Abstract 1719: Dual-profiling of CTC and exosome from the cultured circulating tumor cells using stimuli-responsive degaradable hydrogels
Authors: Yoon-Tae Kang, Young Jun Kim, Tae Hee Lee, Jae-Eul Shim, Young-Ho Cho
Journal: Cancer Research (2017)

Abstract 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