作者要感谢 Wade Koba 在显微计算机断层扫描 （S10RR029545） 方面的帮助，感谢分析成像设施的 Vera DesMarais 和 Hillary Guzik 在显微镜方面的培训和协助，感谢 Einstein Montefiore 癌症中心、国家癌症研究所 （P30CA013330、R01CA21248、R01CA255153）、Gruss Lipper 生物光子学中心、癌症研究综合成像计划、 亨利·惠康爵士博士后奖学金 （221647/Z/20/Z） 和 METAvivor 职业发展奖。

本协议中描述的所有程序均已按照脊椎动物使用的指南和规定执行，包括阿尔伯特爱因斯坦医学院机构动物护理和使用委员会的事先批准。
在手术前，使用表达光转换/光开关蛋白质的癌细胞在小鼠中产生肺转移;已经发表了几种肺转移产生的方案 26,27,28。
1. 手术准备
<ol><li>为小鼠准备（脱毛和插管）、手术和恢复准备不同的工作区域。</li>
	<li>在高压灭菌器中对所有手术器械进行消毒。 注意：由于此手术仅使用尖端技术，因此可以使用热珠灭菌器对器械进行再灭菌以进行后续手术。</li>
	<li>打开加热的手术平台，使其达到并稳定在37-40°C。</li>
	<li>用5%异氟烷诱导麻醉，然后将异氟烷降低至3%。在整个手术过程中定期进行脚趾捏合测试，以评估麻醉充分性。</li>
	<li>将麻醉的小鼠置于右侧卧位。通过在该区域涂抹脱毛膏去除左上胸部/侧身的毛发（见 图 1A）。用水蘸湿一块纱布或纸巾，然后擦去头发和脱毛膏。必要时重复，直到从手术区域去除所有毛发。 注意： 不要将脱毛霜留在鼠标上超过 20 秒，因为这会损坏皮肤。</li>
	<li>用 2-0 丝缝合线在 22 G 导管底部上方 ~3 毫米处打一个双结。留下 2 英寸长的尾巴。</li>
	<li>如前所述，用该导管插管小鼠29,30。为了确认插管成功，将充气球连接到导管末端并轻轻挤压。 注意：成功插管的小鼠将经历双侧胸部隆起并伴有球茎挤压。</li>
	<li>用双结将 2-0 丝缝合线紧紧固定在小鼠鼻子周围，以将插管导管固定到位。</li>
</ol>2.手术暴露肺部
注意：在罩或层流柜中执行手术的所有步骤（图1），包括光转换，以避免污染手术区域。
<ol><li>用消毒肥皂洗手，并戴上新的无菌手套。 注意：由于此手术仅使用尖端技术，因此也可以使用非无菌手套。建议使用含酒精的消毒剂对非无菌手套进行消毒。</li>
	<li>准备0.1mg / kg剂量的丁丙诺啡（0.03mg / mL）并皮下注射镇痛。 注意：多模式镇痛药，包括局部止痛剂和非甾体抗炎药，如果预计不会干扰感兴趣的生物学，则应与丁丙诺啡联合使用。</li>
	<li>将眼药膏涂抹在两只小鼠眼睛上，以防止角膜损伤。</li>
	<li>将鼠标置于加热手术平台上的右侧卧位。</li>
	<li>将呼吸机连接到插管导管。注意保持导管稳定，因为即使是轻微的运动也会干扰导管的位置并导致通风不良。</li>
	<li>观察稳定的、由呼吸机控制的双侧胸部起伏。</li>
	<li>用标签胶带将四肢固定在手术平台上。通过在小鼠背部放置另一条胶带并将背侧皮肤和毛皮固定到手术平台上来稳定手术区域（图1A）。</li>
	<li>打开消毒过的手术器械。</li>
	<li>将洗必泰溶液涂在小鼠的皮肤上以对手术部位进行消毒。</li>
	<li>确定胸骨左侧 ~7 毫米和肋下缘上方 ~7 毫米的区域。用镊子将皮肤抬起该区域，并使用锋利的显微解剖剪刀做一个~10毫米的圆形切口，注意只切开皮肤。</li>
	<li>去除胸腔上的软组织（图1B）。用烧灼笔烧灼两端的任何主要血管。 注意：除了切除皮肤上 10 毫米圆形切口下的所有软组织外，从周边去除一些额外的非肌肉软组织有助于手术的未来步骤。</li>
	<li>用一只手，用镊子抬高第6或第7肋骨。用另一只手，使用钝显微解剖剪刀的单刀片 - 平行于胸壁的角度，圆形边缘朝下 - 并小心地刺穿第6或第7肋间肌肉进入胸腔（图1C）。根据需要轻轻旋转剪刀，在肋间隙切开~10毫米的切口，注意避免接触肺组织和割伤肋骨。</li>
	<li>将压缩空气轻轻排出到肋间切口，以增加肺和胸壁之间的空间。首先在手腕或手上排出空气，以找到合适的流动强度并清除喷嘴上的任何碎屑，然后在切口处短时间排出以防止肺损伤。</li>
	<li>用一只手，用镊子抬高第6或第7肋骨。另一只手握住牵开器并将其插入肋间切口。将牵开器的叶片定向到与胸壁平行，注意避免接触肺本身（图1D）。</li>
	<li>牵开器就位后，慢慢松开手柄，让牵开器打开并暴露肺部（图1E）。 图2A 显示了光转换前暴露肺转移瘤的代表性图像，包括它们在肉眼中的样子，以及使用宽场照明时在FITC（绿色，非光转换）和TRITC（红色，光转换）荧光通道中的图像。</li>
</ol>3.肺转移光转化
注意：有关以下步骤的详细信息和变体，请参阅讨论。
<ol><li>轻轻地将2-3滴加热至37°C的无菌PBS涂抹在暴露的肺组织上，以防止干燥。</li>
	<li>在鼠标上放置一块带有小切口的无菌铝箔。将切口直接放置在暴露的肺组织上，并调整其大小以仅暴露胸部的开放部分和周围几毫米的软组织和皮肤，以确保仅暴露的肺进行光转换。</li>
	<li>在鼠标和铝箔上放置一个带有切口的盒子。确保切口直接位于暴露的肺部上方。</li>
	<li>将光转换灯直接放在盒子上的切口上（图 1F）。</li>
	<li>打开光转换灯，让暴露的肺组织连续照明6分钟。 注意：使用呼吸机上的生理监测程序监测小鼠的生命体征。</li>
	<li>光转换后，关闭灯，从鼠标上取下灯、盒子和铝箔面罩。</li>
</ol>4. 闭合胸壁的程序
<ol><li>重复步骤 2.13 将肺与胸壁分开。</li>
	<li>小心地握住卷开器手柄并挤压以关闭刀片。</li>
	<li>将闭合牵开器移出肋间隙，注意避免接触肺组织。</li>
	<li>使用 5-0 真丝缝合线，用简单的连续缝合模式闭合肋间切口，该缝合模式包括切口两侧的肋骨（图 1G）。拉紧缝合线以确保伤口边缘是并列的，并重新建立胸壁的完整性（图1H）。注意不要过度收紧缝合线，因为这可能会撕裂肋间肌肉。</li>
	<li>通过将缝合线的两端打结 4 倍来固定缝合线。将缝合线尾部剪得尽可能靠近结。</li>
	<li>用手术钉闭合皮肤。</li>
	<li>用 1 mL 胰岛素注射器和 28 G 针头从胸腔中去除多余的空气。用触觉定位剑突，将针插入正下方，向左肩推进，通过横膈膜进入胸腔。将注射器抽回至~1mL;从鼠标上取下针头并排出注射器中的空气。重复此过程 3-4 次。 注意： 注意不要刺穿任何内脏器官。此外，重要的是使用 1 mL 注射器并重复该过程 3-4 次，而不是使用更大容量的注射器并试图一次去除所有空气。使用更大容量的注射器可能会导致胸腔真空过大，并导致肺组织膨胀和损伤。</li>
	<li>关闭异氟烷。</li>
	<li>继续用 100% 氧气通气，直到小鼠出现苏醒迹象。</li>
	<li>一旦小鼠出现觉醒迹象，将插管导管与呼吸机断开，切断鼻子周围的缝合线，然后拔管小鼠。</li>
	<li>解开鼠标的胶带并将其转移到放置在加热灯下方 ~2 英尺处的干净笼子中。监控直到完全恢复。如果小鼠出现呼吸困难或缺乏活动能力的迹象，请用 5% 异氟醚麻醉 5 分钟，通过观察脚趾捏合时反射丧失来确保充分麻醉，并通过颈椎脱位实施安乐死。</li>
	<li>一旦小鼠从麻醉中完全苏醒并开始活动，再准备0.1mg / kg剂量的丁丙诺啡并皮下注射用于术后镇痛。</li>
	<li>手术后，确保单独饲养小鼠。在饮用水中提供抗生素（恩诺沙星，终浓度 0.4 mg/mL）。</li>
	<li>在手术后的 3 天内，每天检查鼠标两次是否有痛苦迹象。对于疼痛管理，每 4-6 小时皮下注射 0.1 mg/kg 丁丙诺啡 （0.03 mg/mL）。如果小鼠出现感染、不动或呼吸困难的迹象，则对小鼠实施安乐死。第三天后，每天可以检查一次小鼠。</li>
</ol>5. 使用组织清除对光转化细胞进行样品处理和检测
<ol><li>在光转换手术后3至5天（或所需的时间长度）之间，用5%异氟烷麻醉小鼠，诱导后将异氟烷从5%减少到2.5%，通过观察脚趾捏伤时反射的丧失来确保充分麻醉，并用10mL室温PBS进行终末经心灌注，然后用10mL冷却至4°C的10mL4%多聚甲醛，如前所述31。</li>
	<li>如前所述，收集感兴趣的器官并对其进行光学清除31.</li>
	<li>如前所述，用光片显微镜对清除的组织进行成像31.或者，将清除的组织放入玻璃底培养皿中，并在 FITC 和 TRITC 通道中成像，以使用共聚焦、转盘或多光子显微镜观察绿色（非光转换）和红色（光转换）细胞。</li>
</ol>6. 使用组织分解对光转化细胞进行样品处理和检测
<ol><li>用5%异氟醚麻醉小鼠5分钟，通过观察脚趾捏合时反射丧失来确保充分麻醉，并在光转换后3-5天通过颈椎脱位处死。</li>
	<li>如前所述收集和消化感兴趣的器官13.</li>
	<li>如前所述，将消化的组织铺板并将它们放入培养箱中过夜粘附过夜。</li>
	<li>第二天，对 FITC 和 TRITC 通道中的接种细胞进行成像，以可视化绿色（非光转换）和红色（光转换）细胞，如前所述13。</li>
</ol>

检查肺转移瘤的肿瘤细胞播散

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作者没有利益冲突需要声明。

在本文中，我们描述了一种用于肺中肿瘤细胞选择性光转化的手术方案。该技术使研究人员能够选择性地标记肺部的肿瘤细胞，并通过在以后的时间点在整个身体中重新识别它们来跟踪它们的命运，从而促进对肺转移转移的研究。使用该方案，可以可视化接受光转换手术的小鼠的大脑，肝脏和非光转换右肺中的光转换细胞，表明这些细胞从光转换的肺转移瘤重新形成到这些三级部位。我们在小鼠?...

转移是癌症相关死亡的主要原因1.当原发肿瘤的细胞扩散到全身并增殖到远处器官中临床可检测到的肿瘤时，就会发生转移性癌症 2,3。
尽管转移通常被认为是肿瘤细胞从原发肿瘤扩散并定植在远处器官的单向过程4，但越来越多的临床和实验证据表明，一个更复杂的多向过程正在发挥作用。已经表明，循环肿瘤细胞可以重新接种原发肿瘤（如果仍然存在）5,6,7,8,9，来自现有转移灶的肿瘤细胞可以移动到三级部位并产生新的病变10,11,12,13.事实上，最近的基因组分析证据表明，一些转移性病变不是来自原发性肿瘤，而是来自其他转移瘤——这种现象被称为“转移灶”或“转移到转移播种”14,15,16。即使在切除原发肿瘤后，转移到转移播种仍会使疾病过程永久化，从而增加转移负担，并降低患者的生活质量和生存率。因此，了解转移到转移播种背后的过程对于完善转移性疾病患者的治疗策略至关重要。
尽管具有潜在的严重临床影响，但对转移到转移播种知之甚少，部分原因是后勤和技术限制。人体研究受到临床样本匮乏的限制。转移性病灶的临床切除和活检并不常见，看似健康的器官的活检也不常见，单个播散性肿瘤细胞可能潜伏其中。这意味着人体研究通常只能使用原发肿瘤仍然存在或以前被切除但仍可供研究人员使用的个体的尸检样本。当有此类样本时，必须使用测序方法14进行癌症进展的谱系分析。然而，匹配的原发肿瘤和转移瘤的批量测序不具备全面谱系追踪所需的灵敏度。例如，对一个病灶进行批量测序可能会发现在其任何匹配病灶中都无法检测到的亚克隆。在这种情况下，人们将无法确定该亚克隆的起源。它可能以低于检测限的频率存在于原发肿瘤或其他转移灶中，或者它可能在发现它的转移性病灶最初定植后出现。单细胞测序提供了更高的灵敏度，但其高成本限制了该技术的大规模应用。这些研究的回顾性也意味着它们对不同时间点的短暂转移事件和疾病状况的见解有限。
在动物模型中，最近的技术进步现在允许具有高空间和时间分辨率的前瞻性系统发育图谱17,18,19,20。这些技术利用CRISPR / Cas9基因组编辑来设计具有进化条形码的细胞 - 随着时间的推移积累的可遗传突变。测序后，可以根据其条形码17、18、19、20 的突变谱来追踪每个细胞的谱系。事实上，这种技术已经被用于绘制转移到转移的播种图。在最近的一篇论文中，Zhang等人证明，骨转移中的乳腺癌和前列腺癌细胞从骨骼重新播散到多个器官中播种继发性转移21。
虽然这些新方法在生成详细、高分辨率的癌症进展系统发育图谱方面具有巨大潜力，但对于那些研究转移到转移播种事件的确切时间以及促进或预防它们的原因的人来说，它们是非常不切实际的。填补这些知识空白对于完善我们对转移性癌症的理解和治疗至关重要，但明显缺乏促进此类研究的技术。为了满足这一需求，我们最近开发了一种新技术，并允许我们通过转移部位（肺）的光转换来特异性标记肿瘤细胞，并随后在三级器官中重新识别它们。使用这种技术，我们最近发现乳腺癌细胞确实从肺转移和种子三级器官重新播散13。该技术还可用于在狭窄的窗口内确定再播散事件的时间并量化红化肿瘤细胞，从而促进研究红化细胞的有机趋向性以及促进/阻止再播散的因素。
虽然以前曾使用光转换和局部诱导的 cre/lox 系统永久替换一种荧光蛋白来标记和跟踪肿瘤细胞 11,22,23，但据我们所知，没有针对肿瘤细胞的时空标记方法经过优化以靶向肺 - 这是被诊断患有 14 种最常见癌症中任何一种的男性和女性中最常见的转移部位之一 24.任何癌细胞类型和任何肺转移产生的方案都可以与我们的程序一起使用，使其对转移研究人员广泛有用。所有用于产生肺转移的癌细胞都应该表达一种光转换或光切换的蛋白质，研究人员可以根据他们的特定需求和资源选择使用哪种蛋白质。在这项研究中，我们使用了 6DT1 乳腺癌细胞，这些细胞稳定地表达标记在组蛋白 H2B 上的光转换绿到红荧光蛋白 Dendra2（6DT1-Dendra2 细胞）25。我们将 5.0 × 104 6DT1-Dendra2 细胞注射到雌性 Rag2-/- 小鼠的第四个乳腺脂肪垫中。原发性肿瘤在注射后 12 至 16 天之间可触及，并且在实验期间未切除。肿瘤细胞注射后 19 至 26 天发生自发性肺转移。在肿瘤细胞注射后 26 至 29 天进行光转换手术。由于肺转移负荷，小鼠在手术后72小时处死。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0-30 V, 0-3 A Power Supply</td>
			<td>MPJA</td>
			<td>9616 PS</td>
			<td></td>
		</tr>
		<tr>
			<td>12 VDC, 1.2 A Unregulated Plug Supply</td>
			<td>MPJA</td>
			<td>17563 PD</td>
			<td></td>
		</tr>
		<tr>
			<td>28 G 1 mL BD Insulin Syringe</td>
			<td>BD</td>
			<td>329410</td>
			<td></td>
		</tr>
		<tr>
			<td>400 nm light emitting diode array lamp</td>
			<td>LedEngin Inc.</td>
			<td>897-LZPD0UA00</td>
			<td>Photoconversion lamp, custom-built (individual parts included below)</td>
		</tr>
		<tr>
			<td>5-0 braided silk suture with RB-1 cutting needle</td>
			<td>Ethicon, Inc.</td>
			<td>774B</td>
			<td></td>
		</tr>
		<tr>
			<td>9 cm 2-0 silk tie</td>
			<td>Ethicon, Inc.</td>
			<td>LA55G</td>
			<td></td>
		</tr>
		<tr>
			<td>Baytril 100 (enrofloxacin)</td>
			<td>Bayer (Santa Cruz Biotechnology)</td>
			<td>sc-362890Rx</td>
			<td>Antibiotic used in drinking water</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Hospira</td>
			<td>0409-2012-32</td>
			<td>Analgesic</td>
		</tr>
		<tr>
			<td>Cables (Cable Assemblies) 2.1 DC JACK-STRAIGHT 72&#34;&#160; BLACK/ZIP CORD</td>
			<td>Mouser</td>
			<td>172-7426-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cables (Cable Assemblies) 2.5 JK-ST 72&#34; ZIP CD</td>
			<td>Mouser</td>
			<td>172-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorhexidine solution</td>
			<td>Durvet</td>
			<td>7-45801-10258-3</td>
			<td>Chlorhexidine Disinfectant Solution</td>
		</tr>
		<tr>
			<td>Compressed air canister</td>
			<td>Falcon</td>
			<td>DPSJB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Micro Dissecting Scissors 4&#34; Straight Sharp/Sharp 24 mm</td>
			<td>Roboz Surgical</td>
			<td>RS-5912</td>
			<td>Sharp Micro Dissecting Scissors</td>
		</tr>
		<tr>
			<td>Fiber-optic illuminator</td>
			<td>O.C. White Company</td>
			<td>FL3000</td>
			<td>Used during mouse intubation</td>
		</tr>
		<tr>
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			<td></td>
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			<td>Isoflurane vaporizer</td>
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			<td>VCT302</td>
			<td></td>
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			<td>Jacobson needle holder with lock</td>
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			<td>T1-140</td>
			<td></td>
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			<td>S68702</td>
			<td></td>
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			<td>LED Lighting Reflectors CREE MP-L SNGL LENS REFLECTOR &#38; LOC PIN</td>
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			<td>Masscool / Soccket 478 / Intel Pentium 4/Celeron up to 3.4GHz / Ball Bearing / Copper Core / CPU Cooling Fan</td>
			<td>CompUSA</td>
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			<td>Micro Dissecting Scissors 4&#34; Straight Blunt/Blunt</td>
			<td>Roboz Surgical</td>
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			<td>Murine ventilator</td>
			<td>Kent Scientific&#160;</td>
			<td>PS-02</td>
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			<td>Nair Hair Removal Lotion</td>
			<td>Amazon</td>
			<td>B001RVMR7K</td>
			<td>Depilatory cream</td>
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			<td>Personnet mini retractor</td>
			<td>Roboz</td>
			<td>RS-6504</td>
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			<td>Phosphate Buffered Saline 1x</td>
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			<td>14190144</td>
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			<td>pLenti.CAG.H2B-Dendra2.W</td>
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			<td>SurgiSuite Multi-Functional Surgical Platform for Mice, with Warming</td>
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			<td>SURGI-M02</td>
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			<td>565-1440-48-0</td>
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			<td>565-1440-48-2</td>
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			<td>26746</td>
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		</tr>
	</tbody>
</table>

tracking tumor cell dissemination from lung metastases using photoconversion

转移 - 癌症的全身扩散 - 是癌症相关死亡的主要原因。虽然转移通常被认为是一个单向过程，其中来自原发肿瘤的细胞播散和播种转移，但现有转移中的肿瘤细胞也可以 在称为“转移转移”或“转移转移播种”的过程中重新播散并在三级部位产生新的病变。转移到转移播种可能会增加转移负担，降低患者的生活质量和生存率。因此，了解这种现象背后的过程对于完善转移性癌症患者的治疗策略至关重要。
对转移到转移播种知之甚少，部分原因是后勤和技术限制。关于转移到转移播种的研究主要依赖于测序方法，这对于研究转移到转移播种事件的确切时间或促进或预防它们的研究人员来说可能不实用。这凸显了缺乏促进转移到转移播种研究的方法。为了解决这个问题，我们开发并描述了一种用于肺转移的选择性光转化的小鼠手术方案，允许对从肺到三级部位的肿瘤细胞进行特异性标记和命运跟踪。据我们所知，这是研究肿瘤细胞再扩散和从肺部转移到转移的唯一方法，不需要基因组分析。

Metastasis is the leading cause of cancer-related deaths1.&#160;Metastatic cancer arises when cells from the primary tumor disseminate throughout the body and proliferate into clinically detectable tumors in distant organs2,3.
Although metastasis is commonly thought of as a unidirectional process wherein tumor cells disseminate from the primary tumor and colonize distant organs4, increasing clinical and experimental evidence suggests that a more complex, multi-directional process is at play. It has been shown that circulating tumor cells can reseed the primary tumor (if still in place)5,6,7,8,9, and tumor cells from existing metastatic foci can travel to tertiary sites and give rise to new lesions10,11,12,13. Indeed, evidence from recent genomic analyses suggests that some metastatic lesions arise not from the primary tumor, but from other metastases-a phenomenon known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding&#34;14,15,16. Metastasis-to-metastasis seeding can perpetuate the disease process even after removal of the primary tumor, increasing metastatic burden, and decreasing patient quality of life and survival. Therefore, understanding the processes behind metastasis-to-metastasis seeding is crucial to refining treatment strategies for patients with metastatic disease.
Despite the potentially severe clinical implications, little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Human studies are limited by a paucity of clinical samples. Clinical resection and biopsy of metastatic lesions are uncommon, as is the biopsy of seemingly healthy organs, where single disseminated tumor cells may lurk. This means that human studies are typically only possible using autopsy samples from individuals whose primary tumors are either still in place or were previously resected but are still available to researchers. When such samples are available, lineage analyses of cancer progression must be performed using sequencing methods14. However, bulk sequencing of matched primary tumors and metastases does not have the sensitivity needed for comprehensive lineage tracing. For instance, bulk sequencing of one lesion may reveal a subclone that is undetectable in any of its matched lesions. In this case, one would be unable to determine the origin of this subclone. It may have been present in the primary tumor or another metastasis at a frequency below the limit of detection, or it may have arisen after the initial colonization of the metastatic lesion it was found in. Single-cell sequencing provides increased sensitivity, but its high cost limits the large-scale application of this technique. The retrospective nature of these studies also means that they provide limited insight into transient metastatic events and the disease landscape at different time points.
In animal models, recent technological advances now allow for prospective phylogenetic mapping with high spatial and temporal resolution17,18,19,20. These techniques utilize CRISPR/Cas9 genome editing to engineer cells with an evolving barcode - heritable mutations that accumulate over time. Upon sequencing, the lineage of each cell can be traced based on the mutational profile of its barcode17,18,19,20. Indeed, such technology is already being used to map metastasis-to-metastasis seeding. In a recent paper, Zhang et al. demonstrated that breast and prostate cancer cells in bone metastases redisseminate from the bone to seed secondary metastases in multiple organs21.
While these novel methods have great potential to generate detailed, high-resolution phylogenetic maps of cancer progression, they are highly impractical for those studying the exact timing of metastasis-to-metastasis seeding events and what promotes or prevents them. Filling these knowledge gaps is crucial to refining our understanding and treatment of metastatic cancer, but there is a noticeable lack of technologies to facilitate such studies. To address this need, we recently developed - and present herein - a novel technique that allows us to specifically mark tumor cells via photoconversion in a metastatic site (the lung) and subsequently reidentify them in tertiary organs. Using this technique, we recently showed that breast cancer cells do redisseminate from lung metastases and seed tertiary organs13. This technique may also be used to determine the timing of redissemination events within a narrow window and quantify redisseminated tumor cells, facilitating the study of organotropism of redisseminated cells and what promotes/prevents redissemination.
While photoconversion and locally inducible cre/lox systems that permanently replace one fluorescent protein with another have been previously used to mark and track tumor cells11,22,23, to our knowledge, no approach for spatiotemporal marking of tumor cells has been optimized to target the lung - one of the most common sites of metastasis among men and women diagnosed with any of the 14 most common cancers24. Any cancer cell type and any protocol for lung metastasis generation may be used with our procedure, making it broadly useful for metastasis researchers. All cancer cells used to generate lung metastases should express a photoconvertible or photo-switchable protein, and researchers may choose which protein to use based on their specific needs and resources. In this study, we used 6DT1 breast cancer cells that stably expressed the photoconvertible green-to-red fluorescent protein Dendra2 (6DT1-Dendra2 cells)25 tagged to the histone H2B. We injected 5.0 &#215; 104 6DT1-Dendra2 cells into the fourth mammary fat pad of female Rag2-/- mice. Primary tumors were palpable between 12 and 16 days after injection and were not resected for the duration of the experiment. Spontaneous lung metastases developed between 19 and 26 days after tumor cell injection. Photoconversion surgeries were performed between 26 and 29 days after tumor cell injection. Mice were sacrificed by 72 h post surgery due to lung metastasis burden.

作者聚焦：使用新的体内技术解码转移转移播种以追踪乳腺癌扩散 

使用光转换追踪肺转移瘤的肿瘤细胞播散

Recent evidence suggests that existing metastatic lesions can act as a source of disseminating tumor cells that give rise to new lesions. This process is called metastasis to metastasis seeding and can increase tumor burden and decrease the quality of life and survival of cancer patients. Our method was developed to study metastasis to metastasis seeding and the factors that influence it.This technique has helped our lab establish that breast cancer cells can spread from lung metastases and enabled us to investigate factors that promote or prevent metastasis. These findings may guide future research on slowing the progression of metastatic cancer. The lungs are a common site of metastasis, but very few techniques allow for the study of tumor cell dynamics in vivo.This technique enables the tracking of tumor cells in live mice facilitating the study of lung metastasis specific dissemination pathways and therapies. Metastatic cancer is often an incurable and fatal disease. This technique can be used to study ways to reduce its spread leading to therapies that extend the lives of patients.

该方案中描述的手术步骤如 图1所示。简而言之，将小鼠麻醉，并从左胸部去除毛发。然后对小鼠进行插管和通气，这允许小鼠在胸腔打开时接受氧气。去除软组织以暴露胸腔，并在第6或第 7肋间肌 上切开一个切口。将牵开器插入肋间裂口并松开以扩散相邻肋骨并暴露左肺及其上的任何光转换转移。然后将肺和转移瘤暴露在蓝光下，以光转换暴露的病变。光...

Watch this Scientific Journal Video about Decoding Metastasis-to-Metastasis Seeding Using a New In Vivo Technique for Tracking Breast Cancer Spread at JoVE.com

我们提出了一种研究肺转移肿瘤细胞再播散的方法，涉及肺转移的选择性光转化手术方案，然后鉴定三级器官中的红化肿瘤细胞。

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

最近的证据表明，现有的转移性病灶可以作为播散肿瘤细胞的来源，从而产生新的病灶。这个过程称为转移到转移播种，会增加肿瘤负荷，降低癌症患者的生活质量和生存率。我们开发的方法是为了研究转移到转移播种以及影响它的因素。这项技术帮助我们的实验室确定乳腺癌细胞可以从肺转移扩散，并使我们能够研究促进或预防转移的因素。这些发现可能指导未来减缓转移性癌症进展的研究。肺是转移的常见部位，但很少有技术可以研究体内肿瘤细胞动力学。该技术能够追踪活小鼠中的肿瘤细胞，促进肺转移特异性播散途径和疗法的研究。转移性癌症通常是一种无法治愈的致命疾病。该技术可用于研究减少其传播的方法，从而延长患者生命的治疗方法。

tracking-tumor-cell-dissemination-from-lung-metastases-using

Research

JoVE Journal

Cancer Research

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

822 次观看.  Albert Einstein College of Medicine/Montefiore Medical Center. 最近的证据表明，现有的转移性病灶可以作为播散肿瘤细胞的来源，从而产生新的病灶。这个过程称为转移到转移播种，会增加肿瘤负荷，降低癌症患者的生活质量和生存率。我们开发的方法是为了研究转移到转移播种以及影响它的因素。这项技术帮助我们的实验室确定乳腺癌细胞可以从肺转移扩散，并使我们能够研究促进或预防转移的因素。这些发现可能指导未来减?...