Capture And Identification Of Heterogeneous Circulating Tumor Cells Using Double Antibodies Nanosubstrate And Quantum Dots- Based Multiplexed Imaging

Part 1 Optimized nanofilm substrate and imaging method for cancer cells capture and identificationObjective To realize efficiently capture and identification of different tumor cell lines by optimizing the fabrication and capture process of nanofilm substrate and introducing a new pattern quantum dots (QDs)-based imaging technology.Methods Hydroxyapatite-chitosan (HA-CTS) nanofilm substrates were modified based on previous work to increase the capture efficiency of epithelial cell adhesion molecule (EpCAM)-negative cancer cells by changing baking time of film fabrication and incubating time of capture process. New pattern QDs-based imaging technology was introduced here and compared with traditional organic dyes in terms of fluorescence intensity, photostability and detection sensitivity.Results Comparisons of capture efficiencies of cell lines at different baking time and incubating time revealed the highest capture efficiencies were achieved at baking time of 90 min and incubating time of 3 h (the capture efficiencies of EpCAM-positive cell lines were more than 80%, and that of EpCAM-negative cell lines were more than 50%). Compared to tranditional organic dyes, QDs-based imaging exhibited comparable detection ability but stronger photostability and higher fluorescence intensity.Conclusion This study established a capture and detection system for tumor cells with well operation, strong stability and high capture efficiency by optimizing HA-CTS nanofilm substrates and combining with QDs-based imaging technology, which laid a good foundation for capture and identification circulating tumor cells (CTCs).Part 2 Capture and identification of heterogeneous circulating tumor cells from cancer patients using nanofilm substrates and QDs-based multiplexed imaging technologyObjective This study aimed at capturing and identifing heterogeneous circulating tumor cells from cancer patients with different tumor types and pathological stage using the optimized HA-CTS nanofilm substrates and QDs-based multiplexed imaging technology.Methods QDs-based multiplexed imaging technology was established and verified the feasibility of multiplexed imaging with different tumor cell lines. Optimized HA-CTS nanofilm substrates in part 1 were used to capture CTCs from cancer patients. QDs-based multiplexed imaging technology was applied to label epithelial CTCs marker CK, mesenchymal CTCs marker vimentin and white blood cells maker CD45 in order to detect and analysis heterogeneous CTCs.Results QDs-based multiplexed imaging technology accurately described the molecular expression of tumor cell lines with different phenotypes. Capture and identification system based on the optimized HA-CTS nanofilm substrates and QDs-based multiplexed imaging successfully captured CTCs from cancer patients blood samples, then specifically identified the existence of heterogeneous CTCs with CK-positive, vimentin-positive and CK/vimentin-positive phenotypes.Conclusion This study successfully established a technology platform based on the optimized HA-CTS nanofilm substrates and QDs-based multiplexed imaging, which could capture and identification CTCs with heterogeneous phenotypes from cancer patients at high performance.Part 3 Capture and identification of circulating tumor cells and circulating tumor stem cells using double antibodies nanofilm substrates and QDs-based multiplexed imagingObjective This study aimed at establishing a new system to simultaneously capture and identification of CTCs and circulating tumor stem cells (CTSCs) from cancer patients by combining double antibodies optimized nanofilm substrates and QDs-based multiplexed imaging technology.Methods Optimized HA-CTS nanofilm substrates were coupled with double antibodies of EpCAM and CD 133 to capture CTCs and CTSCs from cancer patients using immune affinity reaction and physical absorption principle. QDs-based multiplexed imaging labeled CK, vimentin, CD45 was used to recognize and identify the captured cells.Results The optimized nanofilm substrates with double antibodies had higher capture efficiencies than substrates with single antibody for EpCAM-middle and EpCAM-low expression cell lines (A549: P=0.003; HeLa: P=0.001; HepG2:P=0.003). QDs-based multiplexed imaging demonstrated CK-positive, vimentin-positive and CK-positive/vimentin-positive CTCs existed in 32 cancer patients. In addition, percentage of CTCs with vimentin-positive and CK-positive/vimentin-positive in advanced stage (Ⅲ/Ⅳ) cancer (68.4%,52/76; 78.3%,47/60) was higher than that in early stage (Ⅰ/Ⅱ) cancer (31.6%,24/76; 21.7%,13/60).Conclusions The system based on double antibodies optimized nanofilm substrates and QDs-based multiplexed imaging significantly improved the capture efficiency of EpCAM-low expression cell lines, and could simultaneously capture and identify CTCs and CTSCs, providing technical support for CTCs further research.