Laser transfer for circulating tumor cell isolation in liquid biopsy
Cancer research has found in the recent years a formidable ally in liquid biopsy, a noninvasive technique that allows the study of circulating tumor cells (CTCs) and biomolecules involved in the dynamics of cancer spread like cell-free nucleid acids or tumor-derived extracellular vesicles. However, single-cell isolation of CTCs with high viability for further genetic, phenotypic, and morphological characterization remains a challenge. We present a new approach for single CTC isolation in enriched blood samples using a liquid laser transfer (LLT) process, adapted from standard laser direct write techniques. In order to completely preserve the cells from direct laser irradiation, we used an ultraviolet laser to produce a blister-actuated laser-induced forward transfer process (BA-LIFT). Using a plasma-treated polyimide layer for blister generation, we completely shield the sample from the incident laser beam. The optical transparency of the polyimide allows direct cell targeting using a simplified optical setup, in which the laser irradiation module, standard imaging, and fluorescence imaging share a common optical path. Peripheral blood mononuclear cells (PBMCs) were identified by fluorescent markers, while target cancer cells remained unstained. As a proof of concept, we were able to isolate single MDA-MB-231 cancer cells using this negative selection process. Unstained target cells were isolated and culture while their DNA was sent for single-cell sequencing (SCS). Our approach appears to be an effective approach to isolate single CTCs, preserving cell characteristics in terms of cell viability and potential for further SCS.
1. Ashworth TR, 1869, A case of cancer in which cells similar to those in the tumours were seen in the blood after death. Aust Med J, 14: 146–149.
2. Ferreira MM, Ramani VC, Jeffrey SS, 2016, Circulating tumor cell technologies. Mol Oncol, 10:374–394.
3. Poudineh M, Sargent EH, Pantel K, et al., 2018, Profiling circulating tumour cells and other biomarkers of invasive cancers. Nat Biomed Eng, 2:72–84.
4. Wan JCM, Massie C, Garcia-Corbacho J, et al., 2017, Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer, 17:223–238.
5. Alix-Panabières C, Pantel K, 2014, Challenges in circulating tumour cell research. Nat Rev Cancer, 14:623–631.
6. Massagué J, Obenauf AC, 2016, Metastatic colonization by circulating tumour cells. Nature, 529:298–306.
7. Pantel K, Brakenhoff RH, Brandt B, 2008, Detection, clinical relevance and specific biological properties of disseminating tumour cells. Nat Rev Cancer, 8:329–340.
8. Pantel K, Alix-Panabières C, Riethdorf S, 2009, Cancer micrometastases. Nat Rev Clin Oncol, 6:339–351.
9. Kim MY, Oskarsson T, Acharyya S, et al., 2009, Tumor self-seeding by circulating cancer cells. Cell, 139:1315–1326.
10. Lin D, Lesang S, Meng L et al., 2021, Circulating tumor cells: biology and clinical significance Signal Trans Targ Ther, 6(1):404
11. Alix-Panabières C, Pantel K, 2013, Circulating tumor cells: Liquid biopsy of cancer. Clin Chem, 59:110–118.
12. Wills QF, Mead AJ, 2015, Application of single-cell genomics in cancer: Promise and challenges. Hum Mol Genet, 24: R74–R84.
13. Shapiro E, Biezuner T, Linnarsson S, 2013, Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet, 14:618–630.
14. Wang Y, Navin NE, 2015, Advances and applications of single-cell sequencing technologies. Mol Cell, 58:598–609.
15. Su H, Xingxing L, Hunag L, et al., 2021, Plasmonic alloys reveal a distinct metabolic phenotype or early gastric cancer Adv Mater, 33:2007978.
16. Li X, Kulkarni AS, Liu X, et al., 2021, Metal-organic framework hybrids aid metabolic profiling for colorectal cancer. Small Methods, 5:2001001.
17. Wu L, Zhang, Z, Tang M, et al., 2020, Spectrally combined encoding for profiling heterogeneous circulating tumor cells using a multifunctional nanosphere-mediated microfluidic platform. Angew Chem Int Ed, 59:11240–11244.
18. Chen XX, Bai F, 2015, Single-cell analyses of circulating tumor cells. Cancer Biol Med, 12:184–192.
19. Ortiz V, Yu M, 2018, Analyzing circulating tumor cells one at a time. Trends Cell Biol, 28:764–775.
20. Lawson DA, Kessenbrock K, Davis RT, et al., 2018, Tumour heterogeneity and metastasis at single-cell resolution. Nat Cell Biol, 20:1349–1360.
21. Kowalik A, Kowalewska M, Gózdz S, 2017, Current approaches for avoiding the limitations of circulating tumor cells detection methods—Implications for diagnosis and treatment of patients with solid tumors. Transl Res, 185: 58–84.e15.
22. Yu M, Stott S, Toner M, et al., 2011, Circulating tumor cells: Approaches to isolation and characterization. J Cell Biol, 192:373–382.
23. Esmaeilsabzali H, Beischlag TV, Cox ME, et al., 2013, Detection and isolation of circulating tumor cells: Principles and methods. Biotechnol Adv, 31:1063–1084.
24. Hong B, Zu Y, 2013, Detecting circulating tumor cells: Current challenges and new trends. Theranostics, 3:377–394.
25. Ramos-Medina R, Moreno F, Lopez-Tarruella S, et al., 2016, Review: Circulating tumor cells in the practice of breast cancer oncology. Clin Transl Oncol, 18:749–759.
26. Ramos-Medina R, Lopez-Tarruella S, Del Monte-Millan M, et al., 2021, Technical challenges for CTC implementation in breast cancer. Cancers, 13(18):46192021.
27. Yu-Ping Y, Giret TM, Cote R et al., 2021, Circulating tumor cells from enumeration to analysis: Current challenges and future opportunities. Cancers, 13:2723.
28. Hoeppener AELM, Swennenhuis JF, Terstappen LWMM, 2012, Immunomagnetic separation technologies, in Minimal Residual Disease and Circulating Tumor Cells in Breast Cancer (Eds. Ignatiadis M, Sotiriou C, Pantel K), Springer, Berlin, Heidelberg, 195, 43–58.
29. Andree KC, van Dalum G, Terstappen LWMM, 2016, Challenges in circulating tumor cell detection by the CellSearch system. Mol Oncol, 10:395–407.
30. Austin RG, Huang TJ, Wu M, et al., 2018, Clinical utility of non-EpCAM based circulating tumor cell assays. Adv Drug Deliv Rev, 125:132–142.
31. Hao SJ, Wan Y, Xia YQ, et al., 2018, Size-based separation methods of circulating tumor cells. Adv Drug Deliv Rev, 125:3–20.
32. Hajba L, Guttman A, 2014, Circulating tumor-cell detection and capture using microfluidic devices. Trends Anal Chem, 59:9–16.
33. Parker SG, Yang Y, Ciampi S, et al., 2018, A photoelectrochemical platform for the capture and release of rare single cells. Nat Commun, 9:2288.
34. Zhou J, Tu C, Liang Y, et al., 2018, Isolation of cells from whole blood using shear-induced diffusion. Sci Rep, 8:9411.
35. Valihrach L, Androvic P, Kubista M, 2018, Platforms for single-cell collection and analysis. Int J Mol Sci, 19:807.
36. Miccio L, Cimmino F, Kurelac I, et al., 2020, Perspectives on liquid biopsy for label-free detection of “circulating tumor cells” through intelligent lab-on-chips. VIEW, 1:20200034.
37. Hou J, Liu X, Zhou S, 2021, Programmable materials for efficient CTCs isolation: From micro/nanotechnology to biomimicry. VIEW, 2:20200023.
38. Millner LM, Linder MW, Valdes R, 2013, Circulating tumor cells: A review of present methods and the need to identify heterogeneous phenotypes. Ann Clin Lab Sci, 43: 295–304.
39. Bhagwat N, Dulmage K, Pletcher CH, et al., 2018, An integrated flow cytometry-based platform for isolation and molecular characterization of circulating tumor single cells and clusters. Sci Rep, 8:5035.
40. Emmert-Buck MR, Bonner RF, Smith PD, et al., 1996, Laser capture microdissection. Science, 274:998–1001.
41. Espina V, Wulfkhule JD, Calvert VS, et al., 2006, Laser-capture microdissection. Nat Protoc, 1:586–603.
42. Bevilacqua C, Ducos B, 2018, Laser microdissection: A powerful tool for genomics at cell level. Mol Aspects Med, 59:5–27.
43. Park ES, Yan JP, Ang RA, et al., 2018, Isolation and genome sequencing of individual circulating tumor cells using hydrogel encapsulation and laser capture microdissection. Lab Chip, 18:1736–1749.
44. Vogel A, Horneffer, V, Lorenz K, et al., 2007, Principles of laser microdissection and catapulting of histologic specimens and live cells. Methods Cell Biol, 82:153–205.
45. Kim O, Lee D, Lee AC, et al., 2019, Single cell genomics: Whole genome sequencing of single circulating tumor cells isolated by applying a pulsed laser to cell capturing microstructures, Small, 15:1902607.
46. Schiele NR, Corr DT, Huang Y, et al., 2010, Laser-based direct-write techniques for cell printing. Biofabrication, 2:032001.
47. Deng Y, Renaud P, Guo Z, et al., 2017, Single cell isolation process with laser induced forward transfer. J Biol Eng, 11:2.
48. Arnold CB, Serra P, Piqué A, 2007, Laser direct-write techniques for printing of complex materials. MRS Bull, 32:23–32.
49. Hon KKB, Li L, Hutchings IM, 2008, Direct writing technology— Advances and developments. CIRP Ann, 57:601–620.
50. Serra P, Duocastella M, Fernández-Pradas JM, et al., 2009, Liquids microprinting through laser-induced forward transfer. Appl Surf Sci, 255:5342–5345.
51. Fernández-Pradas JM, Florian C, Caballero-Lucas F, et al., 2017, Laser-induced forward transfer: Propelling liquids with light. App Surf Sci, 418:559–564.
52. Morales M, Munoz-Martin D, Marquez A, et al., 2018, Advances in Laser Materials Processing: Technology, Research and Applications, Ed. Jonhatan Lawrence, Woodhead Publishing, an imprint of Elsevier.
53. Piqué A, Serra P, 2018, Laser Printing of Functional Materials: 3D Microfabrication, Electronics and Biomedicine, Wiley- VCH Verlag GmbH & Co, KGaA.
54. Barron JA, Wu P, Ladouceur HD, et al., 2004, Biological laser printing: A novel technique for creating heterogeneous 3-dimensional cell patterns. Biomed Microdevices, 6:139–147.
55. Hopp B, Smausz T, Kresz N, et al., 2005, Survival and proliferative ability of various living cell types after laser-induced forward transfer. Tissue Eng, 11:1817–1823.
56. Colina M, Serra P, Fernández-Pradas JM, et al., 2005, DNA deposition through laser induced forward transfer. Biosens Bioelectron, 20:1638–1642.
57. Duocastella M, Colina M, Fernández-Pradas JM, et al., Study of the laser-induced forward transfer of liquids for laser bioprinting. Appl Surf Sci, 253:7855–7859.
58. Ovsianikov A, Gruene, M, Pflaum M, et al., 2010, Laser printing of cells into 3D scaffolds. Biofabrication, 2:014104.
59. Guillemot F, Souquet A, Catros S, et al., 2010, Laser-assisted cell printing: Principle, physical parameters versus cell fate and perspectives in tissue engineering. Nanomedicine (Lond), 5:507–515.
60. Ali M, Pages E, Ducom A, et al., 2014, Controlling laser-induced jet formation for bioprinting mesenchymal stem cells with high viability and high resolution. Biofabrication, 6:045001.
61. Gruene M, Pflaum M , Deiwick A, et al., 2011, Adipogenic differentiation of laser-printed 3D tissue grafts consisting of human adipose-derived stem cells. Biofabrication, 3:015005.
62. Zhang J, Geiger Y, Sotier F, et al., 2021, Extending single cell bioprinting from femtosecond to picosecond laser pulse durations. Micromachines, 12:1172.
63. Karakaidos P, Kryou C, Simigdala N, et al., 2022, Laser bioprinting of cells using UV and visible wavelengths: A comparative DNA damage study. Bioengineering, 9:378.
64. Catros S, Fricain JC, Guillotin B, et al., 2011, Laser-assisted bioprinting for creating on-demand patterns of human osteoprogenitor cells and nano-hydroxyapatite. Biofabrication, 3:025001.
65. Bourget JM, Kerouredan O, Medina M, et al., 2016, Patterning of endothelial cells and mesenchymal stem cells by laser-assisted bioprinting to study cell migration. Biomed Res Int, 3569843.
66. Vinson BT, Phamduy TB, Shipman J, et al., 2017, Laser direct-write based fabrication of a spatially-defined, biomimetic construct as a potential model for breast cancer cell invasion into adipose tissue. Biofabrication, 9:025013.
67. Koch L, Deiwick A, Franke A, et al., 2018, Laser bioprinting of human induced pluripotent stem cells—The effect of printing and biomaterials on cell survival, pluripotency, and differentiation. Biofabrication, 10(3):035005.
68. Kattamis NT, Purnick PE, Weiss R, et al., 2007, Thick film laser induced forward transfer for deposition of thermally and mechanically sensitive materials. Appl Phys Lett, 91:171120.
69. Brown MS, Kattamis NT, Arnold CB, 2010, Time-resolved study of polyimide absorption layers for blister-actuated laser-induced forward transfer. J Appl Phys, 107:083103.
70. Brown MS, Kattamis NT, Arnold CB, 2011, Time-resolved dynamics of laser-induced micro-jets from thin liquid films. Microfluid Nanofluid, 11:199–207.
71. Brown MS, Brasz CF, Ventikos Y, et al., 2012, Impulsively actuated jets from thin liquid films for high-resolution printing applications. J Fluid Mech, 709:341–370.
72. Turkoz E, Perazzo A, Kim H, et al., 2018, Impulsively induced jets from viscoelastic films for high-resolution printing. Phys Rev Lett, 120:074501.
73. Márquez A, Gomez-Fontela M, Lauzurica S, et al., 2020, Fluorescence enhanced BA-LIFT for single cell detection and isolation. Biofabrication, 12(2):025019.
74. Warawdekar UM, Parmar V, Prabhu A, et al., 2017, A versatile method for enumeration and characterization of circulating tumour cells from patients with breast cancer. J Cancer Metastasis Treat, 3:23.
75. Zhu Z, Qiu S, Shao K, et al., 2018, Progress and challenges of sequencing and analyzing circulating tumor cells. Cell Biol Toxicol, 34:405–415.
76. Kou R, Zhao J, Gogoi P, et al., 2018, Enrichment and mutation detection of circulating tumor cells from blood samples. Oncol Rep, 39:2537–2544.
77. Ikediobi ON, Davies H, Bignell G, et al., 2006, Mutation analysis of 24 known cancer genes in the NCI-60 cell line set. Mol Cancer Ther, 5:2606–2612.
78. Muir W, Hildebrandt A, Riker A, 1958, Am J Botany, 45(8):589–597.