Lateral Magnetophoresis Research Articles

Abstract 5378: A comparative analysis on the expression of PSA protein in serum and PSA mRNA in circulating tumor cells in prostate cancer

Abstract Introduction Unlike other cancers, prostate cancer has a distinguished biomarker for diagnosis and prognosis which is known as prostate specific antigen (PSA) protein in serum. In the early stage of prostate cancer, PSA protein in serum is highly expressed from primary tumor sites and thereby, the expression of PSA protein can be used in prognosis of prostate cancer during whole progression. However, due to low specificity of serum PSA protein, it raised concerns in over-diagnosis. Due to the advantages of noninvasive, real-time and pain-free for patients, circulating tumor cells (CTCs) is being researched as complementary alternative biomarkers for clinical and molecular analysis of cancer. Some studies reported that the levels of PSA protein and PSA mRNA in prostate cancer tissue are proportional to each other. Other studies showed that the expression level of PSA mRNA in peripheral blood and biochemical recurrence of PSA protein in serum are significant relationship. However, the relevance between PSA protein in serum and PSA mRNA in CTCs has not been clearly researched. In this study, we first report the relevance of the expression levels of PSA protein in serum and PSA mRNA in CTCs. Experimental method and analysis Prostate cancer patients were recruited from healthy donor (HD, n=5), local with non-metastatic stage (n=25), metastatic hormone sensitive prostate cancer (mHSPC, n=9) and metastatic castration-resistance prostate cancer (mCRPC, n=28). CTCs were isolated by high efficiency and purity using microfluidic technology based on lateral magnetophoresis, developed in our laboratory. Total mRNA was extracted from isolated CTC and the expression level of PSA mRNA in CTCs was measured by droplet digital PCR (ddPCR). Then, the expression levels of PSA protein (ng/ml) in serum and PSA mRNA (copies/µl) in CTCs were compared by statistical analysis. Results and discussion The expression of PSA mRNA in CTCs was observed in 40% of localized cancer, 55.56% of mHSPC, 89.29% of mCRPC patients and 0% of HD group. It indicates that the detection rate of PSA mRNA increases with stage of prostate cancer. The expression level of PSA mRNA in CTCs also increased from localized to metastatic stages. Interestingly, it increased very rapidly in mHSPC and mCRPC stages. In 7 of the blood samples of localized stage, the expression levels of PSA mRNA in CTCs were detected and they were not related to serum PSA protein. In mHSPC and mCRPC stage, the expression levels of PSA mRNA in CTCs were respectively detected in 5 and 25 samples, implicating that their expression levels are significantly related to the level of PSA protein in serum. Conclusions This result shows that the expression level of PSA mRNA in CTCs could be used as a new marker along with serum PSA protein in metastatic stages of prostate cancer, and that high-quality CTCs can be used as complementary alternative biomarkers for precision medicine in prostate cancer. Citation Format: Hyungseok Cho, Ki-Ho Han, Jae-Seung Chung. A comparative analysis on the expression of PSA protein in serum and PSA mRNA in circulating tumor cells in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5378.

Abstract 774: Highly effective isolation of circulating tumor cells using lateral magnetophoretic technology for precise genetic analysis of prostate cancer

Abstract Introduction Prostate cancer is the second leading cancer among men in the world and known as the most frequently diagnosed cancer in more than half of countries with estimated 1.3 million new emerging cases. Because of low specificity of serum PSA screening for prostate cancer, it raised concerns in keep management of clinical prognosis and therapeutic decision and thereby, there are still needed precise real-time biomarkers. Many recent studies have reported that circulating tumor cells (CTCs) can be used as important biomarkers for prostate cancer at cellular and molecular levels. However, due to their extreme rarity in the circulatory system and the lack of technology to isolate them precisely, it is still difficult to isolate them with high purity and efficiency that can be applied for clinical purposes. In this study, we introduce a microfluidic technology using lateral magnetophoresis for isolating CTCs from blood of prostate cancer patients (n=62) with high purity and efficiency and demonstrate clinical usefulness of isolated CTCs at cellular and molecular levels. Materials and methods By the lateral magnetophoretic technology using immunomagnetic nanobeads, highly pure CTCs were isolated from 5 ml blood, drawn from patients with localized (n=25) and metastatic (n=37) prostate cancer. Then, prostate cancer related genes (AR, AR-V7, EpCAM, KRT-19, PSA and PSMA) were quantified by droplet digital PCR (ddPCR) method using mRNA in CTCs. Results and discussion CTCs were detected from all blood samples (n=62). In localized cases, the average number of isolated CTCs and purity were 6.58 per ml and 2.09%. In metastatic cases, the average number of isolated CTCs and purity were 27.55 per ml and 6.00%, higher than those of localized cases. Then, mRNA was extracted from isolated CTCs and used for detecting prostate cancer specific genes. Their detection rate and expression level were increased from localized to metastatic stages. AR was detected at a similar rate of 76.00% in both localized and mHSPC stages and increased to 100% in mCRPC stage. AR-V7 was detected only in metastatic stages, mainly in mCRPC stages. EpCAM showed the highest detection rate in all stages, such as 96.00% in localized, 88.89% in mHSPC and 100% in mCRPC. This result indicates that CTCs are isolated from all blood of patients. KRT-19 showed slightly lower detection rate than EpCAM, but tend to be similar to EpCAM expression for cancer stages. PSA and PSMA were expressed very high in metastatic stage. PSA expression level in metastatic stage was 198.28 times higher than that in localized stage. Interestingly, the expression of PSA mRNA measured from CTCs was linearly proportional to serum PSA protein level, which is a commonly used diagnostic biomarker for prostate cancer. Conclusions In all stages of prostate cancer patients, the proposed microfluidic technology using the lateral magnetophoresis is a high-performance method for isolating CTCs with high detection rate and purity that can be used for precision genetic analysis. Citation Format: Hyungseok Cho, Ki-Ho Han, Jae-Seung Chung. Highly effective isolation of circulating tumor cells using lateral magnetophoretic technology for precise genetic analysis of prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 774.

Abstract A089: Detection of circulating tumor cells (CTC) by microfluidic technology based on lateral magnetophoresis and CTC-based multigene profiling analysis in prostate cancer patient

Abstract Introduction By using the circulating tumor cells (CTCs), we can get useful genomic information that enable monitoring of patient’s prognosis or selecting of appropriate treatment over time by repeat biopsy. Despite their usefulness, current CTC separation techniques are limited in terms of their ability to separate high recovery and purity CTCs from peripheral blood. We aimed to separate CTCs from prostate cancer patients using microfluidic technique that based on disposable lateral magnetophoretic microseparator (named ‘assembly-disposable CTC-μChip’) and assess the gene expression that are specific for prostate cancer using mRNA from isolated CTCs measured by droplet digital PCR (ddPCR) method. Patients and Methods Total 54 samples of 45 patients who underwent treatment for prostate cancer between 06/2018 and 03/2019 were prospectively enrolled for CTC collection. Approximately 10 ml of whole blood was drawn from prostate cancer patients; Group A, 12 samples (22.2%) with localized stage II-III cancer, Group B, 5 samples (9.3%) with stage IV, Group C, 6 samples (11.1%) with metastatic hormone-sensitive prostate cancer (mHSPC) and Group D, 31 samples (57.4%) with metastatic castration-resistant prostate cancer (mCRPC). CTCs were positively isolated by using Assembly-disposable CTC-μChip. CTCs from 5 mL of blood were used for counting to identify isolated CTC numbers with immunofluorescence detection procedure. CTCs from the remaining 5 mL of blood were used for gene analysis by ddPCR. For the gene expression analysis, selected 13 patient’s samples were examined for 6 prostate cancer-related genes, such as androgen receptor (AR), androgen receptor splice variant 7 (AR-V7), prostate specific membrane antigen (PSMA), prostate specific antigen (PSA), cytokeratin-19 (CK-19) and epithelial cell adhesion molecule (EpCAM). Results We identified the average of 3, 16, 14 and 26 CTCs per 1ml in group A, B, C and D, respectively (P< 0.01). Background contaminated white blood cells are indispensably isolated with CTCs that counted on average 2245.4 cells, thereby CTC purity rate was 2.56 %. The gene positive rate of AR, AR-V7, PSA, PSMA, KRT-19, and EpCAM were 2.3 % (12/13), 15.4 % (2/13), 38.5 % (5/13), 69.2 % (9/13), and 100 % (13/13), respectively. Especially, the copy number of PSA, PSAM and CK-19 increased in proportion as the clinical stage increased. All samples showed EpCAM positive, whereas AR-V7 was rare events, with only in 2 out 5 patients positive with mCRPC, but not in other stages. Conclusions This study demonstrates that assembly-disposable CTC-μChip is a suitable for CTC isolation from prostate cancer patients. Our data also showed that CTC-based multigene profiling can be analyzed using ddPCR, making it a clinically relevant biomarker. Interrogating tumor expression via CTCs isolation may allow for enhancing precision-based treatment. Citation Format: Hyungseok Cho, Ki-Ho Han, Jae-Seung Chung. Detection of circulating tumor cells (CTC) by microfluidic technology based on lateral magnetophoresis and CTC-based multigene profiling analysis in prostate cancer patient [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A089.

Evaluation of Positive and Negative Methods for Isolation of Circulating Tumor Cells by Lateral Magnetophoresis.

We developed an epithelial cell adhesion molecule (EpCAM)-based positive method and CD45/CD66b-based negative method for isolating circulating tumor cells (CTCs) by lateral magnetophoresis. The CTC recovery rate, white blood cell depletion rate, and purity of CTCs isolated using the positive and negative methods were analyzed using blood samples spiked with cancer cells with different expression levels of EpCAM. The aim was to assess the strengths and weaknesses of the positive and negative isolation methods for CTC-based diagnostics, prognostics, and therapeutics for cancer. The EpCAM-based positive method yielded CTCs of high purity, while the CD45/CD66b-based negative method yielded a large number of CTCs. In conclusion, the positive method shows promise for detecting somatic oncogenic mutations and the negative method shows promise for discovery of cellular and transcriptomic biomarkers of cancer.

Electrical Detection Method for Circulating Tumor Cells Using Graphene Nanoplates.

This paper presents a microfluidic device for electrical discrimination of circulating tumor cells (CTCs) using graphene nanoplates (GNPs) as a highly conductive material bound to the cell surface. For two-step cascade discrimination, the microfluidic device is composed of a CTC-enrichment device and an impedance cytometry. Using lateral magnetophoresis, the CTC-enrichment device enriches rare CTCs from millions of background blood cells. Then, the impedance cytometry electrically identifies CTCs from the enriched sample, containing CTCs and persistent residual blood cells, based on the electrical impedance of CTCs modified by the GNPs. GNPs were used as a highly conductive material for modifying surface conductivity of CTCs, thereby improving the accuracy of electrical discrimination. The experimental results showed that a colorectal cancer cell line (DLD-1) spiked into peripheral blood was enriched by nearly 500-fold by the CTC-enrichment device. The phase of the electrical signal measured from DLD-1 cells covered by GNPs shifted by about 100° in comparison with that from normal blood cells, which allows the impedance cytometry to identify CTCs at a rate of 94% from the enriched samples.

Microdevice for Separation of Circulating Tumor Cells using Embedded Magnetophoresis with V-shaped Ni-Co Nanowires and Immuno-nanomagnetic Beads

The novelty of this study resides in a 6”-wafer-level microfabrication protocol for a microdevice with a fluidic control system for the separation of circulating tumor cells (CTCs) from human whole blood cells. The microdevice utilizes a lateral magnetophoresis method based on immunomagnetic nanobeads with anti-epithelial cell adhesive molecule antibodies that selectively bind to epithelial cancer cells. The device consists of a top polydimethylsiloxane substrate for microfluidic control and a bottom substrate for lateral magnetophoretic force generation with embedded v-shaped soft magnetic microwires. The microdevice can isolate about 93% of the spiked cancer cells (MCF-7, a breast cancer cell line) at a flow rate of 40/100 mL/min with respect to a whole human blood/buffer solution. For all isolation, it takes only 10 min to process 400 mL of whole human blood. The fabrication method is sufficiently simple and easy, allowing the microdevice to be a mass-producible clinical tool for cancer diagnosis, prognosis, and personalized medicine.

Circulating Tumor Cell Microseparator Based on Lateral Magnetophoresis and Immunomagnetic Nanobeads

This paper presents a circulating tumor cell (CTC) microseparator for isolation of CTCs from human peripheral blood using immunomagnetic nanobeads with bound antiepithelial cell adhesive molecule (EpCAM) antibodies that specifically bind to epithelial cancer cells. The isolation is performed through lateral magnetophoresis, which is induced by high-gradient magnetic separation technology, involving a ferromagnetic wire array inlaid in the bottom substrate of a microchannel. Experimental results showed that the CTC microseparator isolates about 90% of spiked CTCs in human peripheral blood at a flow rate of up to 5 mL/h and purifies to approximately 97%. The overall isolation procedure was completed within 15 min for 200 μL of peripheral blood. CTCs from peripheral blood of patients with breast and lung cancers were isolated with the CTC microseparator, and the results were compared with those of healthy donors. Using a fluorescence-based viability assay, the viability of CTCs isolated from peripheral blood of patients with cancer was observed. In addition, the usefulness of the CTC microseparator for subsequent genetic assay was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of cancer-specific genes using CTCs isolated from patients with cancer.

A high-speed, high-performance on-chip integrated reverse transcription (RT)-microchip

This report introduces an on-chip integrated reverse transcription (RT)-microchip, which includes two genetic functionalities of RNA extraction and cDNA synthesis. In the RNA extraction compartment, RNA is extracted from peripheral blood lysate within 1min, by lateral magnetophoresis using magnetic oligo-dT beads. The extracted RNA is then collected and used directly to produce cDNA in the cDNA synthesis microchamber, which is monolithically integrated with the RNA extraction compartment. To verify the superiority of the proposed RT-microchip, RT-PCR amplification was performed using cDNA harvested from the RT-microchip, and the results were compared with those obtained using typical RNA extraction methods such as a silica matrix column and magnetic oligo-dT beads. The RT-PCR amplification results using 100μl of blood showed that the intensity of the bands in gel electrophoresis of the RT-microchip was 2-fold stronger than that of the silica matrix column and 2.65-fold stronger than that of the magnetic oligo-dT beads. The results demonstrate that the RT-microchip technique is the most sensitive of the tested methods.

올리고-dT 자성입자와 측면방향 자기영동을 이용한 초고속 RNA 추출 기술

This paper presents a high-speed RNA microextractor for the direct isolation of RNA from blood lysate using magnetic oligo(dT) beads. The extraction is performed through lateral magnetophoresis, which is induced by a ferromagnetic wire array inlaid. With this RNA microextractor, more than 80% of the magnetic beads could be separated at a flow rate up to 20 ml/h, and the overall extraction procedure was completed within 1 min. The absorbance ratio of RNA to protein(A260/A280) was greater than 1.7, indicating that the extraction technique yields pure RNA. The feasibility of using this technique in reverse transcription polymerase chain reaction procedures was investigated by cDNA synthesis and PCR processes. The results confirmed that the RNA microextractor is a practical device for easy, fast, and high-precision RT-PCR using minimal amounts of reagent.

High-speed RNA microextraction technology using magnetic oligo-dT beads and lateral magnetophoresis

This paper presents a high-speed RNA microextractor for the direct isolation of RNA from peripheral blood lysate using magnetic oligo-dT beads. The extraction is achieved through lateral magnetophoresis, generated by a ferromagnetic wire array inlaid on a glass substrate. This RNA microextractor separated more than 80% of magnetic beads with a flow rate up to 20 ml h(-1), and the overall extraction procedure was completed within 1 min. The absorbance ratio of RNA to protein (A(260)/A(280)) was >1.7, indicating that the extraction technology yielded nearly pure RNA. The feasibility of this technique was evaluated further for its applicability to reverse transcription polymerase chain reaction (RT-PCR) procedures by performing cDNA synthesis and PCR. The analysis verified that the RNA microextractor is a practical method for easy, rapid, and high-precision RT-PCR using minimal reagent volumes without requiring highly trained personnel. In addition, it can be readily incorporated into genetic analysis procedures for realizing automated on-chip genetic platforms in a micro format.