Circulating tumor cells (CTCs) are extraordinarily rare in blood samples and represent a real-time “liquid biopsy” of tumors. Although genetic and transcriptional sequencing of single CTCs has been reported, these methods fail to provide phenotypic and functional information of CTCs such as protein levels of surface proteins. Studies of single-cell proteomic assays of CTCs have been rare because of a lack of single-cell proteomic methods to handle and analyze rare cells in a high background of non-target cells with high sensitivity, throughput, and multiplexing capacity. Here, we develop a microchip-assisted single-cell proteomic method for profiling surface proteins of CTCs based on antibody and cellular DNA barcoding strategy. We combine DNA-encoded antibody tags and cell indexes to profile 15 proteins in ~ 100 single rare cells simultaneously, and use high-throughput sequencing as the readout to generate surface protein profiles of CTCs according to their cell indexes and antibody-derived protein barcodes. A 6400-well microchip and the automated puncher are used to rapidly retrieve single CTCs from enriched CTC population with minimal cell loss (~ 10%). This technological platform integrates reliable isolation and proteomic analysis of single CTCs and can be extendable to ~ 100 proteins in hundreds of rare cells with single-cell precision.
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This study received financial support from the National Key Research and Development Program Grant 2016YFC0900200 (to Q.S.) and National Natural Science Foundation of China Grants 81371712 and 21775103 (to Q.S.) and 81701852 (to L.Y.).
Conflict of interest
The authors declare that they have no conflicts of interest.
Research involving human participants and/or animals
This study measured peripheral blood samples from lung adenocarcinoma patients. The clinical sample collection and experiment were carried out in accordance with guidelines and protocols that were approved by the Ethics and Scientific Committees of Shanghai Chest Hospital.
Peripheral blood samples were obtained from healthy donors and lung adenocarcinoma patients in Shanghai Chest Hospital (Shanghai, China) with written informed consent.
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Published in the topical collection Ultrasmall Sample Biochemical Analysis with guest editors Ryan Kelly and Ying Zhu.
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Wang, C., Yang, L., Wang, Z. et al. Highly multiplexed profiling of cell surface proteins on single circulating tumor cells based on antibody and cellular barcoding. Anal Bioanal Chem 411, 5373–5382 (2019). https://doi.org/10.1007/s00216-019-01666-9
- Circulating tumor cells
- DNA barcoding
- High-throughput sequencing
- Single-cell proteomic analysis