Yoon-Tae Kang

Poly(ethylene glycol)-Modified Tapered-Slit Membrane Filter for Efficient Release of Captured Viable Circulating Tumor Cells

The grafting of poly(ethylene glycol) (PEG) onto an SU8 microfilter has been demonstrated for efficient capture and release of circulating tumor cells (CTCs). Previous CTC filters showed low cell release efficiency due to hydrophobic surfaces, even though their capture efficiency was considerable. PEG, a hydrophilic polymeric compound mainly used to form nonfouling thin films on silicon surfaces, induces repulsive force so that the nonspecific adsorption of the surface is incomparably reduced in comparison with unmodified filter surfaces. The effectiveness of PEG-modified CTC filters was verified through lung (H358) and colorectal (SW620) cancer cells spiked, respectively, in phosphate-buffered saline (PBS) and unprocessed whole blood. The modified SU8 filters achieved approximately 37.7% and 22.8% improvement in release efficiency without significant changes in cell viability and capture efficiency. In order to verify the filters potential for clinical applications, we extended our experiments using cancer patient blood samples. Six blood samples from colorectal and lung cancer patients were processed, and captured CTCs were efficiently released. From these experiments, the present PEG-modified filter captures and releases on average 14 ± 7.4 CTCs/mL, including EpCAM-negative CTCs, which could not be captured by previous single antibody-based methods. The antibody-free isolation with enhanced release efficiency facilitates viable cell retrieval, which is significant to CTC culture and comprehensive molecular study for verifying the mechanism of metastasis and cancer.

Label-free Rapid Viable Enrichment of Circulating Tumor Cell by Photosensitive Polymer-based Microfilter Device

We present a clinical device for simple, rapid, and viable isolation of circulating tumor cells (CTCs) from cancer patient bloods. In spite of the clinical importance of CTCs, the lack of easy and non-biased isolation methods is a big hurdle for implementing CTC into clinical use. The present device made of photosensitive polymer was designed to attach to conventional syringe to isolate the CTCs at minimal resources. Its unique tapered-slits on the filter are capable not only to isolate the cell based on their size and deformability, but also to increase sample flow rate, thus achieving label-free rapid viable CTC isolation. We verified our device performance using 9 different types of cancer cells at the cell concentration from 5 to 100cells/ml, showing that the device capture 77.7% of the CTCs while maintaining their viability of 80.6%. We extended our study using the 18 blood samples from lung, colorectal, pancreatic and renal cancer patients and captured 1-172 CTCs or clustered CTCs by immunofluorescent or immunohistochemical staining. The captured CTCs were also molecularly assayed by RT-PCR with three cancer-associated genes (CK19, EpCAM, and MUC1). Those comprehensive studies proved to use our device for cancer study, thereby inaugurating further in-depth CTC-based clinical researches.

Lab on a fabric: Mass producible and low-cost fabric filters for the high-throughput viable isolation of circulating tumor cells

Circulating tumor cells (CTCs) play an important role in estimating the presence and the metastatic relapse of tumor. Despite of their importance, isolation of viable CTCs is still struggling, since chemical or mechanical damages are unavoidable when separating less than 1000 of CTCs out of billions of other blood components. Furthermore, the current CTC isolation devices show low productivity, since they are produced after a series of complicated fabrication processes. Here, we present a low-cost and mass-producible fabric filters for the viable CTC isolation and the further molecular assay for profiling cancer-associated markers. The fabric filter, produced by polyester monofilament yarns, can be massively produced at extremely low-cost, by showing productivity of ~22filters/s at ~59filters/USD. By utilizing size-based sorting method, the fabric filter is capable to isolate both epithelial and mesenchymal CTCs, while slots with curved walls are beneficial for preventing the cell rupture by reducing 21.6% of mechanical stress compared to the conventional straight-walled slots. We applied our filter to 11 human blood samples and found that the number of CTCs was closely related to the expression level of Ki-67, which is highly overexpressed in proliferative tumors. The fabric filter might be an appropriate caner-screening tool in developing countries, where people suffer from insufficient healthcare services.

Circulating tumor cells in the differential diagnosis of adnexal masses

The aim of this study was to evaluate circulating tumor cell (CTC) detection in the differential diagnosis of adnexal masses. A total of 87 preoperative women with an indeterminate adnexal mass were prospectively enrolled. Preoperative diagnostic modalities including CTC detection, risk of ovarian malignancy algorithm, risk of malignancy index, and computed tomography or magnetic resonance imaging were compared. Forty-three (49.4%) benign tumors, 13 (14.9%) borderline malignant masses, and 31 (35.7%) cancers were pathologically confirmed. Forty-nine (56.3%) cases were positive for CTCs: 19/43 (44.2%) benign, 10/10 (100%) early-stage, and 14/21 (66.7%) advanced-stage cancer. CTC detection had sensitivities of 77.4%, 100%, and 100% for benign vs. all stage cancer (n = 74), benign vs. stage I–II cancer (n = 53), and benign vs. stage I cancer (n = 49), respectively. CTC detection had a specificity of 55.8% across all comparisons. The sensitivities of the other modalities assayed were decreased in stage I–II cancer and stage I cancer vs. benign masses. Receiver operating characteristic curves showed that CTCs, of which the area under the curve was modest in all stage cancer (0.655), had the widest area under the curve among the evaluated modalities in stage I–II cancer and stage I cancer (0.768 for both). In conclusion, our study findings suggest that preoperative CTCs could have a substantial role in differentiating early stage cancer from benign tumors for adnexal masses.

Abstract 1719: Dual-profiling of CTC and exosome from the cultured circulating tumor cells using stimuli-responsive degaradable hydrogels

Introduction: Liquid biopsy based on sub-micron or nanosized particles in human body fluid have been received vast attention due to their non-invasive characteristics and enabling multiple check-up. Circulating tumor cells (CTCs), as well as exosome are the most promising markers in liquid biopsy, however, dual isolation and profiling have been hampered due to their size difference and limited quantity for analysis. We proposed the novel and simple methods for both isolation and study their similarity between them. Using the label-free CTC filtration device and anti-CD63 antibody-conjugated degradable hydrogel, the CTCs and the CTCs-derived exosome are specifically isolated, and each samples were followed by molecular study after recovery. This versatile platform facilitates the comprehensive study of two biomarkers with reflecting their inherent characteristics, thus paving the way for revealing their roles in cancer progression and metastasis. Methods: In order to make stimuli-responsive degradable hydrogel, poly (vinyl alcohol) and alginate were mixed under constant stirring at 85 °C. The mixture was poured into the mold and dried for 24 hours. Then, the dried sheet was immersed into 100 mM calcium chloride solution to achieve gelation through ionic interaction. Subsequently, the anti-CD63 antibody was immobilized onto the prepared hydrogels via cross-linking. For the dual-profiling, the hydrogel and the filters containing the captured breast cancer cells by microfiltration were incubated with the exosome-depleted cell culture media for 6 hours. The captured cells were released from the device and the hydrogels were degraded by adding EDTA. The cell and exosome lysate were prepared using RIPA buffer at 4 °C. The supernatant was collected by centrifugation followed by western-blot assay. Four different markers, including exosome-specific marker (CD63), cancer-associated markers (EpCAM, vimentin), and a housekeeping marker (β-actin), were used. Results: All exosome and cell samples highly expressed the housekeeping marker. Especially, two exosome samples dissociated from the hydrogel showed CD63 predominantly, which support the secretion of exosome from the cancer cells. The samples from the released cancer cells from the device did not express CD63 remarkably. To verify the phenotypical similarity between cell and exosome, expressions of the epithelial marker (EpCAM) and mesenchymal marker (vimentin) were examined. The exosome and cell from MCF-7, epithelial cancer cell, showed higher expression of EpCAM then vimentin. On the contrary to this, the samples from MDA-MB-231, mesenchymal cell, showed higher vimentin expression then EpCAM. Discussion and conclusion: We showed that exosome follow the phenotypical characteristics of mother-cells. This dual profiling would be helpful for in-depth study of cancer with consideration of its heterogeneity and complexity. Citation Format: Yoon-Tae Kang, Young Jun Kim, Tae Hee Lee, Jae-Eul Shim, Young-Ho Cho. Dual-profiling of CTC and exosome from the cultured circulating tumor cells using stimuli-responsive degaradable hydrogels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1719. doi:10.1158/1538-7445.AM2017-1719

Abstract 3775: Hydrogel-assisted pathological study of the circulating tumor cells filtered from the renal cell carcinoma patients’ blood

Introduction: Circulating tumor cells (CTCs), defined as tumor cells detached from the primary tumor site and circulating in the peripheral blood, are a promising marker to get the information about tumor status and metastatic potential. However, the limited detection ability as well as their biased specificity of current CTC isolation struggle from understanding the comprehensive characteristics of CTCs. In addition, previous CTC isolation depending on epithelial cell adhesion molecule (EpCAM) antibody make their cytological and pathological study hard. We developed the methods which capture CTCs based on their size and deformability, and construct a hydrogel-assisted cell block for verifying the diagnostic utility for various cancer-associated immuno-markers and their pathological studies. Methods: Two renal cell carcinoma cell lines, SN12C and 769P, and 7 different cancer patients’ blood with renal cell carcinoma were used. To find the applicable markers for diagnosing renal cell carcinoma, we constructed the cell blocks of two cell lines. For the encapsulation of the cells, 4 % alginate was prepared by dissolving in deionized water under constant stirring at 85 °C. Then, the separately prepared renal cancer cells were gently mixed into the dissolved alginate solution. After careful mixing, the solution was loaded into calcium chloride solution drop by drop using volume pipette, followed by 15 min of further incubation with constant stirring. Next, the cell-containing beads were applied to the commonly used procedure for paraffin tissue blocks, and 8 different cancer-associated immuno-markers (EpCAM, CK (AE1/AE3), CAM5.2, EMA, CD10, CA IX, RCC, and vimentin) were stained with each dissected cell blocks. The CTCs from the 7 clinical samples were isolated by tapered-slit filter, and cell containing slides were immunohistochemically stained and examined by pathologist. Results: The four markers, EpCAM, CK (AE1/AE3), CD10, and vimentin in the SN12C cell block were highly expressed. The three markers, CK (AE1/AE3), CD10, and vimentin in the 769-P cell block were predominantly expressed. The EpCAM, CK (AE1/AE3), and CD10 are chosen as the potential immunomarkers for CTC-based diagnosing for RCC. In the clinical study using the 0.5-3.0 ml of blood samples, CTCs were successfully isolated and detected immunohistochemically in 57.1 % (4 of 7) of patients and ranged from 1 to 5. Of 4 CTC positive samples, two had CK (AE1/AE3) positive, each one had EpCAM and CD10 positive CTCs, respectively. Discussion and conclusion: Although the vimentin is highly expressed in both cell blocks, due to diffuse positivity for leukocyte, single use of vimentin for cancer diagnosis is limited. This comprehensive study including immuno-markers screening and their applicability test with clinical samples demonstrates the clinical utility of the present device and hydrogel-assisted cell block for CTC isolation and cancer studies. Citation Format: Yoon-Tae Kang, Young Jun Kim, Tae Hee Lee, Hee Jin Chang, Hyun-Moo Lee, Young-Ho Cho. Hydrogel-assisted pathological study of the circulating tumor cells filtered from the renal cell carcinoma patients’ blood [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3775. doi:10.1158/1538-7445.AM2017-3775