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Effects of RALA/B Knockdown on Extracellular Vesicle Biogenesis and Isolation of CD63+ Vesicles with Microfluidic Device of Triple-Negative Breast Cancer

Gladkiy, Yevgeniy Vyacheslavovich
http://orcid.org/0000-0002-3522-4800
http://rave.ohiolink.edu/etdc/view?acc_num=osu1620635356371349

Abstract Details

2021, Master of Science, Ohio State University, Biomedical Engineering.
Triple negative breast cancer (TNBC) is the deadliest breast cancer subtype occurring with highest frequency in women of African ancestry. Less access to affordable health screening and insurance in women of low socio-economic status limits early diagnosis of TNBC contributing to increased mortality in this group. New, affordable diagnostic methods are urgently needed for earlier detection of TNBC in disadvantaged populations. Our group has determined that the two closely related RAL GTPases, RALA and RALB, have disparate roles in TNBC tumor growth. These G proteins have also recently been implicated in the production and secretion of extracellular vesicles (EVs). EVs secreted by breast cancer cells may serve as valuable diagnostic tools. We have developed microfluidic devices for the capture of EVs and analysis of EV RNA and protein contents. In this study, we optimize these devices for the capture of EVs secreted by TNBC and determine the consequences of RALA/RALB knockdown on total and CD63+ EV production and composition. MDA-MB-231 TNBC cells stably expressing shRNAs targeting RALA, RALB or a control shRNA were used in this study. EVs were collected from serum-free conditioned medium by ultracentrifugation (UC) or ultrafiltration and purified through a microfluidic device. Herringbone-patterned PDMS microfluidic devices were prepared using soft lithography. In a vacuum plasma chamber, PDMS and a glass slide were activated and bonded to create a sealed chamber. Devices were functionalized by introduction of 0.5 % [3-(2-aminoethylamino)propyl]trimethoxysilane in ethanol, followed by 2.5% glutaraldehyde in DI water. Finally, CD63 antibody was flowed through the device. Modeling in COMSOL was utilized to optimize the flow rate for EV capture. UC-purified EV solution was passed through the device to capture CD63+ vesicles which were released with a pH 2.2 glycine-HCL buffer, and immediately neutralized with a pH 8.5 Tris-HCL buffer. EVs from UC were characterized with NanoSight to determine concentration and size distribution. Optimization based on COMSOL modeling was required to reduce shear stress and maximize capture of CD63+ EVs. Knockout of RALB resulted in production of significantly different median of EVs. We predict knockout of RALA will also result in increased production of CD63+ EVs. No difference in EV concentration was noted in cells lacking either RALA or RALB relative to control cells. We have developed an inexpensive, liquid biopsy microfluidics device for the quantification and characterization of EVs from TNBC samples. Application of these devices may lead to earlier detection of TNBC, particularly in patients for whom affordable care is limited. Further implementation of these devices may also lead to improved outcome for women with TNBC by identification of the critical pro-tumorigenic components carried by tumor EVs.
Steven Sizemore (Advisor)
Derek Hansford (Advisor)
70 p.
Biomedical Engineering
Breast Cancer; Microfluidics; RAL-GTPases; Extracellular Vesicles; Exosomes

Recommended Citations

Citations

  • Gladkiy, Y. V. (2021). Effects of RALA/B Knockdown on Extracellular Vesicle Biogenesis and Isolation of CD63+ Vesicles with Microfluidic Device of Triple-Negative Breast Cancer [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1620635356371349

    APA Style (7th edition)

  • Gladkiy, Yevgeniy. Effects of RALA/B Knockdown on Extracellular Vesicle Biogenesis and Isolation of CD63+ Vesicles with Microfluidic Device of Triple-Negative Breast Cancer. 2021. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1620635356371349.

    MLA Style (8th edition)

  • Gladkiy, Yevgeniy. "Effects of RALA/B Knockdown on Extracellular Vesicle Biogenesis and Isolation of CD63+ Vesicles with Microfluidic Device of Triple-Negative Breast Cancer." Master's thesis, Ohio State University, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=osu1620635356371349

    Chicago Manual of Style (17th edition)

osu1620635356371349
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