用于鉴定黑色素瘤转移新介质的强大发现平台
1Department of Pathology,NYU Grossman School of Medicine, 2Interdisciplinary Melanoma Cooperative Group,Perlmutter Cancer Center, 3Department of Radiology,NYU Grossman School of Medicine, 4Preclinical Imaging Core,NYU Grossman School of Medicine, 5Ronald Perelman Department of Dermatology,NYU Grossman School of Medicine
Summary
本文介绍了用于测试黑色素瘤转移的新型候选介质及其作用机制的技术工作流程。
Abstract
转移是一个复杂的过程，需要细胞克服仅通过 体外 测定不完全建模的障碍。使用稳健，可重复 的体内 模型和标准化方法建立了系统工作流程，以识别黑色素瘤转移的新参与者。这种方法允许在特定实验阶段进行数据推断，以精确表征基因在转移中的作用。通过将转基因黑色素瘤细胞通过心内，皮内或皮下注射引入小鼠，然后用连续 体内 成像进行监测来建立模型。一旦达到预先确定的终点，就会收获和处理原发性肿瘤和/或携带转移瘤的器官以进行各种分析。肿瘤细胞可以进行分类并经受几种“组学”平台中的任何一种，包括单细胞RNA测序。器官接受影像学和免疫组织病理学分析，以量化转移的总体负担并绘制其特定的解剖位置。这种优化的管道，包括用于移植，监测，组织收获，处理和分析的标准化方案，可以用于患者来源的短期培养物以及各种固体癌症类型的已建立的人和小鼠细胞系。
Introduction
与转移性黑色素瘤相关的高死亡率加上全球黑色素瘤发病率的增加1 （预计到2025年将增加7.86%），需要新的治疗方法。靶标发现的进步取决于可重复的转移模型，这是一个高度复杂的过程。在转移级联反应的整个步骤中，黑色素瘤细胞必须克服无数障碍才能实现免疫系统的规避和远处组织的定植2。黑色素瘤细胞的弹性和适应性来自多种因素，包括它们的高基因突变负担3 和它们的神经嵴起源，这赋予了关键的表型可塑性3，4，5。在每个步骤中，转录程序允许转移性黑色素瘤细胞根据与微环境的串扰的线索从一种状态切换到另一种状态，包括免疫系统6，细胞外环境7，8和它们接触的物理屏障9 的细胞结构。例如，黑色素瘤细胞通过下调重要的免疫启动肿瘤分泌因子6的表达来逃避免疫监视。
研究描述了一种“前转移生态位”，其中黑色素瘤细胞分泌趋化因子和细胞因子来启动远处的“靶”器官进行转移10。这些发现提出了关于转移性黑色素瘤细胞的器官趋向性以及它们进入远处组织的解剖途径的重要问题。已知在静脉内渗后，黑色素瘤细胞通过淋巴管（淋巴扩散）和血管（血源性扩散）转移2，11。虽然大多数患者表现为局限性疾病，但一小部分病例表现为远处转移性疾病且无淋巴播散（阴性淋巴结受累）11，提示存在黑色素瘤的替代转移途径。
当它们定植于转移部位时，黑色素瘤细胞经历表观遗传和代谢适应12，13。为了进入和侵入新的隔室，黑色素瘤细胞使用蛋白酶14 和细胞骨架修饰11，15，使它们能够穿越并在新位置生长。靶向黑色素瘤细胞的困难在于这种适应的复杂性和数量;因此，该领域应该努力在实验中重建尽可能多的步骤和适应。尽管 体外 测定（如类器官和3D培养物）取得了许多进展16，17，但这些模型仅不完全概括 体内 转移级联反应。
小鼠模型通过在可重复性，技术可行性和人类疾病模拟之间取得平衡而显示出价值。血管内，原位和异位植入黑色素瘤细胞从患者来源的异种移植物或短期培养物到免疫受损或人源化小鼠中代表了转移性黑色素瘤靶标发现的骨干。然而，这些系统通常缺乏对转移的关键生物学约束：免疫系统。具有这种约束的同源黑色素瘤转移模型在该领域相对稀缺。这些系统，在免疫功能小鼠中开发，包括B16-F1018、YUMM家族细胞系19、SM120、D4M321、RIM322 或更近、RMS23 和M1（Mel114433）、M3（HCmel1274）、M4（B2905）24 黑色素瘤细胞系，有助于研究宿主免疫应答在黑色素瘤进展中的复杂作用。
这里，提出了黑色素瘤转移靶标鉴定的管道。随着黑色素瘤患者队列中生成的“组学”数据集越来越多，我们假设具有最大临床前景的研究是那些源于大数据集成的研究，从而导致细致的功能和机制询问25，26，27，28。通过使用小鼠模型来研究转移过程中的潜在靶标，可以解释 体内特异性事件和组织相互作用，从而增加临床转化的可能性。概述了多种量化转移负荷的方法，提供了任何给定实验结果的补充数据。描述了从各种器官中的肿瘤中分离单细胞的方案，以帮助转移细胞中基因表达的无偏倚表征，这可能先于单细胞或本体RNA测序。
Protocol
注：以下协议中涉及的动物程序已获得纽约大学机构动物护理和使用委员会（IACUC）的批准。所有程序均在实验室动物护理国际评估和认证协会（AAALAC）批准的设施中进行。 图1 描述了一般的实验方法。 1. 患者来源的黑色素瘤短期培养 （STCs） 将组织置于60mm培养皿中，其中1mL完全RPMI（RPMI 1640补充10%胎牛血清（FBS），2mM L-谷氨酰胺，1…
Representative Results
下图说明了所描述的工作流程如何应用于鉴定黑色素瘤转移的新驱动因素。 图2 总结了一项已发表的研究的结果，其中研究了沉默岩藻糖基转移酶FUT8 在体内 黑色素瘤转移中的作用26。简而言之，对人类患者血糖数据（通过凝集素阵列获得）和转录组学分析的分析显示，与从原发性到转移性黑色素瘤进展相关的α-1，6-岩藻糖水平升高，与相应岩藻糖?…
Discussion
本技术报告的目的是为黑色素瘤转移中潜在参与者的调查提供标准化的自上而下的工作流程。由于 体内 实验可能既昂贵又耗时，因此最大限度地提高效率和增加所获信息价值的策略至关重要。
必须始终使用互补方法在同一实验中交叉验证结果。例如，NuMA免疫组化染色和BLI都是定量转移负荷的补充方法，因为两者都不是全面的。虽然BLI是一种在 体内跟踪肿瘤进?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢纽约大学朗格尼健康高级研究技术部（DART），特别是实验病理学研究实验室，基因组技术中心，细胞术和细胞分选实验室，临床前成像核心，这些实验室部分由Perlmutter癌症中心支持赠款NIH / NCI 5P30CA016087。我们感谢纽约大学跨学科黑色素瘤合作小组（PI：Iman Osman博士）提供患者来源的黑色素瘤短期培养+ （10-230BM和12-273BM）的途径，这些培养物是通过IRB批准的方案获得的（通用同意研究#s16-00122和跨学科黑色素瘤合作小组研究#10362）。我们感谢Robert Kerbel博士（多伦多大学）提供113 / 6-4L和131 / 4-5B1黑色素瘤细胞系*，以及Meenhard Herlyn博士（Wistar研究所）提供WM 4265-2，WM 4257s-1，WM 4257-2黑色素瘤短期培养**。E.H.由NIH / NCI R01CA243446，P01CA206980，美国癌症协会 – 黑色素瘤研究联盟团队科学奖和NIH黑色素瘤孢子（NCI P50 CA225450;PI：I.O.）。 图 1 是使用 Biorender.com 创建的。
Materials
| #15 Scapel Blade | WPI | 500242 | For surgical procedures |
| #3 Scapel Handle | WPI | 500236 | For surgical procedures |
| 1 mL Tuberculin syringe, slip tip | BD | 309626 | Injections |
| 10 mL syringe, slip tip | BD | 301029 | Perfusion |
| 10% Formalin Sodium Buffered | EK Industries | 4499-20L | For perfusion/tissue fixative |
| 15 mL Conical | Corning | 430052 | Cell culture |
| 15 mL Conical Polypropylene Centrifuge Tubes | Falcon | 352196 | Cell culture |
| 200 Proof Ethanol | Deacon Labs | 04-355-223 | Histology |
| 22G – 22mm needle | BD | 305156 | Perfusion |
| 4-0 Vicryl Suture | Ethicon | J464G | Suture |
| 4% Carson's phosphate buffered paraformaldehyde | EMS | 15733-10 | For perfusion/tissue fixative |
| 40µm | Corning | 431750 | Tissue processing |
| 5-0 Absorbable Suture | Ethicon | 6542000 | Closure |
| 50 mL Conical | Corning | 430828 | Cell culture |
| 50mL Conical Polypropylene Centrifuge Tubes | Falcon | 352070 | Cell culture |
| 7-0 Silk suture | FST | 18020-70 | Ligature |
| 70µm | Corning | 431751 | Tissue processing |
| Anti-fade mounting media | Vector Labs | H-1000-10 | Immunofluorescence |
| Approximator applying Forceps, 10cm | WPI | 14189 | For microsurgical procedures |
| Avance | Bruker | 3 HD | NMR Console |
| Biospec 7030 | Bruker | 7030 | Micro MRI |
| BSA | Bioreg | A941 | NuMA Staining |
| Castroviejo suturing forceps, straight tips 5.5mm tying platform, 11cm | WPI | WP5025501 | For microsurgical procedures |
| Coplin Staining Jar | Bel-Art | F44208-1000 | Histology |
| DAPI | Sigma-Aldrich | D9542-1MG | Immunofluorescence |
| dCas9-KRAB | Addgene | 110820 | Genetic manipulation |
| DNase I | NEB | M0303L | Tissue processing |
| DPBS | Corning | 21-030-CM | Tissue processing |
| Extra Sharp Uncoated Single Edge Blade | GEM | 62-0167 | Tissue processing |
| Extracellular Matrix Substrate | Corning | 354234 | Consider the Growth Factor Reduced ( as alternative |
| FBS | Cytiva | SH30910.03 | Cell culture |
| Fiji Image J | Fiji Image J | Software | Immunofluorescence |
| Goat anti-rabbit HRP conjugated multimer | Thermo Fisher | A16104 | NuMA Staining |
| Goat Serum | Gibco | PCN5000 | Immunofluorescence |
| HBSS | Corning | 21-020-CV | Tissue processing |
| Hematoxylin | Richard-Allan Scientific | 7231 | Histology |
| Illumina III | PerkinElmer | CLS136334 | BLI Instrument |
| Insulin syringe 28G – 8mm needle | BD | 329424 | Injections |
| Insulin syringe 31G – 6mm needle | BD | 326730 | Injections |
| Iris Forceps, 10.2cm, Full Curve, serrated | WPI | 504478 | For perfusion and surgical procedures |
| Isoflurane USP | Covetrus | 11695067772 | Anesthesia |
| Jewelers #7 Forceps Titanium 11 cm 0.07 x 0.01 mm Tip | WPI | WP6570 | For microsurgical procedures |
| Ketamine HCl 100mg/mL | Mylan Ind. | 1049007 | Anesthesia |
| lentiCRISPRv2 | Addgene | 98290 | Genetic manipulation |
| Lycopersicon Esculentum (Tomato) Lectin, DyLight 649 | Invitrogen | L32472 | Vascular endothelial cells marker |
| MEM non-essential amino acids X 100 | Corning | 25-025-CI | Cell culture |
| Metzenbaum Scissors | WPI | 503269 | For surgical procedures |
| Microinjection Unit | KOPF | 5000 | Intracardiac injections |
| NaCl | Fisher | S25877 | NuMA Staining |
| Needle 30G x 25mm | BD | 305128 | Intracardiac Injection |
| Needle 33G x 15mm | Hamilton | 7747-01 | Intracarotid Injection |
| Needle holder, Castroviejo, 14cm, with lock, 1.2mm Serrated Jaws | WPI | 14137-G | For microsurgical procedures |
| NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice | The Jackson Laboratory | 005557 | Murine model |
| NU/J mice | The Jackson Laboratory | 002019 | Murine model |
| Nuclear Mitotic Apparatus Protein polyclonal rabbit anti-human | Abcam | 97585 | NuMA Staining |
| Penicillin-Streptomycin 10000U/mL | Gibco | 15140122 | Cell culture |
| Percoll | GE | 0891-01 | density separation solution |
| PI Classic Surgical Gloves | Cardinal Health | 2D72PT75X | Surgery |
| pLKO Tet-On | Addgene | 21915 | Genetic manipulation |
| Povidone-Iodine 10% Solution | Medline | MDS093943 | Surgery |
| Proparacaine Drops 0.5% | Akorn Pharma | AX0501 | Opthalmic local anesthetic |
| Puralube Petrolatum Opthalmic Ointment | Dechra | 83592 | Anesthesia |
| Razor Blade Double Edge Blades | EMS | 72000 | Shaving and Vibrotome Brain Slicing |
| Reflex 9mm EZ Clip | Braintree | EZC- KIT | Wound closure |
| RPMI 1640 | Corning | 10-040-CM | Cell culture |
| Scissors, Spring 10.5cm Str, 8mm Blades | WPI | 501235 | For microsurgical procedures |
| Semi-Automatic Vibrating Blade Microtome | Leica | VT1200 | Brain Slice Immunofluorescence |
| Single Channel Anesthesia Vaporizer System | Kent Scientific | VetFlo-1210S | Anesthesia |
| Smartbox Tabletop Chamber System and Exhaust Blower | EZ Systems | TT4000 | CO2 Euthanasia |
| Sterile Fenestrated Disposable Drape | Medline | NON21002 | Surgery |
| Sterile Non-Reinforced Aurora Surgical Gowns with Set-In Sleeves | Medline | DYNJP2715 | Surgery |
| T25 Flask | Corning | 430639 | Cell culture |
| Tris | Corning | 46-031-CM | NuMA Staining |
| Triton X-100 | Sigma-Aldrich | X100-500ML | Immunofluorescence |
| Troutman tying forceps, 10cm, Curved G pattern, 0.52mm tip with tying platform | WPI | WP505210 | For microsurgical procedures |
| Vessel clips 10G Pressure 5x 0.8mm Jaws, 5/pkg | WPI | 15911 | For microsurgical procedures |
| Visiopharm | Visiopharm | Visiopharm | NuMA Staining Quantification Software |
| Xylasine 100mg/mL | Akorn Pharma | 59399-111-50 | Anesthesia |
| Xylene | Fisher | X3P-1GAL | Histology |
References
- Sung, H., et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 71 (3), 209-249 (2021).
- Adler, N. R., Haydon, A., McLean, C. A., Kelly, J. W., Mar, V. J. Metastatic pathways in patients with cutaneous melanoma. Pigment Cell Melanoma Research. 30 (1), 13-27 (2017).
- Platz, A., Egyhazi, S., Ringborg, U., Hansson, J. Human cutaneous melanoma; a review of NRAS and BRAF mutation frequencies in relation to histogenetic subclass and body site. Molecular Oncology. 1 (4), 395-405 (2008).
- Alonso, S. R., et al. A high-throughput study in melanoma identifies epithelial-mesenchymal transition as a major determinant of metastasis. Cancer Research. 67 (7), 3450-3460 (2007).
- Rowe, C. J., Khosrotehrani, K. Clinical and biological determinants of melanoma progression: Should all be considered for clinical management. Australasian Journal of Dermatology. 57 (3), 175-181 (2016).
- Plebanek, M. P., et al. Pre-metastatic cancer exosomes induce immune surveillance by patrolling monocytes at the metastatic niche. Nature Communications. 8 (1), 1319 (2017).
- Orgaz, J. L., et al. Loss of pigment epithelium-derived factor enables migration, invasion and metastatic spread of human melanoma. Oncogene. 28 (47), 4147-4161 (2009).
- Ladhani, O., Sanchez-Martinez, C., Orgaz, J. L., Jimenez, B., Volpert, O. V. Pigment epithelium-derived factor blocks tumor extravasation by suppressing amoeboid morphology and mesenchymal proteolysis. Neoplasia. 13 (7), 633-642 (2011).
- Ju, R. J., Stehbens, S. J., Haass, N. K. The role of melanoma cell-stroma interaction in cell motility, invasion, and metastasis. Frontiers in Medicine – Dermatology. 5, 307 (2018).
- Wiley, H. E., Gonzalez, E. B., Maki, W., Wu, M. T., Hwang, S. T. Expression of CC chemokine receptor-7 and regional lymph node metastasis of B16 murine melanoma. Journal of the National Cancer Institute. 93 (21), 1638-1643 (2001).
- Meier, F., et al. Metastatic pathways and time courses in the orderly progression of cutaneous melanoma. British Journal of Dermatology. 147 (1), 62-70 (2002).
- Turner, N., Ware, O., Bosenberg, M. Genetics of metastasis: melanoma and other cancers. Clinical & Experimental Metastasis. 35 (5-6), 379-391 (2018).
- Ubellacker, J. M., et al. Lymph protects metastasizing melanoma cells from ferroptosis. Nature. 585 (7823), 113-118 (2020).
- Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. Taking cell-matrix adhesions to the third dimension. Science. 294 (5547), 1708-1712 (2001).
- Cunningham, C. C., et al. Actin-binding protein requirement for cortical stability and efficient locomotion. Science. 255 (5042), 325-327 (1992).
- Unger, C., et al. Modeling human carcinomas: physiologically relevant 3D models to improve anti-cancer drug development. Advanced Drug Delivery Reviews. 79-80, 50-67 (2014).
- Fong, E. L., Harrington, D. A., Farach-Carson, M. C., Yu, H. Heralding a new paradigm in 3D tumor modeling. Biomaterials. 108, 197-213 (2016).
- Nakamura, K., et al. Characterization of mouse melanoma cell lines by their mortal malignancy using an experimental metastatic model. Life Science. 70 (7), 791-798 (2002).
- Meeth, K., Wang, J. X., Micevic, G., Damsky, W., Bosenberg, M. W. The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations. Pigment Cell Melanoma Research. 29 (5), 590-597 (2016).
- Koya, R. C., et al. BRAF inhibitor vemurafenib improves the antitumor activity of adoptive cell immunotherapy. Cancer Research. 72 (16), 3928-3937 (2012).
- Jenkins, M. H. Multiple murine BRaf(V600E) melanoma cell lines with sensitivity to PLX4032. Pigment Cell Melanoma Research. 27 (3), 495-501 (2014).
- Tuncer, E., et al. SMAD signaling promotes melanoma metastasis independently of phenotype switching. The Journal of Clinical Investigation. 129 (7), 2702-2716 (2019).
- Schwartz, H., et al. Incipient Melanoma Brain Metastases Instigate Astrogliosis and Neuroinflammation. Cancer Research. 76 (15), 4359-4371 (2016).
- Perez-Guijarro, E., et al. Multimodel preclinical platform predicts clinical response of melanoma to immunotherapy. Nature Medicine. 26 (5), 781-791 (2020).
- Krepler, C., et al. A Comprehensive Patient-Derived Xenograft Collection Representing the Heterogeneity of Melanoma. Cell Reports. 21 (7), 1953-1967 (2017).
- Agrawal, P., et al. A systems biology approach identifies FUT8 as a driver of melanoma metastasis. Cell. 31 (6), 804-819 (2017).
- Hanniford, D., et al. Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis. Cancer Cell. 37 (1), 55-70 (2020).
- Kim, H., et al. PRMT5 control of cGAS/STING and NLRC5 pathways defines melanoma response to antitumor immunity. Science Translational Medicine. 12 (551), (2020).
- de Miera, E. V., Friedman, E. B., Greenwald, H. S., Perle, M. A., Osman, I. Development of five new melanoma low passage cell lines representing the clinical and genetic profile of their tumors of origin. Pigment Cell Melanoma Research. 25 (3), 395-397 (2012).
- Morsi, A., et al. Development and characterization of a clinically relevant mouse model of melanoma brain metastasis. Pigment Cell Melanoma Research. 26 (5), 743-745 (2013).
- Huynh, C., et al. Efficient in vivo microRNA targeting of liver metastasis. Oncogene. 30 (12), 1481-1488 (2011).
- Zou, C., et al. Experimental variables that affect human hepatocyte AAV transduction in liver chimeric mice. Molecular Therapy Methods and Clinical Development. 18, 189-198 (2020).
- Kleffman, K., et al. Melanoma-secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis. bioRxiv. , 854885 (2019).
- Curtis, A., Calabro, K., Galarneau, J. R., Bigio, I. J., Krucker, T. Temporal variations of skin pigmentation in C57BL/6 mice affect optical bioluminescence quantitation. Molecular Imaging and Biology. 13 (6), 1114-1123 (2011).
- Sil, P., Wong, S. W., Martinez, J. More than skin deep: autophagy is vital for skin barrier function. Frontiers in Immunology. 9, 1376 (2018).
- Chen, S., et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell. 160 (6), 1246-1260 (2015).
- Hart, T., et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell. 163 (6), 1515-1526 (2015).
- Wang, T., et al. Identification and characterization of essential genes in the human genome. Science. 350 (6264), 1096-1101 (2015).
- Edgar, R., Domrachev, M., Lash, A. E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Research. 30 (1), 207-210 (2002).
- Lappalainen, I., et al. The European Genome-phenome Archive of human data consented for biomedical research. Nature Genetics. 47 (7), 692-695 (2015).
- Cerami, E., et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discovery. 2 (5), 401-404 (2012).
- Grossman, R. L., et al. Toward a shared vision for cancer genomic data. New England Journal of Medicine. 375 (12), 1109-1112 (2016).
Cite This Article
Shadaloey, A. A. S., Karz, A., Moubarak, R. S., Agrawal, P., Levinson, G., Kleffman, K., Aristizabal, O., Osman, I., Wadghiri, Y. Z., Hernando, E. A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis. J. Vis. Exp. (181), e63186, doi:10.3791/63186 (2022).