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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</ol>

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 &amp;deg;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&amp;rsquo;s modified Eagle&amp;rsquo;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 (&amp;ndash; 80 &amp;deg;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&amp;reg; 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

Research

JoVE Journal

Cancer Research

2.7K 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

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 ...

科研

癌症研究

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

应用 Fraumeni 综合征诱导多潜能干细胞建模骨肉瘤

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

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, ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

医学

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

胫骨肉瘤细胞注射生成原位骨肉瘤和肺转移小鼠模型

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is essential to understand the process and related mechanisms of prostate cancer bone metastasis for developing new therapeutics. For this, an animal model of bone metastasis is an essential tool. Here, we report detailed procedures to generate a bone metastasis mouse model via intra-cardiac injection of prostate cancer cells. A bioluminescence imaging system can determine whether prostate cancer cells have been accurately injected into the heart and monitor cancer cell metastasis since it has great advantages in monitoring metastatic lesion development. This model replicates the natural development of disseminated cancer cells to form micro-metastases in the bone and imitates the pathological process of prostate cancer bone metastasis. It provides an effective tool for further exploration of the molecular mechanisms and the in vivo therapeutic effects of this disease.

Here, we present a protocol for the intra-cardiac injection of human prostate cancer cells to generate a mouse model with bone metastasis lesions.

心内注射人前列腺癌细胞以创建骨转移异种移植小鼠模型

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

小鼠原位转移性乳腺癌模型的建立及小鼠根治性乳房切除术

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...