Research On Biosensing And Imaging Of Cancer Stem Cells And Orientational Regulation Of Cancer Cells On Fish Scale Micropatterns

Cancer stem cells(CSCs)are the root cause of tumor occurrence,development,recurrence and chemoradiotherapy resistance.Extracellular matrix(ECM)in the tumor microenvironment is an important chemical and physical clue to maintain the survival of CSCs,trigger and regulate the behavior of cancer cells,and has a significant impact on the growth behavior and drug sensitivity of cancer cells.The detection and imaging of CSCs and the orientational regulation of cancer cells which are of great significance for the diagnosis and treatment of cancer and the study of tumor occurrence and development,are major scientific issues in the field of cancer research.In this study,a novel nanosensor based on affinity peptides was constructed for rapid and sensitive detection and imaging of CSCs marker CD133,and the waste fish scale was used as an anisotropic substrate to simulate ECM for the orientational regulation of cancer cells and the evaluation of drug sensitivity.The main findings are as follows:(1)A fluorescence sensing system based on graphene oxide(GO)-peptide was constructed for detecting and imaging CSCs marker CD133 and molecular logic and computing.Fluorescently labeled CD133binding peptide(FLS7,FITC-LQNAPRS)with high CD133 binding affinity(Kd=12.0?1.2 nM)was synthesized.Fluorescence spectroscopy,atomic force microscopy and UV-Vis absorption spectroscopy showed that the fluorescence quenching rate of 0.05 mg/mL of GO to FLS7 was 96%and they were statically quenched;the thickness of the GO-FLS7 complex was significantly greater than that of GO and the absorption peaks of the complex were blue shifted from 493 to 491nm.The fluorescence of GO-FLS7 complex was recovered selectively with increasing CD133 concentration and the linear range was from 0 to630 nM with a detection limit of 7.91 n M.Atomic force microscopy and Zeta potential confirmed that the fluorescence recovery can be attributed to the fact that FLS7 was competitively bound by CD133 and leaved from GO surface.Cell assay showed that,compared with the control group,GO-FLS7 complex had the most significant fluorescence recovery to CT26 CSCs,and they are proportional with a linear range of 1.8×10~4?1.2×10~5 cells/mL and a detection limit of 1.1×10~3 cells/mL.Immunofluorescence showed that GO-FLS7 co-localized with CD133antibody on the plasma membrane of CT26 CSCs.Small animal imaging showed that after injecting GO-FLS7 via the tail vein,the tumor tissue fluorescence gradually increased over time compared with the organs obtained by dissection.The above results indicated that the GO-FLS7fluorescent nano-sensing system can be used for rapid(30 min)and sensitive detection of CSCs marker CD133 and imaging at the cellular and tumor tissues level,which is helpful for the detection and imaging of CSCs.In addition,a comprehensive analysis of the substances(molecular events),energy(fluorescence)and information changes in the above sensing system can construct AND,OR,N-IMPLY molecular logic gates and multi-input-multi-output complex logic circuits for implementing molecular logic and computing,which lay the foundation for logical sensing.(2)Abandoned fish scales were used as anisotropic substrates to simulate ECM for the directional regulation and drug sensitivity assessment of cancer cells.The surface morphology,chemical composition and wettability of Carassius auratus scales were characterized with the help of optical microscope,scanning electron microscope,thermogravimetry-differential thermal analysis,attenuated total reflection-Fourier transform infrared spectroscopy,X-ray photoelectron spectroscopy and water contact angle.The results showed that the embedded part of the outer surface contains groove-like structures and the inner surface has densely arranged ordered fiber structures;the main components of both are collagen and hydroxyapatite,and the outer surface has more hydroxyapatite and less collagen,the inner surface is opposite;both have strong cell adhesion.Live/dead cell viability assay and laser confocal microscopy imaging showed that fish scales can inhibit the proliferation of tumor cells but no cytotoxicity.The growth morphology of CT26 cells cultured on fish scales substrate with different morphologies was observed using an optical microscope.And then through long-to-short axis index,two-dimensional fast Fourier transform and cell orientation angle analysis,it was found that their ability of guiding cell orientation from high to low was:undamaged inner surface>embedded part>damaged inner surface>exposed part,which proved that the inner surface and the embedded part have a significant ability to guide cell alignment due to their special anisotropic topography.After culturing CT26 cells on the inner surface substrate for different time,the dynamic changes of cell morphology were observed,and it was found that although affected by the interaction between the cells,the anisotropic topography of the fish scale surface played a dominant role in cell orientation.The drug sensitivity experiment found that the CT26cells cultured on the inner surface of fish scales with anisotropic topography had significantly increased resistance to irinotecan and cisplatin compared with traditional flat culture plates.This indicated that discarded fish scales can be used as anisotropic natural materials to simulate the biophysical and chemical properties of ECM for cell orientation and drug sensitivity evaluation,which have the advantages of wide material sources,waste recycling and patterning-free.The above research results not only provide a new platform for the rapid and sensitive detection and imaging of CSCs markers based on molecular affinity ligands,which can promote the accurate identification of CSCs,the early diagnosis and efficacy evaluation of cancer.They also provide a new opportunity for the development of in vitro anisotropic culture platform based on natural wastes to simulate ECM,which can help cell directional regulation,eliminate differences in research results in vivo and in vitro,deepen the understanding of the objective laws of tumorigenesis and development,and provide a reliable platform for more accurate and effective drug screening.