Highly multiplexed profiling of cell surface proteins on single circulating tumor cells based on antibody and cellular barcoding

Abstract

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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References

  1. 1.

    Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis. Science. 2011;331:1559–64.

    Article  CAS  Google Scholar 

  2. 2.

    Krebs MG, Metcalf RL, Carter L, Brady G, Blackhall FH, Dive C. Molecular analysis of circulating tumour cells—biology and biomarkers. Nat Rev Clin Oncol. 2014;11:129–44.

    Article  CAS  PubMed  Google Scholar 

  3. 3.

    Ramsköld D, Luo S, Wang YC, Li R, Deng Q, Faridani OR, et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat Biotechnol. 2012;30:777–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Yu M, Ting DT, Stott SL, Wittner BS, Ozsolak F, Paul S, et al. RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis. Nature. 2012;487:510–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Ni X, Zhuo M, Su Z, Duan J, Gao Y, Wang Z, et al. Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients. Proc Natl Acad Sci U S A. 2013;110:21083–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Miyamoto DT, Zheng Y, Wittner BS, Lee RJ, Zhu HL, Broderick KT, et al. RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance. Science. 2015;349:1351–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Lu Y, Yang L, Wei W, Shi QH. Microchip-based single-cell functional proteomics for biomedical applications. Lab Chip. 2017;17:1250–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Su YP, Shi QH, Wei W. Single cell proteomics in biomedicine: high-dimensional data acquisition, visualization, and analysis. Proteomics. 2017;17:1600267.

    Article  CAS  Google Scholar 

  9. 9.

    Shi QH, Qin LD, Wei W, Geng F, Fan R, Shin YS, et al. Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells. Proc Natl Acad Sci U S A. 2012;109:419–24.

    Article  PubMed  Google Scholar 

  10. 10.

    Zhang Y, Tang Y, Sun S, Wang ZH, Wu WJ, Zhao XD, et al. Single-cell codetection of metabolic activity, intracellular functional proteins, and genetic mutations from rare circulating tumor cells. Anal Chem. 2015;87:9761–8.

    Article  CAS  PubMed  Google Scholar 

  11. 11.

    Yang L, Wang ZH, Deng YL, Li Y, Wei W, Shi QH. Single-cell, multiplexed protein detection of rare tumor cells based on a beads-on-barcode antibody microarray. Anal Chem. 2016;88:11077–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Agasti SS, Liong M, Peterson VM, Lee H, Weissleder R. Photocleavable DNA barcode–antibody conjugates allow sensitive and multiplexed protein analysis in single cells. J Am Chem Soc. 2012;134:18499–502.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Ullal AV, Petersin V, Agasti SS, Tuang S, Juric D, Castro CM, et al. Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates. Sci Transl Med. 2014;6:219ra9e.

    Article  CAS  Google Scholar 

  14. 14.

    Swennenhuis JF, Tibbe AG, Stevens M, Katika MP, van Dalum J, Tong HD, et al. Self-seeding microwell chip for the isolation and characterization of single cells. Lab Chip. 2015;15:3039–46.

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    Tang Y, Wang Z, Li Z, Kim J, Deng YL, Li Y, et al. High-throughput screening of rare metabolically active tumor cells in pleural effusion and peripheral blood of lung cancer patients. Proc Natl Acad Sci U S A. 2017;114:2544–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Ma C, Fan R, Ahmad H, Shi QH, Comin-Anduix B, Chodon T, et al. A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells. Nat Med. 2011;17:738–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Wei W, Shi QH, Remacle F, Qin LD, Shackelford D, Shin YS, et al. Hypoxia induces a phase transition within a kinase signaling network in cancer cells. Proc Natl Acad Sci U S A. 2013;110:E1352–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Deng YL, Zhang Y, Sun S, Wang ZH, Wang MJ, Yu BQ, et al. An integrated microfluidic chip system for single-cell secretion profiling of rare circulating tumor cells. Sci Rep. 2014;4:7499.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Pecot CV, Bischoff FZ, Mayer JA, Wong KL, Pham T, Bottsford-Miller J, et al. A novel platform for detection of CK+ and CK- CTCs. Cancer Discov. 2011;1:580–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Yan F, Pang JX, Peng X, Molina JR, Yang P, Liu SJ. Elevated cellular PD1/PD-L1 expression confers acquired resistance to cisplatin in small cell lung cancer cells. PLoS One. 2016;11:e0162925.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Nishino M, Ozaki M, Hegab AE, Hamamoto J, Kagawa S, Arai D, et al. Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma. J Cancer. 2017;8:1774–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Godfroid E, Geuskens M, Dupressoir T, Parent L, Szpirer C. Cytokeratins are exposed on the outer surface of established human mammary carcinoma cells. J Cell Sci. 1991;99:595–607.

    PubMed  Google Scholar 

  23. 23.

    Liu F, Chen Z, Wang JH, Shao XF, Cui ZY, Yang CZ, et al. Overexpression of cell surface cytokeratin 8 in multidrug-resistant MCF-7/MX cells enhances cell adhesion to the extracellular matrix. Neoplasia. 2008;10:1275–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Gibney GT, Weiner LM, Atkins MB. Predictive biomarkers for checkpoint inhibitor-based immunotherapy. Lancet Oncol. 2016;16:e542-e.

    Article  CAS  Google Scholar 

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Funding

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.).

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Correspondence to Qihui Shi.

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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.

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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

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Keywords

  • Circulating tumor cells
  • DNA barcoding
  • High-throughput sequencing
  • Microchip
  • Single-cell proteomic analysis