 手术刀刀片（带或不带手术刀刀片手柄）在肢体内侧的胫骨近端水平创建一个 3-4 mm 的切口。切开皮肤和皮下组织，露出胫骨近端的内侧皮层。</li>
	<li>用 25 G 针尖轻轻按压，同时旋转针尖，在胫骨近端的内侧皮层上形成一个小孔。在颅骨和尾部胫骨皮质之间的等距点处，在膝关节远端约 2 mm 处开孔。根据肿瘤碎片的大小选择针头尺寸。</li>
	<li>使用无菌镊子拾取肿瘤碎片并将其插入胫骨近端的髓腔中。使用 27 至 30 G 针将肿瘤碎片操纵到髓管中。根据肿瘤碎片的大小，将至少0.5mm3 总肿瘤体积植入每个胫骨。这可能需要植入一个或多个肿瘤片段，具体取决于产生的肿瘤片段的大小。 注意:防止或限制移植物移位到骨外的修改是在骨缺损中放置骨蜡或骨水泥，或者在骨孔上放置凝胶泡沫或皮下脂肪移植物。</li>
	<li>用无菌液体组织粘合剂或单条皮肤缝合线贴合皮肤边缘。请勿在本站点使用伤口夹。如果在术后使用荧光成像，请小心，因为组织粘合剂和缝合线都有可能发出荧光。</li>
	<li>在单个笼子的加热垫上恢复小鼠，直到走动。</li>
</ol>5. 串行和终点评估
<ol><li>如前所述，在氧气麻醉中使用异氟醚麻醉小鼠。</li>
	<li>通过每周数字放射成像、生物发光或荧光成像（如果使用表达荧光素酶或荧光报告基因的细胞）无创评估胫骨肿瘤生长。植入部位肢体的卡尺测量也可以在清醒的小鼠中进行。</li>
	<li>除了对荷瘤小鼠的传统监测（体重、活动水平、呼吸频率、梳理、姿势、心态和行为）外，每周监测小鼠后肢跛行、肿胀和手术部位感染的迹象。</li>
	<li>在最初的 10-14 天内监测皮肤手术伤口是否有过度发红、肿胀、引流和伤口裂开，直到皮肤伤口愈合。4-5周后，根据研究结果评估评估小鼠存活或安乐死后。</li>
</ol>

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Hildreth博士由NIH资助，奖励号为K01OD026527。 内容完全由作者负责，不一定代表NIH的官方观点。

本报告记录了我们在胫内植入肿瘤同种异体移植物后创建原发性骨肿瘤或骨转移的模型。我们认为，这一进程有几个关键步骤。应为皮下注射肿瘤细胞悬液和胫内放置所得肿瘤碎片建立安全的麻醉平面。手术部位应无菌准备，以移除皮下同种异体移植物和胫内放置同种异体移植物。应均匀地创建肿瘤同种异体移植片段，以便随后进行胫内植入。应在胫骨近端形成适当大小的缺损，以植入肿瘤碎片?...

人类和动物疾病的小鼠模型在生物医学研究中越来越受欢迎。在这种情况下使用小鼠的效用在于它们的解剖学和生理学与人类非常相似。它们的妊娠期和产后生命达到成熟的时间相对较短，并且主要与相对较低的成本和住房便利性有关，尽管开发或购买成本的增加与更大程度的基因改造、免疫缺陷和/或人源化有关1。使用近交品系导致在研究纳入之前动物种群基本均匀。对它们基因组的完整了解表明与人类高度相似。许多疾病过程的直系同源分子靶标已经在小鼠基因组中鉴定出来，现在有一个广泛的小鼠特异性试剂库，很容易获得。因此，与大型动物模型相比，它们以更快速、更便宜的方式提供了相对高通量分析的机会1。此外，随着基因编辑策略的出现，允许全局或以细胞类型特定方式和/或组成或诱导方式过表达或删除某些基因，它们代表了用于研究人类和动物疾病的非常生物学上有用的模型系统2。
癌症是小鼠模型具有巨大实用性的领域之一。癌症的遗传小鼠模型依赖于单独或组合调节癌基因或肿瘤抑制基因的表达，使细胞经历致癌转化。还进行将原代或已建立的肿瘤细胞系注射到小鼠中。从人类或其他动物物种（包括小鼠）引入细胞系或组织仍然是体内使用最广泛的癌症模型。在免疫功能低下的小鼠中使用来自不同物种（异种移植物）的细胞和组织是最常见的2。然而，当宿主和受体属于同一物种时，使用同种异体移植肿瘤细胞或组织允许与同源系统中的相同宿主小鼠品系结合时与完整的免疫系统相互作用3。
原发性骨肿瘤或实体瘤的骨转移导致疼痛性溶骨性、成骨细胞性或混合性溶骨性/成骨细胞病变3，4。这些肿瘤会损害骨骼结构，增加病理性骨折的风险，并使患者的治疗选择有限。原发性骨肿瘤转移到远处器官，某些类型能够扩散到其他骨骼部位。在乳腺癌患者中，骨是最常见的首次转移部位，也是转移性疾病最常见的第一表现部位5，6。此外，在诊断并预测其他器官转移之前，播散性肿瘤细胞（DTC）存在于骨髓中7。因此，人们认为存在于骨骼中的癌细胞是最终转移到其他器官系统的细胞的来源。存在许多实体瘤转移的小鼠模型，其转移主要发生在肺和淋巴结中，并且根据肿瘤类型和注射技术，潜在的其他器官系统3。然而，骨转移的小鼠模型缺乏可靠，可重复地产生位点特异性骨骼转移并在小鼠达到从原发性肿瘤负担或转移到其他器官的早期去除标准之前发生骨转移。我们已经报道了一种原发性骨肿瘤骨肉瘤模型，该模型依赖于将实体肿瘤同种异体移植物手术植入小鼠的近端胫骨8。骨肿瘤在100%的小鼠中形成，88%的小鼠发生肺转移。这种转移的发生率超过了临床上常见的人群报告（~20-50%），但由于肺是骨肉瘤最常见的转移部位9，10，11，因此引起了极大的兴趣。虽然该模型在模拟原发性骨肿瘤方面具有优势，但它在模拟其他嗜骨实体瘤（如乳腺癌、肺癌、前列腺瘤、甲状腺肿瘤、肝肿瘤、肾肿瘤和胃肠道肿瘤）的骨转移方面也有很大的实用性。
开发该模型的基本原理是开发一种替代传统的骨内注射，通常注射到胫骨近端或股骨远端，以模拟原发性骨肿瘤或骨转移12。我们的主要目标是减轻该技术的已知局限性，即肺肿瘤栓塞。这导致这些栓塞肿瘤细胞的植入和"人工转移"，这些转移不能概括从转移到肺部的已建立原发性骨肿瘤的完全转移级联反应8，13。当已建立的骨转移扩散到远处部位时，也会出现这种情况。此外，还开发了该技术以产生骨转移模型，与原位或血管内注射技术相比，该模型可确保骨中和均匀部位肿瘤的植入和生长发生率更高。与这些所描述的技术相比，此模型具有明显的优势。该模型涉及将肿瘤细胞受控，一致地输送到骨骼中。它还避免了肺栓塞后人为的肺转移，并建立了基线统一的研究人群。该模型具有特定部位肿瘤的好处，而不会因原发性肿瘤或转移到其他器官而导致早期切除标准的风险。最后，该模型在修改方面具有很大的实用性，包括使用患者来源的异种移植物。
所提出的模型与手术方法后直接将细胞悬液注射到骨中相似，然后通过皮层注射或在皮层中出现小缺陷后将其输送到骨髓腔（有或没有扩孔髓腔）8，14，15，16，17.然而，肿瘤同种异体移植物的植入使这种技术明显不同。因此，本报告的目的是证明这种原发性骨肿瘤和实体瘤骨转移的模型，它克服了先前描述的模型的许多局限性。在细胞培养、小鼠模型、小鼠麻醉和手术以及小鼠解剖学方面具有丰富经验的研究小组有能力重现我们的技术来模拟小鼠的原发性骨肿瘤或骨转移。

<table>
	<tbody>
		<tr>
			<td>#15 scalpel blade</td>
			<td>Henry Schein Ltd.</td>
			<td>75614</td>
			<td>None</td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>10578911</td>
			<td>Used for mincing tumor pieces. Can also be used for cell culture</td>
		</tr>
		<tr>
			<td>Abrams osteosarcoma cell line</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Anesthesia machine with isoflurane vaporiser and oxygen tank(s)</td>
			<td>VetEquip</td>
			<td>901805</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Animal weighing scale</td>
			<td>Kent Scientific</td>
			<td>SCL- 1015</td>
			<td>None</td>
		</tr>
		<tr>
			<td>BALB/c nude mouse (nu/nu)</td>
			<td>Charles River Ltd.</td>
			<td>NA</td>
			<td>6-8 weeks of age. Male or female mice</td>
		</tr>
		<tr>
			<td>Bone cement</td>
			<td>Depuy Synthes</td>
			<td>160504</td>
			<td>Optional use instead of bone wax</td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>Ethicon</td>
			<td>W31G</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Animalcare Ltd.</td>
			<td>N/A</td>
			<td>Buprecare 0.3 mg/ml Solution for Injection for Dogs and Cats</td>
		</tr>
		<tr>
			<td>Carbon dioxide euthanasia station</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Should be provided within animal facility</td>
		</tr>
		<tr>
			<td>Cell culture incubator set at 37 &#176;C and 5% carbon dioxide</td>
			<td>Heraeus</td>
			<td>Various</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Chlorhexidine surgical scrub</td>
			<td>Vetoquinol</td>
			<td>411412</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Cryovials (2 ml)</td>
			<td>Thermo Scientific Nalgene</td>
			<td>5000-0020</td>
			<td>Optional if cryopreserving tumor fragments</td>
		</tr>
		<tr>
			<td>D-luciferin (Firefly), potassium salt</td>
			<td>Perkin Elmer</td>
			<td>122799</td>
			<td>Optional if cell line of interest has a bioluminescent reporter gene</td>
		</tr>
		<tr>
			<td>Digital caliper</td>
			<td>Mitutoyo</td>
			<td>500-181-30</td>
			<td>Can be manual</td>
		</tr>
		<tr>
			<td>Digital microradiography cabinet</td>
			<td>Faxitron Bioptics, LLC</td>
			<td>MX-20</td>
			<td>Optional to evaluate bone response to tumor growth</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>1371171000</td>
			<td>Optional if cryopreserving tumor fragments</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>Sigma Aldrich</td>
			<td>2483</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140079</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Forceps, Dumont</td>
			<td>Fine Science Tools, Inc.</td>
			<td>11200-33</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Freezer (&#8211; 80 &#176;C)</td>
			<td>Sanyo</td>
			<td>MDF-794C</td>
			<td>Optional if cryopreserving or snap freezing tumor fragments</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>11704939</td>
			<td>Can also use automated cell counter, if available</td>
		</tr>
		<tr>
			<td>Hypodermic needles (27 gauge)</td>
			<td>Henry Schein Ltd.</td>
			<td>DIS55510</td>
			<td>May also use 25G (DIS55509) and 30G (Catalog DIS599) needles</td>
		</tr>
		<tr>
			<td>Ice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Ideally small pieces in a container for syringe and cell suspension storage</td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Fine Science Tools, Inc.</td>
			<td>14084-08</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein Ltd.</td>
			<td>1182098</td>
			<td>None</td>
		</tr>
		<tr>
			<td>IVIS Lumina III bioluminescence/fluorescence imaging system</td>
			<td>Perkin Elmer</td>
			<td>CLS136334</td>
			<td>Optional if cell line of interest has bioluminescent or fluorescent reporter genes</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermofisher scientifc</td>
			<td>25030081</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>British Oxygen Corporation</td>
			<td>NA</td>
			<td>Optional if cryopreserving or snap freezing tumor fragments</td>
		</tr>
		<tr>
			<td>Liquid nitrogen dewar, 5 litres</td>
			<td>Thermo Fisher Scientific</td>
			<td>TY509X1</td>
			<td>Optional if cryopreserving tumor fragments</td>
		</tr>
		<tr>
			<td>Matrigel&#174; Matrix GFR, LDEV-Free, 5 ml</td>
			<td>Corning Life Sciences</td>
			<td>356230</td>
			<td>Optional. Also available in 10 ml size (354230)</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002549</td>
			<td>Pellet cells at 1200 rpm for 5-6 minutes</td>
		</tr>
		<tr>
			<td>Mr. Frosty freezing containiner</td>
			<td>Fisher Scientific</td>
			<td>10110051</td>
			<td>Optional if cryopreserving tumor fragments</td>
		</tr>
		<tr>
			<td>NAIR Hair remover lotion/oil</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC0132811</td>
			<td>Can alternatively use an electric clipper with fine blade</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Scalpel handle, #7 Short</td>
			<td>Fine Science Tools, Inc.</td>
			<td>10007-12</td>
			<td>User preference as long as it accepts #15 scalpel blade</td>
		</tr>
		<tr>
			<td>Small animal heated pad</td>
			<td>VetTech</td>
			<td>HE006</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>GT Vision Ltd.</td>
			<td>H600BV1</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sterile phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010023</td>
			<td>Use for injections and also as part of the surgical scrub, alternating with chlorhexidine</td>
		</tr>
		<tr>
			<td>Tissue adhesive (sterile)</td>
			<td>3M Corporation</td>
			<td>84-1469SB</td>
			<td>Can alternatively use non-absorbable skin suture (6-0 size)</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Thermo Fisher Scientific</td>
			<td>5250061</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300054</td>
			<td>Use 0.05%-0.25%</td>
		</tr>
		<tr>
			<td>Tuberculin syringe (1 ml with 0.1 ml gradations)</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td>Slip tip preferred over Luer</td>
		</tr>
		<tr>
			<td>Vented tissue culture flasks, T-75</td>
			<td>Corning Life Sciences</td>
			<td>CLS3290</td>
			<td>Can also use smaller or larger flasks, as needed</td>
		</tr>
	</tbody>
</table>

modeling primary bone tumors and bone metastasis with solid tumor graft implantation into bone

原发性骨肿瘤或实体瘤的骨转移会导致疼痛性溶骨性、成骨性或混合性溶骨性/成骨细胞性病变。这些病变会损害骨骼结构，增加病理性骨折的风险，并使患者的治疗选择有限。原发性骨肿瘤转移到远处器官，某些类型能够扩散到其他骨骼部位。然而，最近的证据表明，对于许多实体瘤，已经扩散到骨骼的癌细胞可能是最终转移到其他器官系统的细胞的主要来源。原发性骨肿瘤的大多数同系或异种移植小鼠模型涉及骨内（原位）注射肿瘤细胞悬液。实体瘤骨骼转移的一些动物模型也依赖于直接骨注射，而另一些动物则试图通过血管内或原发肿瘤器官注射细胞来概括骨转移级联的其他步骤。然而，这些模型都没有可靠地发生骨转移或发生率为 100%。此外，骨内直接注射肿瘤细胞已被证明与潜在的肺肿瘤栓塞有关。这些栓塞肿瘤细胞移植但不概括转移级联反应。我们报告了一种骨肉瘤小鼠模型，其中使用微创手术技术将新鲜或冷冻保存的肿瘤碎片（由肿瘤细胞和基质组成）直接植入胫骨近端。这些动物发展出可重复的植入、生长，并随着时间的推移出现骨质溶解和肺转移。该技术具有用于模拟实体瘤骨转移的多功能性，并且可以很容易地使用由一种或多种细胞类型、转基因细胞、患者来源的异种移植物和/或可以通过光学或高级成像跟踪的标记细胞组成的移植物。在这里，我们展示了这种技术，使用实体瘤移植物植入骨来模拟原发性骨肿瘤和骨转移。

阳性结果将与肿瘤植入和随着时间的推移进行性肿瘤生长有关。根据肿瘤类型，骨内肿瘤生长可能与进行性后肢跛行有关，但尽管伴有骨病体征，但许多肿瘤不会引起跛行。通过先进的成像记录了成功的植入，从而在与目标细胞系的骨表型（溶骨性、成骨细胞性或混合性溶骨/成骨细胞转移）相关的胫骨近端会发生进行性放射成像、μCT 或 μMRI 变化（图 1）8...

Watch this Scientific Journal Video about Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone at JoVE.com

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

骨转移模型不会均匀地发生转移或发生 100%。骨内肿瘤细胞直接注射可导致肺栓塞。我们展示了我们的技术，使用实体瘤移植物植入骨骼来模拟原发性骨肿瘤和骨转移，从而实现可重复的植入和生长。

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 ...

modeling-primary-bone-tumors-bone-metastasis-with-solid-tumor-graft

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Cancer Research

2.6K 次观看.  University of Alabama at Birmingham. 骨转移模型不会均匀地发生转移或发生 100%。骨内肿瘤细胞直接注射可导致肺栓塞。我们展示了我们的技术，使用实体瘤移植物植入骨骼来模拟原发性骨肿瘤和骨转移，从而实现可重复的植入和生长。

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

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

新一代测序为可操作的突变在固体和液体瘤检测

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

科研

癌症研究

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

内髂动脉注射显微骨转移的效率和选择性建模

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

原发肿瘤发展小鼠实验模型和腹膜转移<em&gt;通过</em&gt;原位接种卵巢癌细胞

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

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 ...

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are available for studying the significant crosstalk; however, a two-dimensional transwell system for coculture remains a classic, reliable, and easy way for this crosstalk study. Here, we present a detailed protocol that shows the coculture of bone marrow adipocytes and melanoma cells. Nevertheless, such a coculture system could not only contribute to the study of cell signal transductions of cancer cells induced by bone marrow adipocytes, but also to the future mechanistic study of bone metastasis which may reveal new therapeutic targets for bone metastasis.

Here, we present a reliable and straightforward two-dimensional (2D) coculture system for studying the interaction between tumor cells and bone marrow adipocytes, which reveals a dual effect of melanoma cell-derived factors on the bone marrow adipocytes differentiation and also poses a classic method for the mechanistic study of bone metastasis.

黑色素瘤细胞衍生因子对骨髓脂肪分化的双重影响

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

利用卡尔瓦里亚共同培养和教条法测量骨重塑和再造肿瘤-骨微环境

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...