Specific Labeling of Circulating Tumor Cells by full-spectrum fluorescent carbon dots: An Advanced Nanoplatform for Liquid Biopsy Applications

Objective The promise of liquid biopsy in cervical cancer management hinges on circulating tumor cells (CTCs) - these rare cellular messengers could revolutionize diagnosis, track treatment response, and predict outcomes. But reality falls short: our current tools are clumsy scalpels that maul genetic blueprints even as they attempt detection. Methods and materials: A novel full-spectrum β-cyclodextrin carbon dot (β-CDs) exhibiting violet, blue, green, yellow, and red fluorescence emissions was developed previously. To visualize the effects of β-CDs treatment, we performed fluorescence imaging on cervical cancer cells (HeLa) and leukocytes using confocal microscopy. Next, flow cytometry helped us quantify differences in how these two cell types responded to β-CDs exposure. For a more nuanced assessment, we mixed Hela cells with leukocytes and performed dual staining—targeting CD45 for leukocytes and β-CDs for tumor cells—then used confocal imaging to evaluate whether β-CDs could reliably distinguish malignant cells within a leukocyte-dominated background. Beyond cellular labeling, we also investigated potential β-CDs-induced disruptions to tumor cell genetics. Whole-genome sequencing (WGS) and RNA-seq were employed to assess DNA and RNA integrity. Meanwhile, in collaboration with Mindray Bio-Medical, we refined the automated digital cell morphology analyzer (MC-80), ultimately enhancing its capability to identify and image tumor cells. Results In-depth investigations revealed that these full-spectrum β-CDs demonstrate remarkable mitochondrial staining specificity in tumor cells. Fluorescence imaging and flow cytometry analyses confirmed their capability to effectively discriminate tumor cells from leukocytes, enabling highly efficient and selective tumor cell labeling. This breakthrough provides a robust tool for CTCs identification, characterization, and isolation. Whole genome sequencing (WGS) and RNA sequencing of β-CD-labeled tumor cells demonstrated that, compared to the gold standard cytokeratin (CKs) immunofluorescence method for CTCs detection, the full-spectrum fluorescent β-CDs labeling preserves genomic integrity without compromising nucleic acid quality, thereby meeting stringent sequencing requirements and ensuring reliable molecular-level genotyping and analysis. Furthermore, in collaboration with Shenzhen Mindray Bio-Medical Electronics, the MC-80 automated digital cell morphology analyzer has been optimized, which achieves precise cervical cancer cell Hela identification and demonstrates superior detection efficacy in clinically simulated samples. Conclusions In our earlier work, we developed an innovative full-spectrum β-cyclodextrin carbon dot (β-CDs) system with some remarkable properties. These nanoparticles not only selectively target mitochondria but can also clearly discriminate tumor cells from leukocytes, all while preserving genomic integrity for downstream sequencing applications. This unique combination of features suggests strong potential for circulating tumor cells (CTCs) isolation and analysis. We've also made significant improvements to the automated digital cell morphology analyzer (MC-80). The optimized system now achieves remarkably precise tumor cell imaging, addressing previous limitations in cellular identification.