使用光转换追踪肺转移瘤的肿瘤细胞播散
1Department of Pathology,Albert Einstein College of Medicine/Montefiore Medical Center, 2Cancer Dormancy and Tumor Microenvironment Institute,Albert Einstein College of Medicine/Montefiore Medical Center, 3Gruss-Lipper Biophotonics Center,Albert Einstein College of Medicine/Montefiore Medical Center, 4Department of Cell Biology,Albert Einstein College of Medicine/Montefiore Medical Center, 5Montefiore Einstein Cancer Center,Albert Einstein College of Medicine/Montefiore Medical Center, 6Integrated Imaging Program for Cancer Research,Albert Einstein College of Medicine/Montefiore Medical Center, 7Department of Surgery,Albert Einstein College of Medicine/Montefiore Medical Center, 8Department of Cancer and Cellular Biology, Lewis Katz School of Medicine,Fox Chase Cancer Center
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 …
Representative Results
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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 0-30 V, 0-3 A Power Supply | MPJA | 9616 PS | |
| 12 VDC, 1.2 A Unregulated Plug Supply | MPJA | 17563 PD | |
| 28 G 1 mL BD Insulin Syringe | BD | 329410 | |
| 400 nm light emitting diode array lamp | LedEngin Inc. | 897-LZPD0UA00 | Photoconversion lamp, custom-built (individual parts included below) |
| 5-0 braided silk suture with RB-1 cutting needle | Ethicon, Inc. | 774B | |
| 9 cm 2-0 silk tie | Ethicon, Inc. | LA55G | |
| Baytril 100 (enrofloxacin) | Bayer (Santa Cruz Biotechnology) | sc-362890Rx | Antibiotic used in drinking water |
| Buprenorphine | Hospira | 0409-2012-32 | Analgesic |
| Cables (Cable Assemblies) 2.1 DC JACK-STRAIGHT 72" BLACK/ZIP CORD | Mouser | 172-7426-E | |
| Cables (Cable Assemblies) 2.5 JK-ST 72" ZIP CD | Mouser | 172-0250 | |
| Chlorhexidine solution | Durvet | 7-45801-10258-3 | Chlorhexidine Disinfectant Solution |
| Compressed air canister | Falcon | DPSJB-12 | |
| Extra Fine Micro Dissecting Scissors 4" Straight Sharp/Sharp 24 mm | Roboz Surgical | RS-5912 | Sharp Micro Dissecting Scissors |
| Fiber-optic illuminator | O.C. White Company | FL3000 | Used during mouse intubation |
| Gemini Cautery Kit | Harvard Apparatus | 726067 | Cautery pen |
| Germinator 500 | CellPoint Scientific | GER 5287-120V | Bead Sterilizer |
| Graefe forceps | Roboz | RS-5135 | |
| High power LEDs – single color ultraviolet 90 watts | Mouser | LZP-D0UA00 | |
| Infrared heat lamp | Braintree Scientific | HL-1 | |
| Isoflurane SOL 250 mL PVL | Covetrus | 29405 | Anesthetic |
| Isoflurane vaporizer | SurgiVet | VCT302 | |
| Jacobson needle holder with lock | Kalson Surgical | T1-140 | |
| Labeling tape | Fisher Scientific | S68702 | |
| LED Lighting Reflectors CREE MP-L SNGL LENS REFLECTOR & LOC PIN | Mouser | 928-C11395TM | |
| Long cotton tip applicators | Medline Industries | MDS202055 | |
| Masscool / Soccket 478 / Intel Pentium 4/Celeron up to 3.4GHz / Ball Bearing / Copper Core / CPU Cooling Fan | CompUSA | #S457-1023 | |
| Micro Dissecting Scissors 4" Straight Blunt/Blunt | Roboz Surgical | RS-5980 | Blunt Micro Dissecting Scissors |
| Murine ventilator | Kent Scientific | PS-02 | PhysioSuite |
| Nair Hair Removal Lotion | Amazon | B001RVMR7K | Depilatory cream |
| Personnet mini retractor | Roboz | RS-6504 | Retractor |
| Phosphate Buffered Saline 1x | Fisher Scientific | 14190144 | PBS |
| pLenti.CAG.H2B-Dendra2.W | Addgene | 51005 | Dendra2 lentivirus |
| Puralube | Henry Schein Animal Health | 008897 | Eye Lubricant |
| Rodent intubation stand | Braintree Scientific | RIS 100 | |
| Small animal lung inflation bulb | Harvard Apparatus | 72-9083 | |
| SurgiSuite Multi-Functional Surgical Platform for Mice, with Warming | Kent Scientific | SURGI-M02 | Heated surgical platform |
| Test Leads 48" TEST LEAD BANANA – Black | Mouser | 565-1440-48-0 | |
| Test Leads 48" TEST LEAD BANANA – Red | Mouser | 565-1440-48-2 | |
| Tracheal catheter | Exelint International | 26746 | 22 G catheter |
| Wound closing system veterinary kit | Clay Adams | IN015 | Veterinary surgical stapling kit |
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Cite This Article
Friedman-DeLuca, M., Patel, P. P., Karadal-Ferrena, B., Barth, N. D., Duran, C. L., Ye, X., Papanicolaou, M., Condeelis, J. S., Oktay, M. H., Borriello, L., Entenberg, D. Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion. J. Vis. Exp. (197), e65732, doi:10.3791/65732 (2023).