骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义
Summary
直接注射癌症衍生细胞外囊 (EVs) 导致骨髓支持肿瘤进展的重新编程;然而, 哪些细胞调解这种影响是不清楚的。在这里, 我们描述了一步一步的协议来调查 ev 介导的肿瘤间充质干细胞 (MSC) 相互作用的在体内, 揭示了一个关键的作用, 电动汽车教育的 MSCs 在转移。
Abstract
在肿瘤微环境中, 居民或被征募的间充质干细胞 (MSCs) 有助于多种癌症类型的恶性进展。在特定环境信号的影响下, 这些成体干细胞可以释放分泌介质, 从而加速肿瘤的生长和转移。定义肿瘤和骨髓间充质之间的串扰, 对于了解癌症进展的机制和确定治疗干预的新靶点至关重要。
癌细胞产生大量的胞外囊泡 (EVs), 这会深刻地影响肿瘤微环境或远处的靶细胞的行为。肿瘤电动车包围功能生物分子, 包括炎症 rna 和 (onco) 蛋白, 可以教育基质细胞, 以增强癌细胞的转移行为或参与转移前的利基形成。在这篇文章中, 我们描述了一个前肿瘤小鼠模型的发展, 它可以对 EV 介导的肿瘤和间充质干细胞之间的串扰进行具体评估。首先, 我们描述了肿瘤分泌的电动车的纯化和表征, 以及 MSCs 对电动汽车内化的评价。然后利用复用珠基免疫分析法, 对癌症电动车诱导的细胞因子表达谱的改变进行评价。最后, 我们说明了概括骨肉瘤的生物荧光原位异种移植小鼠模型的生成, 并显示了 EV 培养的骨髓间充质干细胞对肿瘤生长和转移形成的贡献。
我们的模型提供了一个机会来定义癌症电动车如何形成肿瘤支持的环境, 并评估是否封锁的 EV 介导的肿瘤和 MSCs 之间的沟通, 以防止癌症进展。
Introduction
肿瘤微环境积极参与了肿瘤发生和肿瘤进展的大部分 (如果不是全部) 方面, 包括转移形成和对治疗的抵抗力的发展1。这强调需要对前体肿瘤小鼠模型, 允许解剖复杂的肿瘤基质相互作用发生在肿瘤的利基。
在肿瘤微环境的许多细胞成分中, 间充质干细胞 (MSCs) 在多种癌症类型 (如乳腺癌、前列腺癌、脑肿瘤、多发性骨髓瘤和骨肉瘤) 中有强烈的促进作用2 ,3,4,5,6,7。MSCs 是驻留在各种成人和胎儿组织中的多能干细胞, 包括骨髓、脂肪组织、胎盘、脐血和其他8,9。为应对癌症产生的炎症信号, MSCs 向肿瘤部位迁移, 纳入肿瘤微环境, 最终分化为肿瘤支持细胞10。这些与癌症相关的 MSCs 提供了重要因素 (例如,生长因子、趋化因子、细胞因子和免疫抑制剂), 用于肿瘤细胞和周围基质的作用2,3,11,12,13. 虽然在众多的癌症模型中研究了癌症相关的 mscs 的促肿瘤作用, 但是肿瘤细胞重新编程 mscs 以形成促进癌症的利基的机制却不甚了解。在这里, 我们描述了一个同种异体原位移植模型的生成, 它特别允许通过细胞外囊泡 (EVs) 研究骨癌细胞与骨髓间充质干细胞之间的亲癌变相互作用。
EVs 是肿瘤与基质细胞间通讯的关键介质14。EVs 携带原细胞的功能生物分子, 包括蛋白质、血脂和调控 rna。一旦在细胞外空间释放, 这些囊泡可以被周围的细胞占用, 或者通过血液或淋巴循环携带到远处的部位, 并能深刻地影响靶细胞的行为。15,16, 17例如, 基质成纤维细胞对癌电动车的吸收可能导致上皮细胞分化支持血管生成和加速肿瘤生长体内 18 19, 内皮内部化细胞可以刺激肿瘤血管生成, 增加血管通透性16,20, 与免疫细胞的相互作用可能导致抑制抗肿瘤免疫应答21。
我们最近用一种生物发光的同种异体骨肉瘤模型证明, 肿瘤细胞释放出大量的电动车, 促使 MSCs 获得亲癌变和支持转移的表型。这种影响是由于 msc 细胞因子表达谱 (称为 “理学硕士教育”) 的戏剧性变化, 并可通过管理治疗 interleukin-6 受体 (IL-6R) 抗体7来预防。我们的研究表明, 癌症电动车是 MSC 行为的关键调节剂, 从而为以微环境为目标的方法阻止骨肉瘤进展提供了理论依据。在此, 我们描述了一个逐步的协议, 以调查 EV 介导的肿瘤理学硕士互动在体内。该模型的目的是: 1) 明确定义的癌症 EV 诱导改变的肿瘤微环境, 2) 评估这种相互作用是如何促进骨肿瘤的生长和转移形成, 3) 研究是否干预EV 介导的串扰在体内防止癌症进展。
Protocol
在机构伦理委员会批准和书面知情同意后, Tergooi 医院 (荷兰 Hilversum) 整形外科部获得了人骨髓间充质干细胞分离的人体脂肪组织。GFP-阳性脂肪间充质干细胞是从医学和外科科学部为儿童和成人 (摩德纳大学和艾米利亚)。 动物实验是按照荷兰动物实验法进行的, 该议定书是荷兰阿姆斯特丹大学医学中心动物实验委员会批准的。 1. 分离肿瘤分泌的胞外囊。 …
Representative Results
在这项研究中, 我们探讨了骨肉瘤分泌的电动车的能力, 以培养骨髓间充质干细胞向癌变和支持转移表型。我们表明, 骨肉瘤细胞释放外切样的电动车, 由 MSCs 内化。我们测量了癌症电动车诱导的细胞因子表达谱的变化, 并评价了 EV 培养的骨髓间充质干细胞对肿瘤生长和转移形成的影响。研究设计的一般表示形式在图 1中进行了说明。 <p class="jove_cont…
Discussion
肿瘤分泌的胞外囊 (EVs) 可以改变局部和远端间充质细胞的生理, 从而产生肿瘤支持的环境。在这里, 我们描述的一代骨肉瘤的前体小鼠模型, 允许解剖的 EV 介导的相互作用的肿瘤细胞和间充质干细胞 (MSCs)在体内。我们表明, 系统注射的人肿瘤 EV 培养的小鼠骨肉瘤移植通过激活 IL-6/STAT3 信号通路7强促进肿瘤的生长和转移形成。
最近的研究表明, 癌症电?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
纳丹 Baglio 得到了由欧洲联盟出资的 Associazione 意大利每 la Ricerca Cancro (AIRC) 的奖学金。此外, 该项目还从欧洲联盟2020《玛丽·斯卡洛多斯卡·居里-居里赠款协定》660200号 (纳丹 Baglio) 下的研究和创新方案中获得资金。
Materials
| Equipment | |||||||
| Ultra Centrifuge | Beckman | Optima L-90K | |||||
| Rotor SW32Ti | Beckman | 369650 | Referred to in the manuscript as ultra-swinging bucket rotor | ||||
| Transmission electron microscope | Zeiss | EM109 | Or similar TEM | ||||
| Digital camera | Nikon | DMX 1200F | Or similar camera | ||||
| Imaging software TEM | Nikon | ACT-1 | |||||
| Fluorescence microscope | Zeiss | Imager.D2 | Or similar Fluorescence microscope | ||||
| Imaging software FM | Zeiss | ZEN Blue | |||||
| Incubator | Nuaire | 4750E | |||||
| Centrifuge | Hettick | ROTANTA 460R | |||||
| -80 Freezer | Thermo electro corporation | n.a. | |||||
| FACS | BD | BD FACScalibur | Or similar flow cytometer | ||||
| Drill | Ferm | FCT-300 | With 0.8 mm drill | ||||
| HSS micro twist drills, 0.8 mm | Proxxon | 28 852 | 0.8 mm drill | ||||
| IVIS camera | Xenogen | Ivis Lumina | Referred to in the manuscript as bioluminescence camera. Xenogen is now part of Perkin Elmer | ||||
| Living image software2.60 | Xenogen / Igor Por | n.a | Xenogen is now part of Perkin Elmer | ||||
| 10 µL Syringe | Hamilton | Neuros Model 1701 RN | |||||
| Needle: Hamilton RN Needle for Syringe, 26 Gauge, Pointstyle AS, custom length 2 cm | Hamilton | n.a. | |||||
| Caliper | Mitutoyo | G08004463 | |||||
| Autoclave | Astell | n.a. | |||||
| Heat Lamp | Philips | n.a. | |||||
| Culture media | |||||||
| Fetal Bovine Serum | Hyclone | RYG35912 | |||||
| Platelet Lysate | n.a. | n.a. | |||||
| IMDM medium | Lonza | BE12-722F | |||||
| alpha-MEM medium | Lonza | BE02-002F | |||||
| DMEM medium | Lonza | BE12-614F | |||||
| pen/strep/glutamine | GIBCO | 10378-016 | |||||
| heparin | LEO | 012866-08 | |||||
| Trypsin/EDTA (10x) | GIBCO | 15400-054 | |||||
| Cells | |||||||
| adipose deriverd MSCs | n.a. | n.a. | |||||
| GFP-positive MSCs | n.a. | n.a. | |||||
| human fibroblasts | n.a. | n.a. | |||||
| 143B cells | ATCC | CRL-8303 | |||||
| FLUC-143B cells | ATCC | CRL-8303 | Transduced | ||||
| Disposables | |||||||
| Culture flasks 175 cm2 | CELLSTAR | 660175 | |||||
| 50 mL tubes | Greiner bio-one | 210261 | |||||
| Freeze tubes | Thermoscientific | 377224 | |||||
| Ultra-Clear tubes | Beckman | 344058 | Referred to in the manuscript as ultra-centrifuge tubes | ||||
| 0,22 µm filter | Millex | SLGV033RS | |||||
| 200 mesh Formvar-carbon-coated nickel grids | EMS (Electron Microscopy Sciences) | ||||||
| 0.5 mL insulin syringes with 29G Needle | Terumo | U-100 | |||||
| Petri dish | Sigma – Aldrich | P7612 | |||||
| Filter paper | Thermo fisher Scientific | 50363215 | |||||
| Reagents / kits | |||||||
| paraformaldehyde | Alfa Aeser | 43368.9M | |||||
| PBS | Braun | 220/12257974/110 | |||||
| glutaraldehyde | EMS (Electron Microscopy Sciences) | 16300 | |||||
| uranyl oxalate | EMS (Electron Microscopy Sciences) | 22510 | |||||
| urany acetate | EMS (Electron Microscopy Sciences) | 22400 | |||||
| methyl cellulose | EMS (Electron Microscopy Sciences) | 1560 | |||||
| PKH67 | Sigma | mini67-1kt | Referred to in the manuscript as GFLD | ||||
| BSA | Sigma | A8412 | |||||
| CBA – human inflammatory cytokine kit | BD | 551811 | |||||
| Formaldehyde 37% | VWR | 104003100 | |||||
| Carbon Steel surgical blades | Swann-Morton | 206 | Referred to in the manuscript as surgical knife | ||||
| anti-human vimentin antibody | Santa Cruz | sc-6260 | Clone V9 | ||||
| Antibody diluent | DAKO | S0809 | |||||
| HRP-labeled anti mouse IgG antibody | Life Technologies | 32230 | |||||
| DAB-kit | DAKO | K500711 | |||||
| hematoxyllin | Sigma | GHS232 | |||||
| EDTA-buffer | n.a. | n.a. | |||||
| Citrate buffer | n.a. | n.a. | |||||
| rabbit polyclonal anti-GFP antibody | Abcam | n.a. | Ab290 | ||||
| DAPI | Life Technologies | D1306 | |||||
| Paracetamol, 120 mg / 5 ml syrup | Bayer | n.a. | Sinaspril, paracetamol solution for kids | ||||
| Isoflurane 1000 mg/g | Vumc pharmacy | n.a. | |||||
| buprenofine hydrochloride, 0.3 mg/ml | Indivior UK Limited | n.a. | |||||
| lidocaine-HCL 2% | Vumc pharmacy | n.a. | |||||
| 70% ethanol | VWR | 93003.1006 | |||||
| Tissue glue | Derma+Flex, formulated medical cyanoacrylate | Vygon | LB604060 | ||||
| Eyedrops: Vidisec Carbogel, 2 mg/ml | Bausch+Lomb | n.a. | |||||
| D-luciferin, potassium salt | Gold Biotechnology | LUCK-1 | |||||
| Glass slides | Thermo scientific | 630-0954 | |||||
| Stainless steel loops | n.a. | n.a. | |||||
| Mice experiments | |||||||
| Mice, Hsd:Athymic Nude-Foxn1nu, female, 6 weeks at arrival, bacterial status conform FELASA | ENVIGO | n.a. | |||||
| Paper-pulp smart home (cage enrichment) | Bio Services | n.a. | |||||
| Alpha-dri bedding material | Shepperd Speciality Papers | n.a. | |||||
| Mouse food: Teklad global 18% protein rodent diet | ENVIGO | 2918-11416M | |||||
| Sutures | Ethicon | V926H | |||||
| Scissors | Sigma-Aldrich | S3146-1EA | (or similar) | ||||
| Tweezers | Sigma-Aldrich | F4142-1EA | (or similar) | ||||
References
- Hanahan, D., Weinberg, R. A. Hallmarks of cancer: The next generation. Cell. 144 (5), 646-674 (2011).
- Karnoub, A. E., et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 449 (7162), 557-563 (2007).
- Jung, Y., et al. Recruitment of mesenchymal stem cells into prostate tumours promotes metastasis. Nat Commun. 4, 1795 (2013).
- Shahar, T., et al. Percentage of mesenchymal stem cells in high-grade glioma tumor samples correlates with patient survival. Neuro Oncol. 19 (5), (2016).
- Behnan, J., et al. Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression. Stem Cells. 32 (5), 1110-1123 (2014).
- Giallongo, C., et al. Granulocyte-like myeloid derived suppressor cells (G-MDSC) are increased in multiple myeloma and are driven by dysfunctional mesenchymal stem cells (MSC). Oncotarget. 7 (52), 85764-85775 (2016).
- Baglio, S. R., et al. Blocking tumor-educated MSC paracrine activity halts osteosarcoma progression. Clin Cancer Res. 23 (14), 3721-3733 (2017).
- Pittenger, M. F., et al. Multilineage potential of adult human mesenchymal stem cells. Science. 284 (5411), 143-147 (1999).
- Shi, Y., Du, L., Lin, L., Wang, Y. Tumour-associated mesenchymal stem/stromal cells: emerging therapeutic targets. Nat Rev Drug Discov. 16 (1), 35-52 (2017).
- Ridge, S. M., Sullivan, F. J., Glynn, S. A. Mesenchymal stem cells: key players in cancer progression. Mol Cancer. 16 (1), 31 (2017).
- Luo, J., et al. Infiltrating bone marrow mesenchymal stem cells increase prostate cancer stem cell population and metastatic ability via secreting cytokines to suppress androgen receptor signaling. Oncogene. 33 (21), 2768-2778 (2013).
- Huang, W. -. H., Chang, M. -. C., Tsai, K. -. S., Hung, M. -. C., Chen, H. -. L., Hung, S. -. C. Mesenchymal stem cells promote growth and angiogenesis of tumors in mice. Oncogene. 32 (37), 4343-4354 (2013).
- Patel, S. A., Meyer, J. R., Greco, S. J., Corcoran, K. E., Bryan, M., Rameshwar, P. Mesenchymal stem cells protect breast cancer cells through regulatory T cells: role of mesenchymal stem cell-derived TGF-beta. J Immunol. 184 (10), 5885-5894 (2010).
- Becker, A., Thakur, B. K., Weiss, J. M., Kim, H. S., Peinado, H., Lyden, D. Extracellular vesicles in cancer: Cell-to-cell mediators of metastasis. Cancer Cell. 30 (6), 836-848 (2016).
- Skog, J., et al. Glioblastoma microvesicles transport RNA and protein that promote tumor growth and provide diagnostic biomarkers. Nat Cell Biol. 10 (12), 1470-1476 (2008).
- Peinado, H., et al. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat Med. 18 (6), 883-891 (2012).
- Zomer, A., et al. In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior. Cell. 161 (5), 1046-1057 (2015).
- Webber, J., Steadman, R., Mason, M. D., Tabi, Z., Clayton, A. Cancer exosomes trigger fibroblast to myofibroblast differentiation. Cancer Res. 70 (23), 9621-9630 (2010).
- Webber, J. P., et al. Differentiation of tumour-promoting stromal myofibroblasts by cancer exosomes. Oncogene. 34 (3), 290-302 (2015).
- Zhou, W., et al. Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell. 25 (4), 501-515 (2014).
- Whiteside, T., Anastasopoulou, E., Voutsas, I., Papamichail, M., Perez, S., Nunes, D. Exosomes and tumor-mediated immune suppression. Expert Rev Mol Diagn. 15 (10), 1293-1310 (2016).
- Verweij, F. J., Van Eijndhoven, M. A. J., Middeldorp, J., Pegtel, D. M. Analysis of viral microRNA exchange via exosomes in vitro and in vivo. Methods Mol Biol. 1024, 53-68 (2013).
- Baglio, S. R., et al. Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species. Stem Cell Res Ther. 6 (1), 127 (2015).
- Naaijkens, B. A., et al. Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications. Cell Tissue Res. 348 (1), 119-130 (2012).
- Grisendi, G., et al. Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy. Cancer Res. 70 (9), 3718-3729 (2010).
- Cosette, J., Abdelwahed, R. B., Donnou-Triffault, S., Sautès-Fridman, C., Flaud, P., Fisson, S. Bioluminescence-based tumor quantification method for monitoring tumor progression and treatment effects in mouse lymphoma models. J Vis Exp. (113), (2016).
- Carbone, L., et al. Assessing cervical dislocation as a humane euthanasia method in mice. J Am Assoc Lab Anim Sci. 51 (3), 352-356 (2012).
- Costa-Silva, B., et al. Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol. 17 (6), 816-826 (2015).
- Hoshino, A., et al. Tumour exosome integrins determine organotropic metastasis. Nature. 527 (7578), 329-335 (2015).
- Clayton, A., Mitchell, J. P., Court, J., Mason, M. D., Tabi, Z. Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2. Cancer Res. 67 (15), 7458-7466 (2007).
- Wieckowski, E. U., Visus, C., Szajnik, M., Szczepanski, M. J., Storkus, W. J., Whiteside, T. L. Tumor-derived microvesicles promote regulatory t cell expansion and induce apoptosis in tumor-reactive activated cd8+ T lymphocytes. J Immunol. 183 (6), 3720-3730 (2009).
- Valenti, R., Huber, V., Iero, M., Filipazzi, P., Parmiani, G., Rivoltini, L. Tumor-released microvesicles as vehicles of immunosuppression. Cancer Res. 67 (7), 2912-2915 (2007).
- Costa-Silva, B., et al. Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol. 17 (6), 816-826 (2015).
- Lin, L. Y., et al. Tumour cell-derived exosomes endow mesenchymal stromal cells with tumour-promotion capabilities. Oncogene. 35 (46), 6038-6042 (2016).
Cite This Article
Lagerweij, T., Pérez-Lanzón, M., Baglio, S. R. A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells. J. Vis. Exp. (135), e56932, doi:10.3791/56932 (2018).