Immobilization of nucleic acid aptamer on macrophages for circurating tumor cell capture

Abstract

Event Abstract Back to Event Immobilization of nucleic acid aptamer on macrophages for circurating tumor cell capture Shunsuke Sugimoto1*, Takeshi Mori2* and Yasuhiko Iwasaki3, 4* 1 Kansai university, Graduate School of Science and Engineering, Japan 2 Kyushu University, Department of Applied Chemistry, Faculty of Engineering, Japan 3 Kansai university, Faculty of Chemistry, Materials and Bioengineering, Japan 4 Kansai university, ORDIST, Japan Introduction: Metastasis is the dominant trigger of cancer deaths. Circulating tumor cells (CTCs) are main components for metastasis [1]. A Therefore, the system to eliminate CTCs from the metastatic routes is required. In the present study, the surface modification of macrophages with nucleic acid aptamers which can bind to membrane protein of cancer cell, was performed to capture CTCs. The cancer cell capture by macrophages was significantly induced by the nucleic acid aptamer immobilization on macrophages. Materials and Methods: In this experiments, N-methacryloyl mannosamine (ManM) [2] was used to deliver methacryloyl groups on macrophages. . ManM was synthesized as previously described. After 1 day cultivation of macrophages (RAW264.7 cells) with ManM, the cells were in contact with thiol-terminated nucleic acid aptamers (sgc8-SH) and Eosin-Y for radical precursor. Subsequently RAW264.7 cells were exposed to visible light (505 nm) for 10 min at room temperature. In order to confirm immobilization of sgc8-SH on the RAW264.7 cells, fluophore-conjugated complementary DNA was added in sgc8-SH treated cells for 1 min. After rinsing with culture medium, the cells was observed using a confocal laser scanning (CLS) and differential interference contrast (DIC) microscope. The fluorescence intensity of the cells was determined by a flow cytometer. The viability of aptamer-immobilized cells was quantitated by WST-8 assay. Cancer cell capture by macrophages was evaluated by using CCRF-CEM cells having membrane proteins, which can bind to sgc8, as a model of CTCs. After surface modification of RAW264.7 cells with sgc8, the macrophages were in contact with CCRF-CEM cells for 15 and 30 min at room temperature. After washing, adherent CCRF-CEM cells on RAW264.7 cells were counted by micrographs. Results: Figure 1 shows CLS and DIC micrographs of RAW264.7 cells contacted with sgc8-SH and complementary-sgc8 Alexa Fluor 488. On native and non ManM-treated cells (ManM (-)), fluorescence probes were not detected. In contrast, the outline of the ManM-treated cells (ManM (+)) was clearly stained. Similar data was obtained from flow cytometric analysis. On WST-8 assay, all cells (native, ManM (-), ManM (+)) show same absorbance derived from WST-8 formazan. Figure 2 proves the number of adherent CCRF-CEM cells on RAW264.7 cells after incubation for 15 and 30 min. On native and ManM (-), the captured CCRF-CEM cells were hardly observed. In contrast, the attached CCRF-CEM cells on sgc8-immobilized RAW264.7 cells (ManM (+)) well occurred. Discussion: According to Figure 1, ManM (+) show high fluorescence because of duplex formation with aptamers and complementary DNA. These results indicate that surface modification with nucleic acid aptamers via thiol-ene click reaction can perform using ManM-treated cells. The result of WST-8 assay means that the visible light-assisted surface immobilization does not influence cell viability. On ManM-treated RAW264.7 cells, a number of CCRF-CEM cells were captured (Figure 2). Thus, it was clarified that intercellular adherent was enhance via aptamer-immobilized cells. Conclusion: Nucleic acid aptamers could be freely desingned for targeting any specific cells, protein and other biosubstances. Therefore, this cell surface engineering would be applied for the elimination of CTCs and other tumor tissue which expressed diagnostic biomolecules for cancer cells. This study was supported by a Grant-in-Aid for Scientific Research on Innovative Areas ‘NanoMedicine Molecular Science’ from Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (#26107719).