Poster Y17 - A One-Step Urinary EV Isolation and EV-derived miRNA Extraction Platform for the Diagnosis of Prostate Cancer.

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Minju Lee

ph.D student
Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, United States

Primary Investigator(s)

YS

Yong Shin, PhD

Professor
Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Republic of Korea

Co-Author(s)

Bk

Bonhan koo

ph.D
Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, United States

Introduction: Extracellular vesicles (EVs) contain a variety of biomarkers, including proteins, microRNAs (miRNAs), and circular RNAs (circRNAs), which can serve as indicators of the status of the parent cell. Cancer-derived EV miRNAs are nucleic acids that are suitable for the diagnosis, metastasis, and prognosis of cancer. However, the conventional method for isolating EV-derived miRNAs is complex and has possibility of miRNA damage, therefore an effective method for isolating EV-derived miRNAs is required. Prostate cancer (PCa) is the second most common type of cancer worldwide. Due to the absence of distinctive symptoms during the early stages of the disease, it is often not recognized until the disease has progressed. Traditional diagnostic procedures for PCa include analysis of prostate-specific antigen (PSA) levels in the blood, tissue biopsy, and digital rectal examination (DRE). However, elevated PSA levels may be indicative of other conditions, and DRE induces significant patient discomfort. Therefore, non-invasive and PCa-specific diagnosis based on the isolation of EVs from patient urine can act as an effective alternative. Urine, containing EVs derived directly from the prostate, is an optimal specimen for PCa diagnosis. In this study, we introduce a platform that enables the isolation of EVs, as well as the extraction of proteins and nucleic acids in a single step, using amine-modified zeolite and carbohydrazide, a homobifunctional hydrazide family (ACE platform). This platform has demonstrated its potential for PCa diagnosis through quantitative analysis of extracted miRNAs.

Materials and

Methods: Amine-modified zeolite (AZ) synthesis
The synthesis of amine-modified zeolite is initiated by sequential washing of zeolite with deionized water and 95% ethanol. After homogenizing the zeolite by washing, we incubate zeolite in a 2% (v/v) APDMS solution in 95% ethanol for 4 hours to synthesize amine-modified zeolite. The synthesized amine-modified zeolite is washed three times with 95% ethanol and dried for at least one day before storage.
Amine-modified zeolite and Carbohydrazide based Extracellular vesicle isolation platform (ACE) platform
The 22Rv1 prostate cancer cell line culture media were harvested and filtered through a PVDF 0.22 µm syringe filter to remove cell debris. 10 mL of cell culture media were mixed with 50 mg of AZ and 400 mg of carbohydrazide (CDH) and incubated for 10 minutes. Transfer the mixture to a syringe and pass through a PVDF 0.45 µm syringe filter. Wash the EV-enriched zeolite on the filter with PBS and isolate it with a high pH elution buffer.
miRNA analysis
Treat the EV-enriched zeolite on the filter with the lysis buffer and CDH mixture and incubate for 20 minutes at room temperature. When the nucleic acid in EV leaks out and binds to AZ-CDH, wash off the lysis buffer with PBS washing and extract the nucleic acid with a high pH elution buffer. The extracted nucleic acid is synthesized into cDNA using a commercial kit and quantitatively analyzed by real-time PCR.

Results, Conclusions, and Discussions: The ACE platform is an extracellular vesicle isolation platform based on amine-modified zeolite and carbohydrazide. Zeolite is treated with APDMS to expose amine groups on its surface. The positive charges of AZ and CDH create electrostatic bonds with EVs, resulting in the enrichment of EVs. This is followed by either high pH elution, which causes AZ and CDH to lose their positive charge and separate the EVs, or lysis of the EVs on a filter, which re-binds the nucleic acids to AZ and elutes them.
To optimize EV isolation conditions, we used the Ct value of miR-122-5p extracted via the platform as a reference. To identify the optimal conditions for EV isolation, we evaluated the pore size of the PVDF syringe filter, the concentration of AZ, CDH, lysis buffer, and the incubation time.
The characterization of the ACE platform was analyzed by scanning electron microscopy (SEM), zeta potential, Fourier-transform infrared (FTIR) spectroscopy, western blotting, nano tracking analysis (NTA) and dynamic light scattering (DLS).
We evaluated the expression of 16 miRNA markers associated with prostate cancer at the cell line level. The results indicated that 9 of these markers exhibited significant differential expression between prostate cancer and normal cells. The next phase of this study involves validating these findings using patient urine samples. By applying the ACE platform to isolate urinary EVs, we will assess miRNA expression differences between prostate cancer patients and healthy controls to verify the diagnostic potential for prostate cancer.

Acknowledgements (Optional):