Construction of Artificial Cells with Application to Investigate Peritoneal Metastasis in Ovarian Cancer

WAN Li-zhou; DU Ru-pei; CHEN Kang-mei

doi:10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0610

您当前的位置：

首页 >

文章列表页 >

Construction of Artificial Cells with Application to Investigate Peritoneal Metastasis in Ovarian Cancer

Preclinic Research | 更新时间：2024-02-19

- Construction of Artificial Cells with Application to Investigate Peritoneal Metastasis in Ovarian Cancer
- Journal of Sun Yat-sen University(Medical Sciences) Vol. 44, Issue 6, Pages: 965-973(2023)
- 作者机构：
  
  1.中山大学孙逸仙纪念医院基础与转化医学研究中心，广东广州 510120
  2.华南理工大学生物医学科学与工程学院，广东广州 511442
  3.中山大学孙逸仙纪念医院检验科，广东广州 510120
- 作者简介：
  
  CHEN Kang-mei； E-mail： chenkm7@outlook.com
- 基金信息：
- DOI：10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0610
  CLC： R737.31
- Published：20 November 2023，
  
  Received：08 August 2023，
扫描看全文
万里舟,杜汝沛,陈康梅.卵巢癌腹腔转移工具细胞的构建及其应用[J].中山大学学报（医学科学版）,2023,44(06):965-973.

WAN Li-zhou,DU Ru-pei,CHEN Kang-mei.Construction of Artificial Cells with Application to Investigate Peritoneal Metastasis in Ovarian Cancer[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(06):965-973.
万里舟,杜汝沛,陈康梅.卵巢癌腹腔转移工具细胞的构建及其应用[J].中山大学学报（医学科学版）,2023,44(06):965-973. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0610.

WAN Li-zhou,DU Ru-pei,CHEN Kang-mei.Construction of Artificial Cells with Application to Investigate Peritoneal Metastasis in Ovarian Cancer[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(06):965-973. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2023.0610.

摘要

目的

构建稳定共表达荧光素酶（Luc）和增强型绿色荧光蛋白（EGFP）的人源卵巢癌细胞SKOV3（SK-Luc-EGFP）并探究其在卵巢癌腹腔转移的体内外研究中的应用。

方法

利用重叠PCR扩增得到的Luc-T2A-EGFP基因片段，构建重组质粒pCDH-Luc-T2A-EGFP-Puro；采用三质粒慢病毒包装系统转染HEK 293T细胞，收集病毒液感染SKOV3细胞；通过嘌呤霉素筛选，流式细胞术检测，筛选高效表达EGFP的细胞（SK-Luc-EGFP），并通过体外生物发光实验验证Luc的表达。对SK-Luc-EGFP细胞进行如下应用的探究：利用流式细胞术和激光共聚焦区分SK-Luc-EGFP细胞与腹水微环境中的非肿瘤细胞，利用荧光显微镜观察SK-Luc-EGFP与间皮细胞的黏附，利用小动物活体成像仪观察SK-Luc-EGFP细胞的大网膜黏附以及体内成瘤的过程。

结果

成功构建慢病毒载体pCDH-Luc-T2A-EGFP-Puro；感染并筛选得到SK-Luc-EGFP单克隆细胞株。通过荧光显微镜和流式细胞术均能检测到EGFP的表达，细胞纯度达100%；通过流式细胞术和激光共聚焦成像可直观辨别SK-Luc-EGFP细胞与腹水微环境细胞。体外生物发光试验成功验证Luc的表达，且细胞数和生物发光信号的线性相关系数为0.997 9。细胞黏附试验中通过荧光成像直接观察到SK-Luc-EGFP细胞对间皮细胞的黏附；经腹腔注射SK-Luc-EGFP细胞观察到细胞在裸鼠大网膜上的黏附；在SK-Luc-EGFP细胞建立的卵巢癌腹腔转移模型中，利用小动物活体成像仪实现对腹腔肿瘤的活体监测。

结论

SK-Luc-EGFP细胞株在卵巢癌腹腔转移的体内外研究中具有多重应用潜力。

Abstract

Objective

To construct a human ovarian cancer cell line SKOV3 （SK-Luc-EGFP） stably co-expressing luciferase （Luc） and enhanced green fluorescent protein （EGFP） and to explore its application in ovarian cancer research both

in vitro

and

in vivo

Methods

The recombinant plasmid pCDH-Luc-T2A-EGFP-Puro was constructed by introducing a Luc-T2A-EGFP fusion gene fragment amplified by Overlap PCR into plasmid vector. The three-plasmid lentivirus packaging system was transfected into HEK 293T cells and the viral supernatant was harvested to infect SKOV3 cells. SK-Luc-EGFP cell line with the highest fluorescence intensity of EGFP was obtained by puromycin selection and flow cytometry assessment， and the Luc expression of the cell line was subsequently validated by

in vitro

bioluminescent assay. SK-Luc-EGFP cells were further explored for the following applications： distinguishing SK-Luc-EGFP cells from non-tumor cells in ascites by flow cytometry and confocal microscopy； visualizing adhesion of SK-Luc-EGFP cells to mesothelial cells or omentum by fluorescence microscopy； monitoring process of SK-Luc-EGFP tumorigenesis by in vivo bioluminescence imaging.

Results

A recombinant lentiviral expression plasmid pCDH-Luc-T2A-EGFP-Puro was constructed and packaged into lentiviral particles that were then transfected into SKOV3 cells to generate SK-Luc-EGFP cell line. The purity of SK-Luc-EGFP cells based on EGFP expression was 100% as validated by fluorescence microscopy and flow cytometry； SK-Luc-EGFP cells could be visually distinguished from non-tumor cells in ascitic fluid by flow cytometry and confocal imaging. Moreover， Luc expression in SK-Luc-EGFP cells was verified by

in vitro

bioluminescence assay， and a linear relationship with a correlation coefficient of 0.997 9 was found between cell number and the bioluminescent signal. Adhesion of SK-Luc-EGFP cells to mesothelial cells was directly observed by fluorescence imaging in

in vitro

adhesion assay； peritoneal adhesion of SK-Luc-EGFP cells to omentum was also observed after intraperitoneal （i.p.） injection of SK-Luc-EGFP cells in nude mice； in the peritoneal metastasis mouse model established by i.p. injection of SK-Luc-EGFP cells， monitoring of tumorigenesis process was achieved by

in vivo

bioluminescence imaging.

Conclusion

SK-Luc-EGFP cell line is a useful tool for investigating ovarian cancer

in vitro

and

in vivo.

关键词

卵巢癌腹腔转移荧光素酶增强型绿色荧光蛋白构建

Keywords

ovarian cancerperitoneal metastasisluciferaseenhanced green fluorescent proteinconstruction

references

Siegel RL， Miller KD， Fuchs HE， et al. Cancer statistics， 2022［J］. CA Cancer J Clin， 2022， 72（1）： 7-33.

Zheng R， Zhang S， Zeng H， et al. Cancer incidence and mortality in China， 2016［J］. J Natl Cancer Cent， 2022， 2（1）： 1-9.

Castellani F， Nganga EC， Dumas L， et al. Imaging in the pre-operative staging of ovarian cancer［J］. Abdom Radiol （NY）， 2019， 44（2）： 685-696.

Bast RC， Han CY， Lu Z， et al. Next steps in the early detection of ovarian cancer［J］. Commun Med （Lond）， 2021，1： 36.

Prat J. Staging classification for cancer of the ovary， fallopian tube， and peritoneum［J］. Int J Gynaecol Obstet， 2014， 124（1）： 1-5.

Hennessy BT， Coleman RL， Markman M. Ovarian cancer［J］. Lancet， 2009， 374（9698）： 1371-1382.

Azaïs H， Vignion-Dewalle AS， Carrier M， et al. Microscopic peritoneal residual disease after complete macroscopic cytoreductive surgery for advanced high grade serous ovarian cancer［J］. J Clin Med， 2020， 10（1）： 41.

Fu SL， Chen CA， Hung LC， et al. Preliminary results of a non-invasive method to measure tumor size and distribution in vivo［J］. Exp Ther Med， 2016， 12（6）： 3614-3620.

Klerk CP， Overmeer RM， Niers TM， et al. Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals［J］. Biotechniques， 2007， 43（1 Suppl）： 7-13；+30.

Kagadis GC， Loudos G， Katsanos K， et al. In vivo small animal imaging： current status and future prospects［J］. Med Phys， 2010， 37（12）： 6421-6442.

Rampurwala M， Ravoori MK， Wei W， et al. Visualization and quantification of intraperitoneal tumors by in vivo computed tomography using negative contrast enhancement strategy in a mouse model of ovarian cancer［J］. Transl Oncol， 2009， 2（2）： 96-106.

Mei SS， Chen X， Wang K， et al. Tumor microenvironment in ovarian cancer peritoneal metastasis［J］. Cancer Cell Int， 2023， 23（1）： 1-13.

Jiang Y， Pu K. Molecular probes for autofluorescence-free optical imaging［J］. Chem Rev， 2021， 121（21）： 13086-13131.

Serkova NJ， Glunde K， Haney CR， et al. Preclinical applications of multi-platform imaging in animal models of cancer［J］. Cancer Res， 2021， 81（5）： 1189-1200.

Alsawaftah N， Farooq A， Dhou S， et al. Bioluminescence imaging applications in cancer： a comprehensive review［J］. IEEE Rev Biomed Eng， 2021， 14： 307-326.

Ohba Y， Fujioka Y， Nakada S， et al. Fluorescent protein-based biosensors and their clinical applications［J］. Prog Mol Biol Transl Sci， 2013， 113： 313-348.

Nieman KM， Kenny HA， Penicka CV， et al. Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth［J］. Nat Med， 2011， 17（11）： 1498-1503.

Lengyel E. Ovarian cancer development and metastasis［J］. The Am J Pathol， 2010， 177（3）： 1053-1064.

Mukherjee A， Chiang CY， Daifotis HA， et al. Adipocyte-induced FABP4 expression in ovarian cancer cells promotes metastasis and mediates carboplatin resistance［J］. Cancer Res， 2020， 80（8）： 1748-1761.

Van Baal J， Van Noorden CJF， Nieuwland R， et al. Development of peritoneal carcinomatosis in epithelial ovarian cancer： a review［J］. J Histochem Cytochem， 2018， 66（2）： 67-83.

Sakuma T， Barry MA， Ikeda Y. Lentiviral vectors： basic to translational［J］. Biochem J， 2012， 443（3）： 603-618.

Scott DJ， Gunn NJ， Yong KJ， et al. A novel ultra-stable， monomeric green fluorescent protein for direct volumetric imaging of whole organs using CLARITY［J］. Sci Rep， 2018， 8（1）： 667.

Tam JM， Upadhyay R， Pittet MJ， et al. Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging［J］. Mol Imaging， 2007， 6（4）： 269-276.

Hall MP， Unch J， Binkowski BF， et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate［J］. ACS Chem Biol， 2012， 7（11）： 1848-1857.

Syed AJ， Anderson JC. Applications of bioluminescence in biotechnology and beyond［J］. Chem Soc Rev， 2021， 50（9）： 5668-5705.

Sfarcic I， Bui T， Daniels EC， et al. Nanoluciferase-based method for detecting gene expression in caenorhabditis elegans［J］. Genetics， 2019， 213（4）： 1197-1207.

Miao Q， Pu K. Organic Semiconducting agents for deep-tissue molecular imaging： second near-infrared fluorescence， self-luminescence， and photoacoustics［J］. Adv Mater， 2018， 30（49）： e1801778.

Yoon S， Cheon SY， Park S， et al. Recent advances in optical imaging through deep tissue： imaging probes and techniques［J］. Biomater Res， 2022， 26（1）： 57.

Chen Y， Wang S， Zhang F. Near-infrared luminescence high-contrast in vivo biomedical imaging［J］. Nat Rev Bioeng， 2023， 1： 60-78.

Yue Y， Zhao T， Wang Y， et al. HSA-Lys-161 covalent bound fluorescent dye for in vivo blood drug dynamic imaging and tumor mapping［J］. Chem sci， 2021， 13（1）： 218-224.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Model Based on Preoperative Inflammatory Markers to Predict the Overall Survival in Patients with Ovarian Cancer

Cellular Localization and Clinical Relevance of GPR30 in Ovarian Cancer

Resistance Reversion of Rapamycin on Ovarian Cancer Cell Line SKOV3/DDP and Its Molecular Mechanisms

Decreased Expression of Long Non-Coding RNA GAS5 in Ovarian Cancer Patients and Its Clinical Significance

Related Author

LI Xi

LIN Min

MA Ben

GU Zheng-tian

KANG Jia-li

XU Guo-cai

WANG Dong-yan

LU Huai-wu

Related Institution

Department of Obstetrics and Gynecology， Guangzhou First People's Hospital

Department of Gynecologic Oncology，Sun Yat-sen Memorial Hospital

Department of Gynecology，The Eastern Hospital of the First Affiliated Hospital，Sun Yat-sen University

Department of Gynecology，The First Affiliated Hospital of Sun Yat-sen University

Postal code：510080
Tel：020-87331643，87330827 Email：xbmed@mail.sysu.edu.cn
Technical support is provided by Beijing Founder electronics co., LTD 京ICP备09064830号-19 京公网安备11010802024621
It is recommended to read the content of this site in Chrome&IE9+. Please switch to extreme mode in browser 360.
Cookies We use cookies to help provide and enhance our service and tailor content. By continuing, you agree to the use of cookies.

⁰