腹膜内移植在小鼠中产生急性髓系白血病
Summary
在这里，腹膜内注射白血病细胞用于在小鼠中建立和繁殖急性髓性白血病（AML）。这种新方法在AML细胞的连续移植中是有效的，并且可以作为那些可能在小鼠静脉注射中遇到困难和不一致的人的替代方案。
Abstract
治疗急性髓系白血病（AML）和涉及持续性白血病干细胞（LSCs）的相关复发的新疗法的需求尚未得到满足。一种实验性AML啮齿动物模型测试基于通过眶后注射在受体小鼠中成功移植这些细胞的疗法，充满了挑战。本研究的目的是开发一种简单、可靠和一致的方法，使用腹膜内途径生成强大的 AML 小鼠模型。在本协议中，骨髓细胞用表达人MLL-AF9融合癌蛋白的逆转录病毒转导。测试了谱系阴性（Lin-）和Lin-Sca-1 + c-Kit+（LSK）群体作为供体LSC在原发性AML发展中的效率，并采用腹膜内注射作为产生AML的新方法。在连续移植中对腹膜内和眶后注射进行比较，以比较和对比两种方法。用人MLL-AF9病毒转导的Lin细胞和LSK细胞都很好地移植在受体的骨髓和脾脏中，导致全面的AML。连续移植时腹膜内注射供体细胞在受者中建立AML，并通过流式细胞术、qPCR和组织学分析在受者的血液、骨髓、脾脏和肝脏中检测到AML细胞浸润。因此，腹膜内注射是一种使用供体白血病细胞连续移植诱导AML的有效方法。
Introduction
急性髓系白血病（AML）是一种病因多样的血液系统恶性肿瘤，预后不良1。AML动物模型的产生为理解其复杂的变异和病理生物学奠定了基础，以努力发现新的疗法2。小鼠的白血病发生涉及表达融合癌蛋白的供体细胞的移植，包括涉及混合谱系白血病（MLL）基因的融合，以有效诱导AML，以模拟人类的疾病3。在MLL基因相关AML4的移植中，已经报道了供体细胞的各种细胞起源，对导致疾病起源的细胞知之甚少。
已经开发了多种途径用于小鼠移植;与将突变供体细胞直接引入骨髓5 的股内注射不同，利用静脉窦丛、尾静脉和颈静脉的静脉注射已被广泛用于生成鼠 AML 模型6，7，8，9。在眶后（r.o.）注射的情况下，各种固有的缺点，如体积限制、高技术要求、重复尝试或错误的机会很少以及潜在的眼部损伤，一直是主要的绊脚石，有限的或没有可行的替代方案7。除了局部损伤外，尾静脉注射还可能存在类似的问题;为了促进手术，小鼠通常需要加热以扩张尾静脉10。如果没有额外的光源，也很难找到尾静脉，特别是在小鼠的C57BL / 6品系中。对于颈静脉注射，研究人员需要足够的培训来定位静脉并限制可能的并发症。此外，静脉窦和颈静脉注射都需要在麻醉下进行，这增加了另一个层次的复杂性。因此，探索新的移植途径以促进AML小鼠模型的建立是很诱人的。
腹膜内（ip）注射通常用于施用药物，染料和麻醉剂11，12，13，14，15;它还被用于引入异位造血的造血细胞16，并在各种小鼠模型中移植骨髓来源的间充质干细胞17，18，19，20，21。然而，它很少用于建立小鼠的造血恶性肿瘤，特别是研究AML疾病的进展。
除了比较谱系阴性（Lin-）和Lin-Sca-1 + c-Kit+（LSK）群体作为供体细胞的移植效率外，本研究还描述了腹腔注射在AML小鼠模型生成中的可行性。这些发现为生成AML和相关髓系白血病的实验模型提供了一种简单有效的方法。这种方法有可能进一步了解疾病机制，并提供一个相对容易的模型来测试实验性疗法。
Protocol
所有实验均由宾夕法尼亚州立大学机构动物护理和使用委员会预先批准。 1. 缓冲液和试剂的制备 准备氨苄青霉素补充（AP）LB琼脂平板（无菌10厘米平板）。为此，将 10 克 LB 肉汤与琼脂溶解在 400 mL 蒸馏水中，搅拌，使体积达到 500 mL。通过高压灭菌对溶液进行灭菌，然后让溶液冷却，向溶液中加入0.5mL氨苄西林（库存：150mg / mL），摇晃混合。立即将18mL溶?…
Representative Results
使用r.o.和ip.移植途径的小鼠AML细胞移植效率的比较以前，在用MLL-AF9转导的LSK细胞进行眶后移植的受体小鼠中报告了1°AML的建立，并且通过连续移植证明了1°AML细胞的可移植性30。本研究首次评估使用骨髓林细胞 进行移植的可能性。异常白细胞增多（图2A）的存在和骨髓和脾脏中白血病细胞（CD45.1+）浸润增加（<strong class="xfig…
Discussion
这些上述研究提供了支持性证据，证明Lin– 细胞的移植在1°鼠AML的产生中与LSK细胞相当。此外，目前的数据还表明，与静脉内（或r.o.）注射相比，腹腔注射是建立鼠AML的一种有效便捷的方法。
除LSK细胞外，据报道，其他群体（如粒细胞-单核细胞祖细胞（GMP）、淋巴总祖细胞（CLP）和髓系总祖细胞（CMP））在产生1° MLL-AF9诱导的AML时被取代为供体细胞，具有不同的孵育…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢哈克研究所的流式细胞术核心设施和宾夕法尼亚州立大学兽医和生物医学科学系动物诊断实验室的组织病理学核心设施提供及时的技术支持。这项工作得到了美国癌症研究所（KSP），宾夕法尼亚州立大学农业科学学院，宾夕法尼亚州癌症研究所，USDA-NIFA项目4771，KSP和RFP的入藏号00000005的资助。
Materials
| a-Select competent cells | Bioline | BIO-85027 | |
| Ammonium chloride (NH4Cl) | Sigma Aldrich | Cat# A-9434 | |
| Ampicillin | Sigma Aldrich | Cat# A0797 | |
| Bovine Serum Albumin (BSA), Fraction V—Low-Endotoxin Grade | Gemini bio-products | Cat# 700-102P | |
| Ciprofloxacin HCl | GoldBio.com | Cat# C-861-100 | |
| DMEM, high glucose, no glutamine | Gibco | Cat# 11960-044 | |
| Dulbecco’s Phosphate-Buffered Saline (PBS) | Corning | Cat# 21-031-CV | |
| EDTA, Disodium Salt (EDTA-2Na), Dihydrate, Molecular Biology Grade | Calbiochem | Cat# 324503 | |
| Fetal Bovine Serum – Premium Select | Atlanta Biologicals | Cat# S11550 | |
| Holo-transferrin, bovine | Sigma Aldrich | Cat# T1283 | |
| Insulin solution human | Sigma | Cat# I-9278 | |
| Iscove's Modified Dulbecco's Medium (IMDM) | Gibco | Cat# 12440-053 | |
| L-glutamine 200 mM (100×) solution | HyClone, Gelifesciences | Cat# SH30034.01 | |
| LB broth, Lennox | NEOGEN | Cat #: 7290A | |
| LB Broth with agar (Miller) | Sigma Aldrich | Cat# L-3147 | |
| Mouse anti-mouse CD45.1 (FITC) | Miltenyi Biotec | Cat# 130-124-211 | |
| Mouse Interleukin-3 (IL-3) | Gemini bio-products | Cat# 300-324P | |
| Mouse Interleukin-6 (IL-6) | Gemini bio-products | Cat# 300-327P | |
| Mouse Stem Cell Factor (SCF) | Gemini bio-products | Cat# 300-348P | |
| Penicillin-Streptomycin Solution, 100x | Corning | Cat# 30-002-CI | |
| Phenix-Eco (pECO) cells | ATCC | CRL-3214 | |
| Potassium Bicarbonate (KHCO3), Granular | JT. Baker | Cat# 2940-01 | |
| Rat anti-mouse CD117 (c-kit) (APC) | BioLegend | Cat # 105812 | |
| Rat anti-mouse Ly-6A/E (Sca-1) (PE-Cy7) | BD Pharmingen | Cat# 558162 | |
| Recombinant Murine Flt3-Ligand | Pepro Tech, INC. | Cat# 250-31L | |
| RetroNectin Recombinant Human Fibronectin Fragment | TaKaRa | Cat# T100A | |
| TransIT-293 Reagent | MirusBio | Cat# MIR 2705 | |
| TRI Reagent | Sigma Aldrich | Cat# T9424 | |
| Trypan Blue Solution, 0.4% | Gibco | Cat # 15250061 | |
| Trypsin-EDTA (0.25%), phenol red | Gibco | Cat# 25200-056 | |
| β-Mercaptoethanol (BME) | Sigma Aldrich | Cat# M3148 | |
| Commercial Assays | |||
| EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit | StemCell technologies | Cat# 19856A | |
| High-Capacity cDNA Reverse Transcription Kit | Thermo Fisher | Cat# 4368813 | |
| PerfeCTa qPCR SuperMix | Quanta Bio | Cat# 95051-500 | |
| Plasmid Maxi Kit (25) | Qiagen | Cat#:12163 | |
| Animals | |||
| Ai14TdTomato mice | Jackson Laboratory | Strain # 007914 | |
| CD45.1 C57BL6/J mice | Jackson Laboratory | Strain # 002014 | |
| CD45.2 C57BL6/J mice | Jackson Laboratory | Strain # 000664 | |
| Instruments and Softwares | |||
| Adobe illustrator | Version 25.2.3 | ||
| BD accuri C6 flow cytometer | BD Biosciences | ||
| FlowJo 10.8.0 | BD | ||
| GeneSys software program | Version 1.5.7.0 | ||
| GraphPad Prism version 6 | GraphPad | ||
| Hemavet 950FS | Drew Scientific | ||
| 7300 Real time PCR system | Applied Biosystems | ||
| Syngene G:BOX Chemi XR5 Chemiluminescence Fluorescence Imaging | G:Box Chemi |
References
- Dohner, H., Weisdorf, D. J., Bloomfield, C. D. Acute myeloid leukemia. The New England Journal of Medicine. 373 (12), 1136-1152 (2015).
- Fortier, J. M., Graubert, T. A. Murine models of human acute myeloid leukemia. Cancer Treatment and Research. 145, 183-196 (2010).
- Ernst, P., et al. Definitive hematopoiesis requires the mixed-lineage leukemia gene. Developmental Cell. 6 (3), 437-443 (2004).
- Fisher, J. N., Kalleda, N., Stavropoulou, V., Schwaller, J. The Impact of the cellular origin in acute myeloid leukemia: learning from mouse models. Hemasphere. 3 (1), 152 (2019).
- Zhan, Y., Zhao, Y. Hematopoietic stem cell transplant in mice by intra-femoral injection. Methods in Molecular Biology. 430, 161-169 (2008).
- Price, J. E., Barth, R. F., Johnson, C. W., Staubus, A. E. Injection of cells and monoclonal antibodies into mice: comparison of tail vein and retroorbital routes. Proceedings of the Society for Experimental Biology. 177 (2), 347-353 (1984).
- Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. Retro-orbital injections in mice. Lab Animal. 40 (5), 155-160 (2011).
- Suckow, M. A., Danneman, P., Brayton, C. . The Laboratory Mouse. , (2001).
- Barr, J. E., Holmes, D. B., Ryan, L. J., Sharpless, S. K. Techniques for the chronic cannulation of the jugular vein in mice. Pharmacology, Biochemistry, and Behavior. 11 (1), 115-118 (1979).
- Kang, Y. Analysis of cancer stem cell metastasis in xenograft animal models. Methods in Molecular Biology. 568, 7-19 (2009).
- Nungestee, W., Wolf, A., Jourdonais, L. Effect of gastric mucin on virulence of bacteria in intraperitoneal injections in the mouse. Proceedings of the Society for Experimental Biology and Medicine. 30 (2), 120-121 (1932).
- Gargiulo, S., et al. Mice anesthesia, analgesia, and part I: anesthetic considerations in preclinical research. ILAR journal. 53 (1), 55-69 (2012).
- Leong, S. -. K., Ling, E. -. A. Labelling neurons with fluorescent dyes administered via intravenous, subcutaneous or intraperitoneal route. Journal of Neuroscience Methods. 32 (1), 15-23 (1990).
- Ma, P., et al. Intraperitoneal injection of magnetic Fe3O4-nanoparticle induces hepatic and renal tissue injury via oxidative stress in mice. International Journal of Nanomedicine. 7, 4809-4918 (2012).
- Schwarze, S. R., Ho, A., Vocero-Akbani, A., Dowdy, S. F. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. 285 (5433), 1569-1572 (1999).
- Muench, M. O., Chen, J. C., Beyer, A. I., Fomin, M. E. Cellular therapies supplement: the peritoneum as an ectopic site of hematopoiesis following in utero transplantation. Transfusion. 51, 106-117 (2011).
- Zhao, W., et al. Intravenous injection of mesenchymal stem cells is effective in treating liver fibrosis. World Journal of Gastroenterology. 18 (10), 1048 (2012).
- Yousefi, F., Ebtekar, M., Soleimani, M., Soudi, S., Hashemi, S. M. Comparison of in vivo immunomodulatory effects of intravenous and intraperitoneal administration of adipose-tissue mesenchymal stem cells in experimental autoimmune encephalomyelitis (EAE). International Immunopharmacol. 17 (3), 608-616 (2013).
- Cheng, K., et al. Transplantation of bone marrow-derived MSCs improves cisplatinum-induced renal injury through paracrine mechanisms. Experimental and Molecular Pathology. 94 (3), 466-473 (2013).
- Castelo-Branco, M., et al. Intraperitoneal but not intravenous cryopreserved mesenchymal stromal cells home to the inflamed colon and ameliorate experimental colitis. PLoS One. 7 (3), 33360 (2012).
- Bazhanov, N., et al. Intraperitoneally infused human mesenchymal stem cells form aggregates with mouse immune cells and attach to peritoneal organs. Stem Cell Research & Therapy. 7, 27 (2016).
- Liu, Q., Chen, L., Atkinson, J. M., Claxton, D. F., Wang, H. G. Atg5-dependent autophagy contributes to the development of acute myeloid leukemia in an MLL-AF9-driven mouse model. Cell Death & Disease. 7 (9), 2361 (2016).
- Wognum, A. W., Eaves, A. C., Thomas, T. E. Identification and isolation of hematopoietic stem cells. Archives of Medical Research. 34 (6), 461-475 (2003).
- Randall, T. D., Weissman, I. L. Characterization of a population of cells in the bone marrow that phenotypically mimics hematopoietic stem cells: resting stem cells or mystery population. Stem Cells. 16 (1), 38-48 (1998).
- Gott, K. M., et al. A comparison of Cs-137 gamma rays and 320-kV X-rays in a mouse bone marrow transplantation model. Dose Response. 18 (2), 1559325820916572 (2020).
- Miner, N. A., Koehler, J., Greenaway, L. Intraperitoneal injection of mice. Applied Microbiology. 17 (2), 250-251 (1969).
- Madisen, L., et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nature Neuroscience. 13 (1), 133-140 (2010).
- Cardiff, R. D., Miller, C. H., Munn, R. J. Manual hematoxylin and eosin staining of mouse tissue sections. Cold Spring Harbor Protocols. 2014 (6), 655-658 (2014).
- Ronan, J. L., Wu, W., Crabtree, G. R. From neural development to cognition: unexpected roles for chromatin. Nature Review Genetics. 14 (5), 347-359 (2013).
- Qian, F., et al. Interleukin-4 treatment reduces leukemia burden in acute myeloid leukemia. FASEB Journal. 36 (5), 22328 (2022).
- Krivtsov, A. V., et al. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature. 442 (7104), 818-822 (2006).
- Chen, W., et al. Malignant transformation initiated by Mll-AF9: gene dosage and critical target cells. Cancer Cell. 13 (5), 432-440 (2008).
- Somervaille, T. C. P., Cleary, M. L. Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell. 10 (4), 257-268 (2006).
Cite This Article
Qian, F., Arner, B. E., Nettleford, S. K., Paulson, R. F., Prabhu, K. S. Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice. J. Vis. Exp. (191), e64834, doi:10.3791/64834 (2023).