Name: real-time quantitative measurement of tumor cell migration and invasion following synthetic mrna transfection
Uploaded: 2023-06-23T13:10:00
Description: Watch this Scientific Journal Video about Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection at JoVE.com

作者感谢堪萨斯城儿童慈善中心的医学写作中心编辑了这份手稿。这项工作得到了娜塔莉的 A.R.T. 基金会（对 TP）的支持，并得到了儿童慈善医院 （CMH） 和堪萨斯大学癌症中心 （KUCC） 的 MCA 合作伙伴咨询委员会资助（对 TP）。

1. mRNA的合成
注意：对于mRNA合成，所有试剂和设备必须在使用前经过特殊处理以灭活RNase。有关本协议中使用的所有材料、仪器和试剂的详细信息，请参阅 材料表 。
<ol><li>DNA的线性化 注：将 CrkI 和 CrkL 的小鼠 cDNA 克隆到 pFLAG-CMV-5a 表达载体中，以在 C 末端添加 FLAG 表位标签，并将亚克隆到 pcDNA3.1/myc-His 载体中以掺入 T7 启动子?...

<ol>
	<li>Palmer, T. D., Ashby, W. J., Lewis, J. D., Zijlstra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+tumor+cell+motility+to+prevent+metastasis.">Targeting tumor cell motility to prevent metastasis.</a> Advanced Drug Delivery Reviews. 63 (8), 568-581 (2011).</li><li>Eccles, S. A., Box, C., Court, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration/invasion+assays+and+their+application+in+cancer+drug+discovery.">Cell migration/invasion assays and their application in cancer drug discovery.</a> Biotechnology Annual Review. 11, 391-421 (2005).</li><li>Kluiver, T. A., Alieva, M., van Vuurden, D. G., Wehrens, E. J., Rios, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invaders+exposed:+Understanding+and+targeting+tumor+cell+invasion+in+diffuse+intrinsic+pontine+glioma.">Invaders exposed: Understanding and targeting tumor cell invasion in diffuse intrinsic pontine glioma.</a> Frontiers in Oncology. 10, 92 (2020).</li><li>Xie, Q., Mittal, S., Berens, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+adaptive+glioblastoma:+An+overview+of+proliferation+and+invasion.">Targeting adaptive glioblastoma: An overview of proliferation and invasion.</a> Neuro-Oncology. 16 (12), 1575-1584 (2014).</li><li>Wee, Y., Wang, T., Liu, Y., Li, X., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pan-cancer+study+of+copy+number+gain+and+up-regulation+in+human+oncogenes.">A pan-cancer study of copy number gain and up-regulation in human oncogenes.</a> Life Sciences. 211, 206-214 (2018).</li><li>Zhao, M., Liu, Y., Qu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+epithelial-mesenchymal+transition-related+genes+increases+with+copy+number+in+multiple+cancer+types.">Expression of epithelial-mesenchymal transition-related genes increases with copy number in multiple cancer types.</a> Oncotarget. 7 (17), 24688-24699 (2016).</li><li>Pijuan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+migration,+invasion,+and+adhesion+assays:+From+cell+imaging+to+data+analysis.">In vitro cell migration, invasion, and adhesion assays: From cell imaging to data analysis.</a> Frontiers in Cell and Developmental Biology. 7, 107 (2019).</li><li>Park, T., Large, N., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+glioblastoma+phenotypes+in+vitro+establishes+cell+migration+as+a+robust+readout+of+Crk+and+CrkL+activity.">Quantitative assessment of glioblastoma phenotypes in vitro establishes cell migration as a robust readout of Crk and CrkL activity.</a> The Journal of Biological Chemistry. 296, 100390 (2021).</li><li>Urcan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+xCELLigence+impedance+analysis+of+the+cytotoxicity+of+dental+composite+components+on+human+gingival+fibroblasts.">Real-time xCELLigence impedance analysis of the cytotoxicity of dental composite components on human gingival fibroblasts.</a> Dental Materials. 26 (1), 51-58 (2010).</li><li>Limame, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+dynamic+cell+viability,+migration+and+invasion+assessments+by+novel+real-time+technology+and+classic+endpoint+assays.">Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.</a> PLoS One. 7 (10), e46536 (2012).</li><li>Mudduluru, G., Large, N., Park, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impedance-based+real-time+measurement+of+cancer+cell+migration+and+invasion.">Impedance-based real-time measurement of cancer cell migration and invasion.</a> Journal of Visualized Experiments. (158), e60997 (2020).</li><li>Feller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+family+adaptors-signalling+complex+formation+and+biological+roles.">Crk family adaptors-signalling complex formation and biological roles.</a> Oncogene. 20 (44), 6348-6371 (2001).</li><li>Park, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+and+CrkL+as+therapeutic+targets+for+cancer+treatment.">Crk and CrkL as therapeutic targets for cancer treatment.</a> Cells. 10 (4), 739 (2021).</li><li>Verbeke, R., Lentacker, I., De Smedt, S. C., Dewitte, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dawn+of+mRNA+vaccines:+The+COVID-19+case.">The dawn of mRNA vaccines: The COVID-19 case.</a> Journal of Controlled Release. 333, 511-520 (2021).</li><li>Heine, A., Juranek, S., Brossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+immunological+effects+of+mRNA+vaccines+in+malignant+diseases.">Clinical and immunological effects of mRNA vaccines in malignant diseases.</a> Molecular Cancer. 20 (1), 52 (2021).</li><li>Kwon, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+synthetic+mRNA:+In+vitro+synthesis+of+mRNA+and+its+applications+in+regenerative+medicine.">Emergence of synthetic mRNA: In vitro synthesis of mRNA and its applications in regenerative medicine.</a> Biomaterials. 156, 172-193 (2018).</li><li>Campillo-Davo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+messenger+RNA+electroporation+for+physical+gene+delivery+in+immune+cell-based+therapy.">The ins and outs of messenger RNA electroporation for physical gene delivery in immune cell-based therapy.</a> Pharmaceutics. 13 (3), 396 (2021).</li><li>Park, T., Koptyra, M., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+requires+CT10+regulator+of+kinase+(Crk)+and+Crk-like+(CrkL).">Fibroblast growth requires CT10 regulator of kinase (Crk) and Crk-like (CrkL).</a> The Journal of Biological Chemistry. 291 (51), 26273-26290 (2016).</li><li>Park, T. J., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+roles+of+Crk+and+CrkL+in+fibroblast+structure+and+motility.">Essential roles of Crk and CrkL in fibroblast structure and motility.</a> Oncogene. 33 (43), 5121-5132 (2014).</li><li>Chou, P. P., Jaynes, P. K., King, B. P., Chapman, E., Bailey, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+comparison+between+conventional+(method-I)+and+reverse+(method-Ii)+pipetting.">Precision comparison between conventional (method-I) and reverse (method-Ii) pipetting.</a> Clinical Chemistry. 30 (6), 958 (1984).</li><li>Pushparaj, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+the+micropipetting+techniques+in+biomedical+sciences:+A+fundamental+prerequisite+in+good+laboratory+practice.">Revisiting the micropipetting techniques in biomedical sciences: A fundamental prerequisite in good laboratory practice.</a> Bioinformation. 16 (1), 8-12 (2020).</li><li>Park, T. J., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+and+Crk-like+play+essential+overlapping+roles+downstream+of+disabled-1+in+the+Reelin+pathway.">Crk and Crk-like play essential overlapping roles downstream of disabled-1 in the Reelin pathway.</a> The Journal of Neuroscience. 28 (50), 13551-13562 (2008).</li><li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRK+proteins+selectively+regulate+T+cell+migration+into+inflamed+tissues.">CRK proteins selectively regulate T cell migration into inflamed tissues.</a> The Journal of Clinical Investigation. 125 (3), 1019-1032 (2015).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+adaptor+protein+Crk+is+indispensable+for+malignant+feature+of+glioblastoma+cell+line+KMG4.">Signaling adaptor protein Crk is indispensable for malignant feature of glioblastoma cell line KMG4.</a> Biochemical and Biophysical Research Communications. 362 (4), 976-981 (2007).</li><li>Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+regulation+of+Abl+kinase+by+Crk+Y251+and+Abi1+controls+invasive+phenotypes+in+glioblastoma.">Reciprocal regulation of Abl kinase by Crk Y251 and Abi1 controls invasive phenotypes in glioblastoma.</a> Oncotarget. 6 (35), 37792-37807 (2015).</li><li>Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+tyrosine+phosphorylation+regulates+PDGF-BB-inducible+Src+activation+and+breast+tumorigenicity+and+metastasis.">Crk tyrosine phosphorylation regulates PDGF-BB-inducible Src activation and breast tumorigenicity and metastasis.</a> Molecular Cancer Research. 16 (1), 173-183 (2018).</li><li>Raudenska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engine+shutdown:+Migrastatic+strategies+and+prevention+of+metastases.">Engine shutdown: Migrastatic strategies and prevention of metastases.</a> Trends in Cancer. 9 (4), 293-308 (2023).</li><li>Decaestecker, C., Debeir, O., Van Ham, P., Kiss, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+anti-migratory+drugs+be+screened+in+vitro?+A+review+of+2D+and+3D+assays+for+the+quantitative+analysis+of+cell+migration.">Can anti-migratory drugs be screened in vitro? A review of 2D and 3D assays for the quantitative analysis of cell migration.</a> Medicinal Research Reviews. 27 (2), 149-176 (2007).</li><li>Farasani, A., Darbre, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+exposure+to+triclosan+increases+migration+and+invasion+of+human+breast+epithelial+cells+in+vitro.">Long-term exposure to triclosan increases migration and invasion of human breast epithelial cells in vitro.</a> Journal of Applied Toxicology. 41 (7), 1115-1126 (2021).</li><li>Hanusova, V., Skalova, L., Kralova, V., Matouskova, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+flubendazole+on+adhesion+and+migration+in+SW480+and+SW620+colon+cancer+cells.">The effect of flubendazole on adhesion and migration in SW480 and SW620 colon cancer cells.</a> Anti-Cancer Agents in Medicinal Chemistry. 18 (6), 837-846 (2018).</li><li>Lago-Baameiro, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARK7/DJ-1+inhibition+decreases+invasion+and+proliferation+of+uveal+melanoma+cells.">PARK7/DJ-1 inhibition decreases invasion and proliferation of uveal melanoma cells.</a> Tumori. 109 (1), 47-53 (2023).</li><li>Escalona, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knock+down+of+TIMP-2+by+siRNA+and+CRISPR/Cas9+mediates+diverse+cellular+reprogramming+of+metastasis+and+chemosensitivity+in+ovarian+cancer.">Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer.</a> Cancer Cell International. 22 (1), 422 (2022).</li><li>Mosca, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+S-adenosyl-L-methionine+on+the+invasion+and+migration+of+head+and+neck+squamous+cancer+cells+and+analysis+of+the+underlying+mechanisms.">Effects of S-adenosyl-L-methionine on the invasion and migration of head and neck squamous cancer cells and analysis of the underlying mechanisms.</a> International Journal of Oncology. 56 (5), 1212-1224 (2020).</li><li>Zhao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGI174+protein+and+its+functional+domain+peptides+exert+antitumour+effects+on+renal+cell+carcinoma.">VEGI174 protein and its functional domain peptides exert antitumour effects on renal cell carcinoma.</a> International Journal of Oncology. 54 (1), 390-398 (2019).</li><li>Witte, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta1-induced+cell+migration+in+pancreatic+carcinoma+cells+is+RAC1+and+NOX4-dependent+and+requires+RAC1+and+NOX4-dependent+activation+of+p38+MAPK.">TGF-beta1-induced cell migration in pancreatic carcinoma cells is RAC1 and NOX4-dependent and requires RAC1 and NOX4-dependent activation of p38 MAPK.</a> Oncology Reports. 38 (6), 3693-3701 (2017).</li><li>Bui, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+aquaporin+3+expression+by+the+AhR+pathway+is+critical+to+cell+migration.">Regulation of aquaporin 3 expression by the AhR pathway is critical to cell migration.</a> Toxicological Sciences. 149 (1), 158-166 (2016).</li><li>Tsuji, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+cell-detaching+methods+on+the+viability+and+cell+surface+antigen+expression+of+synovial+mesenchymal+stem+cells.">Effects of different cell-detaching methods on the viability and cell surface antigen expression of synovial mesenchymal stem cells.</a> Cell Transplantation. 26 (6), 1089-1102 (2017).</li><li>Frandsen, S. K., McNeil, A. K., Novak, I., McNeil, P. L., Gehl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difference+in+membrane+repair+capacity+between+cancer+cell+lines+and+a+normal+cell+line.">Difference in membrane repair capacity between cancer cell lines and a normal cell line.</a> The Journal of Membrane Biology. 249 (4), 569-576 (2016).</li></ol>

迁移和侵袭是肿瘤细胞的重要特征。测量肿瘤细胞的运动性并了解控制肿瘤细胞运动的潜在机制为治疗干预提供了重要的见解2,27。已经开发了几种方法来研究细胞迁移7.使用划痕或培养插入物的伤口愈合测定是一种简单且常用的方法，可提供间隙闭合的对比图像。单个细胞追踪测定需要通过延时成像监测单个细胞，为此可以用荧光染?...

肿瘤细胞运动在转移中起着至关重要的作用 1,2。肿瘤细胞扩散到邻近和遥远的健康组织使癌症治疗变得困难，并导致复发3,4。因此，了解肿瘤细胞运动的机制并制定相关的治疗策略至关重要。由于许多肿瘤细胞改变了基因表达谱，因此了解基因表达谱的哪些变化导致肿瘤细胞运动性改变至关重要 5,6。
已经开发了几种测定法来测量 体外细胞迁移。由于只允许在特定时间点进行测量，一些检测仅提供有限的信息，而另一些则实时提供有关肿瘤细胞运动的全面信息7。尽管这些细胞运动测定中的许多可以在给定时间或终点提供定量结果，但它们无法提供有关实验期间细胞迁移速率动态变化的足够详细的信息。此外，根据实验设计、细胞类型和细胞数量，可能很难检查细胞迁移速率的潜在变化。此外，可以通过传统运动测定的简单定量来研究简单处理的效果，但可能需要更复杂的定量来研究各种联合处理的复杂效果8。
已经开发出一种用于监测覆盖有微电极的微量滴定板孔底电流的仪器9。细胞与孔表面的粘附阻碍了电子流动，阻抗与细胞的定量和定性结合相关。孔底存在微电极，可以测量细胞粘附、扩散和增殖。微电极存在于上腔室的微孔膜下方，允许测量细胞迁移和侵袭到下腔室，上腔室涂有细胞外基质 （ECM） 蛋白以允许侵袭10。
此前，已经证明，基于阻抗的肿瘤细胞迁移和侵袭的实时测量在整个实验过程中提供了实时数据，以及在各种实验条件下的即时比较和量化11。在该方法论文中，诱导基因敲低以测试目标蛋白在肿瘤细胞迁移和侵袭中的作用。由于在测试的实验条件下，使用小干扰 RNA （siRNA） 8 电穿孔后需要 3-4 天才能完全实现基因敲低效应，因此在电穿孔后重新接种细胞，并在 3 天后重新收获，以进行基于阻抗的肿瘤细胞迁移和侵袭实时测量。
激酶 （Crk） 和 Crk 样 （CrkL） 的 CT10 调节因子是介导各种生长因子受体激酶通路和非受体酪氨酸激酶通路下游的蛋白质-蛋白质相互作用的接头蛋白12。Crk 和 CrkL 蛋白水平升高会导致几种人类癌症（包括胶质母细胞瘤13）的预后不良。然而，目前尚不清楚 Crk 和 CrkL 蛋白升高如何导致预后不良。因此，确定 Crk 和 CrkL 过表达对肿瘤细胞功能的影响非常重要。此前，进行了一项基因敲低研究，以证明胶质母细胞瘤细胞迁移和侵袭需要内源水平的 Crk 和 CrkL 蛋白8。在这里，已经开发了一种改进的测定系统来解决 Crk 和 CrkL 过表达对肿瘤细胞迁移和侵袭的影响。
最近，由于针对 SARS-CoV-2 的 mRNA 疫苗的开发，mRNA 的体外合成及其治疗应用再次受到关注（由 Verbeke 等人审查 14）。此外，在癌症和其他疾病中使用合成mRNA方面也取得了显着进展15,16。细胞电穿孔是递送合成 mRNA 和诱导瞬时基因修饰的有效方法（Campillo-Davo 等人 17 评论），使用合成 mRNA 可在永生化成纤维细胞中快速有效地表达基因18。本方法论文将使用合成mRNA的基因过表达与实时细胞分析相结合，以研究肿瘤细胞迁移和侵袭。然而，用于 siRNA 的实验方案不适用于合成 mRNA 转染，因为外源蛋白的水平在合成 mRNA 转染时迅速增加并逐渐降低18。因此，该方法已被修改为在转染后立即进行细胞迁移和侵袭的实时分析，而无需额外培养细胞。
本方法论文表明，将基于阻抗的实时测量与用合成mRNA转染肿瘤细胞相结合，可以快速、全面地分析基因上调对肿瘤细胞迁移和侵袭的影响。本方法论文描述了测量胶质母细胞瘤细胞迁移和侵袭如何受 Crk 和 CrkL 过表达影响的详细程序。通过研究合成mRNA对肿瘤细胞迁移的浓度依赖性影响，该论文清楚地描述了蛋白质水平的增加如何刺激肿瘤细胞迁移。此外，还提出了一种改变ECM凝胶浓度的方法，以评估基因表达变化对肿瘤细胞侵袭的影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AlphaImager HP</td>
			<td>ProteinSimple</td>
			<td>92-13823-00</td>
			<td>Agarose gel imaging system</td>
		</tr>
		<tr>
			<td>&#945;-Tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td>Used to detect &#945;-tubulin protein (dilution 1:3,000)</td>
		</tr>
		<tr>
			<td>CIM-plate 16</td>
			<td>Agilent Technologies, Inc</td>
			<td>5665825001</td>
			<td>Cell invasion and migration plates</td>
		</tr>
		<tr>
			<td>Crk antibody</td>
			<td>BD Biosciences</td>
			<td>610035</td>
			<td>Used to detect CrkI and CrkII proteins (dilution 1:1,500)</td>
		</tr>
		<tr>
			<td>CrkL antibody</td>
			<td>Santa Cruz</td>
			<td>sc-319</td>
			<td>Used to detect CrkL protein (dilution 1:1,500)</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>ATCC</td>
			<td>302002</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td>Buffer used to wash cells</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td>Culture medium supplement</td>
		</tr>
		<tr>
			<td>Heracell VIOS 160i CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td>51030285</td>
			<td>CO2 incubator</td>
		</tr>
		<tr>
			<td>IRDye 800CW goat anti-mouse IgG secondary antibody</td>
			<td>Li-Cor</td>
			<td>926-32210</td>
			<td>Secondary antibody for Western blot analysis (dilution 1:10,000)</td>
		</tr>
		<tr>
			<td>IRDye 800CW goat anti-rabbit IgG secondary antibody</td>
			<td>Li-Cor</td>
			<td>926-32211</td>
			<td>Secondary antibody for Western blot analysis&#160; (dilution 1:10,000)</td>
		</tr>
		<tr>
			<td>Lithium chloride&#160;</td>
			<td>Invitrogen</td>
			<td>AM9480</td>
			<td>Used for RNA precipitation</td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Extracellular matrix (ECM) gel</td>
		</tr>
		<tr>
			<td>MEGAscript T7 transcription kit</td>
			<td>Invitrogen</td>
			<td>AM1334</td>
			<td>Used for RNA synthesis</td>
		</tr>
		<tr>
			<td>Millennium RNA markers</td>
			<td>Invitrogen</td>
			<td>AM7150</td>
			<td>Used for formaldehyde agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>ISC BioExpress</td>
			<td>C1301P-ISC</td>
			<td>Used to spin down cells</td>
		</tr>
		<tr>
			<td>Mouse brain QUICK-Clone cDNA</td>
			<td>TaKaRa</td>
			<td>637301</td>
			<td>Source of genes (inserts) for cloning</td>
		</tr>
		<tr>
			<td>NanoQuant</td>
			<td>Tecan</td>
			<td>M200PRO</td>
			<td>Nucleic acid quantification system</td>
		</tr>
		<tr>
			<td>Neon electroporation system&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK5000</td>
			<td>Electroporation system1</td>
		</tr>
		<tr>
			<td>Neon transfection system 10 &#181;L kit</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK1025</td>
			<td>Electroporation kit</td>
		</tr>
		<tr>
			<td>Neon transfection system 100 &#181;L kit</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK10096</td>
			<td>Electroporation kit</td>
		</tr>
		<tr>
			<td>NorthernMax denaturing gel buffer</td>
			<td>Invitrogen</td>
			<td>AM8676</td>
			<td>Used for formaldehyde agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>NorthernMax formaldehyde load dye</td>
			<td>Invitrogen</td>
			<td>AM8552</td>
			<td>Used for formaldehyde agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>NorthernMax running buffer</td>
			<td>Invitrogen</td>
			<td>AM8671</td>
			<td>Used for formaldehyde agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Teknova</td>
			<td>W3331</td>
			<td>Used for various reactions during mRNA synthesis</td>
		</tr>
		<tr>
			<td>Odyssey CLx Imager</td>
			<td>Li-Cor</td>
			<td></td>
			<td>Imager for Western blot analysis</td>
		</tr>
		<tr>
			<td>pcDNA3.1/myc-His</td>
			<td>Invitrogen</td>
			<td>V80020</td>
			<td>The vector into which inserts (mouse CrkI and CrkL cDNAs) were cloned</td>
		</tr>
		<tr>
			<td>pFLAG-CMV-5a</td>
			<td>Millipore Sigma</td>
			<td>E7523</td>
			<td>Source of the FLAG epitope tag</td>
		</tr>
		<tr>
			<td>Phenol:chloroform:isoamyl alcohol&#160;</td>
			<td>Sigma</td>
			<td>P2069</td>
			<td>Used for DNA extraction</td>
		</tr>
		<tr>
			<td>PmeI</td>
			<td>New England BioLabs</td>
			<td>R0560L</td>
			<td>Used to linearize the plasmids for mRNA synthesis</td>
		</tr>
		<tr>
			<td>Poly(A) tailing kit</td>
			<td>Invitrogen</td>
			<td>AM1350</td>
			<td>Used for poly(A) tail reaction</td>
		</tr>
		<tr>
			<td>Polystyrene tissue culture dish (100 x 20 mm style)</td>
			<td>Corning</td>
			<td>353003</td>
			<td>Used for culturing cells before transfection</td>
		</tr>
		<tr>
			<td>Polystyrene tissue culture dish (35 x 10 mm style)</td>
			<td>Corning</td>
			<td>353001</td>
			<td>Used for culturing transfected cells</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Invitrogen</td>
			<td>25530049</td>
			<td>Used to remove protein in the reaction mixture</td>
		</tr>
		<tr>
			<td>Purifier Axiom Class II, Type C1</td>
			<td>Labconco Corporation</td>
			<td>304410001</td>
			<td>Biosafety cabinet for sterile handling of cells</td>
		</tr>
		<tr>
			<td>Resuspension Buffer R</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>A buffer included in the electroporation kits, MPK1025 and MPK10096. The buffer is used to resupend cells before electroporation, and its composition is proprietary information.</td>
		</tr>
		<tr>
			<td>RNaseZap</td>
			<td>Invitrogen</td>
			<td>AM9780</td>
			<td>RNA decontamination solution</td>
		</tr>
		<tr>
			<td>Scepter</td>
			<td>Millipore</td>
			<td>C85360</td>
			<td>Handheld automated cell counter&#160;</td>
		</tr>
		<tr>
			<td>ScriptCap 2&#39;-O-methyltransferase kit</td>
			<td>Cellscript</td>
			<td>C-SCMT0625</td>
			<td>Used for capping reaction</td>
		</tr>
		<tr>
			<td>ScriptCap m7G capping system</td>
			<td>Cellscript</td>
			<td>C-SCCE0625</td>
			<td>Used for capping reaction</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate solution</td>
			<td>Invitrogen</td>
			<td>15553-035</td>
			<td>Detergent used for the proteinase K reaction</td>
		</tr>
		<tr>
			<td>Sorvall Legend XT centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004532</td>
			<td>Benchtop centrifuge to spin down cells</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Used for dissociation of cells</td>
		</tr>
		<tr>
			<td>U-118MG&#160;</td>
			<td>ATCC</td>
			<td>HTB15</td>
			<td>An adherent cell line derived from a human glioblastoma patient</td>
		</tr>
		<tr>
			<td>Vinculin antibody</td>
			<td>Sigma</td>
			<td>V9131</td>
			<td>Used to detect vinculin protein (dilution 1:100,000)</td>
		</tr>
		<tr>
			<td>xCELLigence RTCA DP</td>
			<td>Agilent Technologies, Inc</td>
			<td>380601050</td>
			<td>Instrument used for real-time cell analysis</td>
		</tr>
		<tr>
			<td>1Electroporation parameters and other related information for various cell lines are available on the manufacturer&#39;s homepage (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/neon-transfection-system/neon-transfection-system-cell-line-data.html?).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time quantitative measurement of tumor cell migration and invasion following synthetic mrna transfection

肿瘤细胞具有高度的运动性和侵袭性，并显示出基因表达模式的改变。了解基因表达的变化如何调节肿瘤细胞迁移和侵袭对于了解肿瘤细胞浸润到邻近健康组织和转移的机制至关重要。以前，已经证明基因敲低，然后基于阻抗的肿瘤细胞迁移和侵袭实时测量能够识别肿瘤细胞迁移和侵袭所需的基因。最近，针对 SARS-CoV-2 的 mRNA 疫苗增加了人们对使用合成 mRNA 进行治疗的兴趣。本文对合成mRNA方法进行了修正，研究了基因过表达对肿瘤细胞迁移和侵袭的影响。这项研究表明，通过合成 mRNA 转染和基于阻抗的实时测量提高基因表达可能有助于识别刺激肿瘤细胞迁移和侵袭的基因。本方法论文提供了有关检查基因表达改变对肿瘤细胞迁移和侵袭影响的程序的重要细节。

Crk 和 CrkL 蛋白在许多细胞类型的运动中起着重要作用，包括神经元22、T 细胞23、成纤维细胞 18,19 和多种肿瘤细胞13。由于已报道 Crk 和 CrkL 蛋白在胶质母细胞瘤24,25,26 中升高，因此本研究研究了 CrkI（Crk 的剪接变体）过表达对胶质母细胞...

Watch this Scientific Journal Video about Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection at JoVE.com

Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

许多上调的基因刺激肿瘤细胞迁移和侵袭，导致预后不良。确定哪些基因调节肿瘤细胞迁移和侵袭至关重要。该协议提出了一种实时研究基因表达增加对肿瘤细胞迁移和侵袭的影响的方法。

合成mRNA转染后肿瘤细胞迁移和侵袭的实时定量测量

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell ...

该协议表明，将感觉信使RNA交易与基于阻抗的细胞迁移和侵袭实时测量相结合，有助于识别刺激肿瘤细胞迁移和侵袭的基因。基于阻抗的实时细胞分析系统能够定量、连续、全面地监测肿瘤细胞在基因表达变化时的迁移。过度表达肿瘤并刺激肿瘤细胞迁移的基因可以被确定为抑制癌症治疗转移的潜在靶点。首先，将 10 μL 的 10x 反应缓冲液、1.5 μL 的 PNE 1 和 10 μg 的质粒 DNA 加入微量离心管中，用限制性内切酶对质粒 DNA 进行线性化。加入不含核酸酶的水，使反应体积达到100微升。在将反应混合物在 37 摄氏度下孵育过夜之前，通过敲击和旋转将其混合。再次，在混合并旋转之前，向反应混合物中加入 5 微升 10% SDS 和 1 微升 20 毫克/毫升 RNA 级蛋白酶 K，然后再将其混合并下降。在 50 摄氏度下孵育 30 分钟后，向微量离心管中加入 2 μL 的 10x 转录缓冲液、ATP、GTP、UTP、CTP 和 T7，以及 1 微克线性化 DNA，用于 T7 RNA 聚合酶介导的 RNA 合成。加入无核酸酶的水，使反应体积达到20微升。离心后，将反应混合物在 37 摄氏度下孵育 2 小时以进行 RNA 合成。接下来，向下旋转并加入 1 微升 DNA，并在 37 摄氏度下孵育 15 分钟前敲击以去除模板 DNA。通过向反应混合物中加入 10 微升 7.5 摩尔氯化锂，开始 RNA 的氯化锂沉淀。敲击并旋转后，将反应混合物在零下 20 摄氏度下孵育 30 分钟。盖帽时，将RNA样品在65摄氏度下加热10分钟，然后将其放在冰上冷却。接下来，添加封端反应组分并通过敲击混合。将反应混合物在 37 摄氏度下旋转并孵育 1 小时。对于poly-A拖尾，将样品旋转并加入poly-A拖尾反应组分。然后敲击、旋转，并将反应混合物在 37 摄氏度下孵育 2 小时。对于氯化锂沉淀和合成mRNA的定量，加入50微升7.5摩尔氯化锂并进行氯化锂沉淀。从冰箱中取出等分试样的ECM凝胶原液，并将其放在冰上。通过在微量离心管中将 990 微升 DMEM 与 10 微升 ECM 凝胶混合，将每升 10 毫克 ECM 凝胶稀释至工作浓度。通过轻柔的移液混合。将 60 微升稀释的 ECM 凝胶加入 CIM 板上室的 16 个孔中的每一个。将CIM板的上腔室与板盖脱开，放在二氧化碳培养箱内的保护性塑料片上约四小时，以形成凝胶层。对于电穿孔，将一毫升DPBS添加到每个试管中的肿瘤细胞悬液中。轻敲细胞悬液并向下旋转 3 次，持续 10 秒钟。使用微量移液管除去上清液。向细胞沉淀中加入 110 微升重悬缓冲液 R，以获得 10 微升的 10 万个细胞。将合成的 mRNA 添加到细胞沉淀中，以获得 0.2 至 20 ng/μL 浓度，具体取决于所需的表达水平。通过敲击轻轻混合并重悬细胞沉淀。接下来，用 1， 350 伏的电穿孔系统电穿孔 10 微升细胞悬液，持续 10 毫秒，每次三个脉冲。完成后，将电穿孔细胞转移到含有 1.1 毫升含有 0.5% FBS 的 DMEM 的新微量离心管中。双击实时细胞分析软件图标打开分析程序。打开默认实验模式设置选项后，选择用于分别运行三个实验的选项。通过单击数字选项卡打开每个支架。接下来，单击实验注释选项卡。选择文件夹，然后通过输入实验的名称来保存数据。单击布局选项卡，一次选择四个孔，为每个处理条件设置四重孔。输入示例信息，然后单击应用。单击计划选项卡，然后添加一个步骤以设置电池阻抗测量的两步模式。然后，单击“应用”以设置第一步。再次单击“添加步骤”，然后输入 10 分钟作为间隔，输入 48 小时作为迁移和入侵的持续时间。然后，单击“应用”以设置第二步。在细胞阻抗测量前一小时，将含有10%血清或其他化学引诱剂的160微升DMEM加入CIM板下腔室的孔中。组装包含用于侵入的ECM凝胶包被孔的上腔室或用于与下腔室迁移的未包被孔的上室。对于细胞迁移测定，向CIM板上室的孔中加入50μL低血清培养基。将CIM板和系统的支架放入CO 2培养箱中。单击消息选项卡。连接正常后，将显示消息，CIM 板已准备好进行实验。在进行实时细胞分析之前，将组装好的 CIM 板在二氧化碳培养箱中预孵育 30 至 60 分钟，以使 CIM 板适应培养条件。对于基线读数，请单击每个底座的开始按钮。当“另存为”窗口出现时，保存实验文件以执行基线读数。对于细胞就座，从支架上取下 CIM 板并将其放入板支架上的生物安全柜中。然后将含有100,000个细胞的100微升电穿孔细胞添加到CIM板上室的孔中，并将CIM板在室温下置于生物安全柜下30分钟。通过将完全组装好的CIM板移回相应的支架来测量电池阻抗。通过单击底座开始按钮和绘图选项卡开始第二步的电池阻抗测量。然后单击“全部添加”按钮并选择“平均值”和“标准差”框以实时可视化数据。使用不同浓度的合成裂纹 1 mRNA 对 U 118 mg 胶质母细胞瘤细胞进行电穿孔，导致转染后一天标记的裂纹蛋白的浓度依赖性增加。每微升 mRNA 0.2 和 2 纳克导致外源性裂纹蛋白的检测不到或适度表达。20 ng/μL mRNA 导致比内源性裂解 1 蛋白更高的表达水平。细胞迁移试验表明，电穿孔为0.2或2纳克/微升裂纹，一个mRNA对细胞迁移影响不大。然而，用每微升裂纹 20 纳克的 mRNA 进行电穿孔导致细胞迁移的明显刺激，更多的细胞在 2 到 13 小时之间迁移，这表明胶质母细胞瘤细胞迁移是由裂纹蛋白水平的增加刺激的。使用合成裂纹 L mRNA 进行电穿孔，导致标记标记的裂纹 L 蛋白在转染后 1 天出现稳健表达。随着ECM蛋白浓度的增加，对照细胞的侵袭速度减慢。裂纹L过度表达细胞显示出细胞侵袭的ECM凝胶浓度依赖性降低。对照组和裂纹L在不同ECM凝胶浓度下过表达细胞的比较表明，裂纹L过表达通常刺激细胞通过ECM凝胶层侵袭。结果还表明，在不同时间点增加ECM凝胶浓度对两种细胞群的影响不同。细胞应快速、温和、完全地重悬于重悬的气泡 R 中，进行电穿孔，因为细胞在重悬气泡 R 中长时间孵育会使细胞不健康。可以分离快速和缓慢迁移的肿瘤细胞，以表征两个细胞群之间的差异。

Research

JoVE Journal

Cancer Research

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

细胞侵袭和迁移过程的评价: 视频 Microscope-based 刮伤法与博伊登室法的比较

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

科研

癌症研究

Serum and Plasma Copy Number Detection Using Real-time PCR

应用实时 PCR 技术检测血清和血浆拷贝数

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

三维流状入侵检测中的全人肿瘤基基质

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

多端根结核细胞迁移的实时监测

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

通过高分辨率共聚焦显微镜对3D肿瘤球体入侵的影响表征

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

测量组织肿瘤组织切片的实时药物反应

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...