Summary

从小鼠横纹肌瘤分离的原发性肿瘤细胞的肿瘤球衍生和治疗

Published: September 13, 2019
doi:

Summary

该协议描述了一种从肿瘤层培养开始分离小鼠横纹肌瘤原发细胞、肿瘤球的形成和治疗以及异体移植的可重复方法。

Abstract

横纹肌瘤(RMS)是儿童最常见的软组织肉瘤。尽管已作出重大努力,能够识别与 RMS 相关的常见突变,并允许对不同 RMS 亚型进行区分,但开发新疗法以进一步改善预后仍面临重大挑战。虽然通过表达的致词标记,RMS是有致血的还是非造血的,由于对起源细胞的了解仍然不足,因此仍然存在重大争议。本研究为小鼠RMS的肿瘤球测定提供了可靠的方法。该测定基于肿瘤细胞的功能特性,允许识别肿瘤中具有肿瘤生成功能的稀有种群。还介绍了测试重组蛋白、将转染方案与肿瘤球测定相结合以及评估参与肿瘤发育和生长的候选基因的程序。进一步描述是一种将肿瘤球移植到受体小鼠中以验证体内肿瘤生成功能的程序。总体而言,所述方法允许可靠地识别和测试可在不同环境中产生的稀有 RMS 肿瘤群。最后,该协议可作为药物筛选和治疗学未来发展的平台。

Introduction

癌症是一种异质疾病;此外,同一类型的肿瘤可以在不同的患者中呈现不同的基因突变,在患者体内,肿瘤由多个细胞群组成。异质性在识别负责引发和传播癌症的细胞方面提出了挑战,但其特性对于开发有效治疗至关重要。肿瘤传播细胞(TPC)的概念,一种罕见的细胞群,有助于肿瘤的发展,已经广泛审查2。尽管TPC在多种癌症中具有特征,但识别其可靠分离的标记物仍然是几种肿瘤类型3、4、5、6的挑战。,7,8,9.因此,一种不依赖于分子标记,而是依赖于TPC功能特性(高自我更新和低附着条件下生长能力)的方法,称为肿瘤层形成测定,可以广泛应用于从大多数肿瘤的TPC的识别。重要的是,这种测定也可用于扩大三氯图C,从而直接应用于抗癌药物筛选和研究抗癌1,10。

横纹肌瘤(RMS)是一种罕见的软组织肉瘤,在幼儿11中最为常见。Althoug RMS可以通过对造血标记物表达的评估进行组织学鉴定,由于肿瘤发育刺激的多种肿瘤亚型和高异质性,原源RMS细胞尚未被单体特征化。事实上,最近的研究已经产生了关于RMS是致幻或非造血源的重要科学讨论,这表明RMS可能来自不同的细胞类型,这取决于上下文12,13。14,15,16,17.对RMS细胞系进行了大量研究,利用肿瘤层形成测定来鉴定肿瘤发育的通路,并鉴定与高度自我更新人群相关的标记物的表征18,19,20,21.

然而,尽管肿瘤圈形成测定具有识别原源RMS细胞的潜力,但一种可用于原发RMS细胞的可靠方法尚未得到描述。在此背景下,我们小组最近的一项研究采用了优化的肿瘤球形成测定,用于鉴定Duchenne肌肉萎缩症(DMD)小鼠模型22中的原源RMS细胞。从肌肉组织分离出的多种肿瘤前细胞类型,测试其在低附着条件下生长的能力,从而能够将肌肉干细胞识别为营养不良环境中RMS的原源细胞。这里描述的是肿瘤层形成测定的可重复和可靠的协议(图1),它已被成功地用于识别负责小鼠RMS发育的极其罕见的细胞群。

Protocol

小鼠的住宿、治疗和牺牲均按照桑福德·伯纳姆·普雷比斯医学发现研究所批准的IACUC协议进行。 1. 试剂制备 制备100 mL的细胞分离培养基:F10培养基辅以10%马血清(HS)。 制备50 mL的胶原酶 II 型溶液:在 50 mL 的细胞分离介质中溶解 1 g 胶原酶 II 型粉末(注意酶的单位,每 1 mL 培养基,因为单位数量会根据批次而变化)。将溶液与溶液加数,并储存在-20°C冷冻箱?…

Representative Results

肿瘤球检测细胞分离得到优化,以获得肿瘤组织中细胞群的最大异质性。首先,由于分离组织呈现形态上不同的区域,为了增加分离均匀稀有细胞群的机会,从肿瘤的多个区域进行了取样(图1A,左侧第一面板)。第二,对采集的样品进行机械解离,同时保持切碎组织大小的均质性,尽管样品中可能存在不同的电阻(?…

Discussion

已采用多种方法从肿瘤异质细胞群中分离和表征TPC:肿瘤克隆测定、FACS分离和肿瘤球层形成测定。肿瘤克隆测定在1971年首次被描述,用于干细胞研究,后来才应用于癌症生物学29,30。该方法是以癌症干细胞的内在特性为基础,在软胶囊培养中不受约束地扩展31。该方法自开发以来,已广泛应用于癌症研究,包括肿瘤细胞异质性研究、…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

这项工作得到了埃里森医学基金会拨款AG-NS-0843-11的支持,以及NCI癌症中心支持赠款P30CA030199授予A.S.的NIH试点补助金。

Materials

Accutase cell dissociation reagent Gibco A1110501 Detach adherent cells and dissociate tumorspheres
Celigo Nexcelom Celigo Microwell plate based image cytometer for adherent and suspension cells
Collagenase, Type II Life Technologies 17101015 Tissue digestion enzyme
Dispase II, protease Life Technologies 17105041 Tissue digestion enzyme
DMEM high glucose media Gibco 11965092 Component of tumor cells media
DMEM/F12 Media Gibco 11320033 Component of tumosphere media
EDTA ThermoFisher S312500 Component of FACS buffer
EGF recombinant mouse protein Gibco PMG8041 Component of tumosphere media
FACSAria II Flow Cytometry BD Biosciences 650033 Fluorescent activated cell sorter
Fetal Bovine Serum Omega Scientific FB-11 Component of tumor cells media
Fluriso (Isofluornae) anesthetic agent MWI Vet Supply 502017 Anesthetic reagent for animals
FxCycle Violet Stain Life Technologies F10347 Discriminate live and dead cells
Goat Serum Life Technologies 16210072 Component of FACS buffer
Ham's F10 Media Life Technologies 11550043 Component of FACS buffer
Horse Serum Life Technologies 16050114 Component of cell isolation media
Lipofectamine 3000 transfection reagent ThermoFisher L3000015 Transfection Reagent
Matrigel membrane matrix Corning CB40234 Provides support to trasplanted cells
N-2 Supplemtns (100X) Gibco 17502048 Component of tumosphere media
Neomycin-Polymyxin B Sulfates-Bacitracin Zinc Ophthalmic Ointment MWI Vet Supply 701008 Eyes ointment
PBS Gibco 10010023 Component of FACS buffer and used for washing cells
pEGFP-C1 Addgene 6084-1 GFP plasmid
Penicillin – Streptomyocin Life Technologies 15140163 Component of tumosphere and tumor cells media
Recombinant Human βFGF-basic Peprotech 10018B Component of tumosphere media
Recombinant mouse Flt-3 Ligand Protein R&D Systems 427-FL-005 Recombinant protein
Trypan blue ThermoFisher 15250061 Discriminate live and dead cells

Referencias

  1. Dagogo-Jack, I., Shaw, A. T. Tumour heterogeneity and resistance to cancer therapies. Nature Reviews Clinical Oncology. 15 (2), 81-94 (2018).
  2. Wicha, M. S., Liu, S., Dontu, G. Cancer stem cells: an old idea–a paradigm shift. Investigación sobre el cáncer. 66 (4), 1883-1890 (2006).
  3. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. Prospective identification of tumorigenic breast cancer cells. Proceedings National Academy of Science of the United States of America. 100 (7), 3983-3988 (2003).
  4. Oishi, N., Yamashita, T., Kaneko, S. Molecular biology of liver cancer stem cells. Liver Cancer. 3 (2), 71-84 (2014).
  5. Crous, A. M., Abrahamse, H. Lung cancer stem cells and low-intensity laser irradiation: a potential future therapy. Stem Cell Research & Therapy. 4 (5), 129 (2013).
  6. Tomao, F., et al. Investigating molecular profiles of ovarian cancer: an update on cancer stem cells. Journal of Cancer. 5 (5), 301-310 (2014).
  7. Zhan, H. X., Xu, J. W., Wu, D., Zhang, T. P., Hu, S. Y. Pancreatic cancer stem cells: new insight into a stubborn disease. Cancer Letters. 357 (2), 429-437 (2015).
  8. Sharpe, B., Beresford, M., Bowen, R., Mitchard, J., Chalmers, A. D. Searching for prostate cancer stem cells: markers and methods. Stem Cell Reviews and Reports. 9 (5), 721-730 (2013).
  9. Lapidot, T., et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 367 (6464), 645-648 (1994).
  10. Lee, C. -. H., Yu, C. -. C., Wang, B. -. Y., Chang, W. -. W. Tumorsphere as an effective in vitro platform for screening anti- cancer stem cell drugs. Oncotarget. 7 (2), 1215-1226 (2015).
  11. Sultan, I., Qaddoumi, I., Yaser, S., Rodriguez-Galindo, C., Ferrari, A. Comparing adult and pediatric rhabdomyosarcoma in the surveillance, epidemiology and end results program. Journal of Clinical Oncology. 27 (20), 3391-3397 (1973).
  12. Blum, J. M., et al. Distinct and overlapping sarcoma subtypes initiated from muscle stem and progenitor cells. Cell Reports. 5 (4), 933-940 (2013).
  13. Rubin, B. P., et al. Evidence for an unanticipated relationship between undifferentiated pleomorphic sarcoma and embryonal rhabdomyosarcoma. Cancer Cell. 19 (2), 177-191 (2011).
  14. Keller, C., et al. Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity. of Ink4a/ARF and Trp53 loss of function. Genes & Development. 18 (21), 2614-2626 (2004).
  15. Tremblay, A. M., et al. The Hippo transducer YAP1 transforms activated satellite cells and is a potent effector of embryonal rhabdomyosarcoma formation. Cancer Cell. 26 (2), 273-287 (2014).
  16. Hatley, M. E., et al. A mouse model of rhabdomyosarcoma originating from the adipocyte lineage. Cancer Cell. 22 (4), 536-546 (2012).
  17. Drummond, C. J., et al. Hedgehog Pathway Drives Fusion-Negative Rhabdomyosarcoma Initiated From Non-myogenic Endothelial Progenitors. Cancer Cell. 33 (1), 108-124 (2018).
  18. Almazan-Moga, A., et al. Hedgehog Pathway Inhibition Hampers Sphere and Holoclone Formation in Rhabdomyosarcoma. Stem Cells International. , (2017).
  19. Walter, D., et al. CD133 positive embryonal rhabdomyosarcoma stem-like cell population is enriched in rhabdospheres. PLoS One. 6 (5), (2011).
  20. Ciccarelli, C., et al. Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population. Molecular Cancer. 15, (2016).
  21. Deel, M. D., et al. The Transcriptional Coactivator TAZ Is a Potent Mediator of Alveolar Rhabdomyosarcoma Tumorigenesis. Clinical Cancer Research. 24 (11), 2616-2630 (2018).
  22. Boscolo Sesillo, F., Fox, D., Sacco, A. Muscle Stem Cells Give Rise to Rhabdomyosarcomas in a Severe Mouse Model of Duchenne Muscular Dystrophy. Cell Reports. 26 (3), 689-701 (2019).
  23. Chamberlain, J. S., Metzger, J., Reyes, M., Townsend, D., Faulkner, J. A. Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma. The FASEB Journal. 21 (9), 2195-2204 (2007).
  24. Kessel, S., et al. High-Throughput 3D Tumor Spheroid Screening Method for Cancer Drug Discovery Using Celigo Image Cytometry. SLAS Technology. 22 (4), 454-465 (2017).
  25. Johnson, S., Chen, H., Lo, P. K. In vitro Tumorsphere Formation Assays. Bio-Protocol. 3 (3), (2013).
  26. Zhu, Z. W., et al. A novel three-dimensional tumorsphere culture system for the efficient and low-cost enrichment of cancer stem cells with natural polymers. Experimental and Therapeutic. 15 (1), 85-92 (2018).
  27. Takahashi, S. Downstream molecular pathways of FLT3 in the pathogenesis of acute myeloid leukemia: biology and therapeutic implications. Jornal of Hematology and Oncology. 4, (2011).
  28. Laouar, Y., Welte, T., Fu, X. Y., Flavell, R. A. STAT3 is required for Flt3L-dependent dendritic cell differentiation. Immunity. 19 (6), 903-912 (2003).
  29. Ogawa, M., Bergsagel, D. E., McCulloch, E. A. Differential effects of melphalan on mouse myeloma (adj. PC-5) and hemopoietic stem cells. Investigación sobre el cáncer. 31 (12), 2116-2119 (1971).
  30. Hamburger, A. W., Salmon, S. E. Primary bioassay of human tumor stem cells. Science. 197 (4302), 461-463 (1977).
  31. Hamburger, A. W. The human tumor clonogenic assay as a model system in cell biology. The International Journal of Cell Cloning. 5 (2), 89-107 (1987).
  32. Jimenez-Hernandez, L. E., et al. NRP1-positive lung cancer cells possess tumor-initiating properties. Oncology Reports. 39 (1), 349-357 (2018).
  33. Singh, S. K., et al. Identification of human brain tumour initiating cells. Nature. 432 (7015), 396-401 (2004).
  34. Kimlin, L. C., Casagrande, G., Virador, V. M. In vitro three-dimensional (3D) models in cancer research: an update. Molecular Carcinogenesis. 52 (3), 167-182 (2013).
  35. Salerno, M., et al. Sphere-forming cell subsets with cancer stem cell properties in human musculoskeletal sarcomas. International Journal of Oncology. 43 (1), 95-102 (2013).

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Boscolo Sesillo, F., Sacco, A. Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas. J. Vis. Exp. (151), e59897, doi:10.3791/59897 (2019).

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