Real time visualization of cancer cell death, survival and proliferation using fluorochrome-transfected cells in an IncuCyte® imaging system
Main Article Content
Keywords
cancer, fluorescence, transduction
Abstract
Cancer immunotherapy is a rapidly advancing and viable approach to treating cancer along with more traditional forms of therapy. Real-time cell analysis technologies that examine the dynamic interactions between cancer cells and the cells of the immune system are becoming more important for assessment of novel therapeutics. In this report, we use the IncuCyte® imaging system to study the killing potential of various immune cells on cancer cell lines. The IncuCyte® system tracks living cells, labeled by a red fluorescent protein, and cell death, as indicated by the caspase-3/7 reagent, which generates a green fluorescent signal upon activation of apoptotic pathways. Despite the power of this approach, obtaining commercially fluorescent cancer cell lines is expensive and limited in the range of cell lines that are available. To overcome this barrier, we developed an inexpensive method using a lentiviral construct expressing nuclear localized mKate2 red fluorescent protein to stably label cancer cells. We demonstrate that this method is effective in labeling a wide variety of cell lines, allowing for analyses of different cancers as well as different cell lines of the same type of cancer.
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References
1. Zhang H, Chen J. Current status and future directions of cancer immunotherapy. J Cancer. 2018;9(10):1773-81. PubMed PMID: 29805703; PubMed Central PMCID: PMCPMC5968765.
2. Boussiotis VA. Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway. N Engl J Med. 2016;375(18):1767-78. PubMed PMID: 27806234; PubMed Central PMCID: PMC5575761.
3. Postow MA, Sidlow R, Hellmann MD. Immune-Related Adverse Events Associated with Immune Checkpoint Blockade. N Engl J Med. 2018;378(2):158-68. PubMed PMID: 29320654.
4. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature. 2011;480(7378):480-9. PubMed PMID: 22193102; PubMed Central PMCID: PMCPMC3967235.
5. Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy. Cell. 2017;168(4):707-23. PubMed PMID: 28187290; PubMed Central PMCID: PMCPMC5391692.
6. Sambi M, Bagheri L, Szewczuk MR. Current Challenges in Cancer Immunotherapy: Multimodal Approaches to Improve Efficacy and Patient Response Rates. J Oncol. 2019;2019:4508794. PubMed PMID: 30941175; PubMed Central PMCID: PMCPMC6420990.
7. Wlodkowic D, Skommer J, Darzynkiewicz Z. Cytometry of apoptosis. Historical perspective and new advances. Exp Oncol. 2012;34(3):255-62. PubMed PMID: 23070010; PubMed Central PMCID: PMCPMC3476471.
8. Cen H, Mao F, Aronchik I, Fuentes RJ, Firestone GL. DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells. FASEB J. 2008;22(7):2243-52. PubMed PMID: 18263700.
9. Komoriya A, Packard BZ, Brown MJ, Wu ML, Henkart PA. Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J Exp Med. 2000;191(11):1819-28. PubMed PMID: 10839799; PubMed Central PMCID: PMCPMC2213522.
10. Liu L, Chahroudi A, Silvestri G, Wernett ME, Kaiser WJ, Safrit JT, et al. Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates. Nat Med. 2002;8(2):185-9. PubMed PMID: 11821904.
11. Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M, et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem. 1997;272(29):17907-11. PubMed PMID: 9218414.
12. Poreba M, Szalek A, Kasperkiewicz P, Rut W, Salvesen GS, Drag M. Small Molecule Active Site Directed Tools for Studying Human Caspases. Chem Rev. 2015;115(22):12546-629. PubMed PMID: 26551511; PubMed Central PMCID: PMCPMC5610424.
13. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991;19(14):4008. PubMed PMID: 1861999; PubMed Central PMCID: PMCPMC328507.
14. Teschendorf C, Warrington KH, Jr., Siemann DW, Muzyczka N. Comparison of the EF-1 alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-associated virus. Anticancer Res. 2002;22(6A):3325-30. PubMed PMID: 12530082.
15. Qin JY, Zhang L, Clift KL, Hulur I, Xiang AP, Ren BZ, et al. Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One. 2010;5(5):e10611. PubMed PMID: 20485554; PubMed Central PMCID: PMCPMC2868906.
16. Yue X, Zhao Y, Xu Y, Zheng M, Feng Z, Hu W. Mutant p53 in Cancer: Accumulation, Gain-of-Function, and Therapy. J Mol Biol. 2017;429(11):1595-606. PubMed PMID: 28390900; PubMed Central PMCID: PMCPMC5663274.
17. Hobbs GA, Der CJ, Rossman KL. RAS isoforms and mutations in cancer at a glance. J Cell Sci. 2016;129(7):1287-92. PubMed PMID: 26985062; PubMed Central PMCID: PMCPMC4869631.
18. Neubert NJ, Soneson C, Barras D, Baumgaertner P, Rimoldi D, Delorenzi M, et al. A Well-Controlled Experimental System to Study Interactions of Cytotoxic T Lymphocytes with Tumor Cells. Front Immunol. 2016;7:326. PubMed PMID: 27625650; PubMed Central PMCID: PMCPMC5003846.
19. Nelson N, Lopez-Pelaez M, Palazon A, Poon E, De La Roche M, Barry S, et al. A cell-engineered system to assess tumor cell sensitivity to CD8(+) T cell-mediated cytotoxicity. Oncoimmunology. 2019;8(8):1599635. PubMed PMID: 31413906; PubMed Central PMCID: PMCPMC6682348.
1. Zhang H, Chen J. Current status and future directions of cancer immunotherapy. J Cancer. 2018;9(10):1773-81. PubMed PMID: 29805703; PubMed Central PMCID: PMCPMC5968765.
2. Boussiotis VA. Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway. N Engl J Med. 2016;375(18):1767-78. PubMed PMID: 27806234; PubMed Central PMCID: PMC5575761.
3. Postow MA, Sidlow R, Hellmann MD. Immune-Related Adverse Events Associated with Immune Checkpoint Blockade. N Engl J Med. 2018;378(2):158-68. PubMed PMID: 29320654.
4. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature. 2011;480(7378):480-9. PubMed PMID: 22193102; PubMed Central PMCID: PMCPMC3967235.
5. Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy. Cell. 2017;168(4):707-23. PubMed PMID: 28187290; PubMed Central PMCID: PMCPMC5391692.
6. Sambi M, Bagheri L, Szewczuk MR. Current Challenges in Cancer Immunotherapy: Multimodal Approaches to Improve Efficacy and Patient Response Rates. J Oncol. 2019;2019:4508794. PubMed PMID: 30941175; PubMed Central PMCID: PMCPMC6420990.
7. Wlodkowic D, Skommer J, Darzynkiewicz Z. Cytometry of apoptosis. Historical perspective and new advances. Exp Oncol. 2012;34(3):255-62. PubMed PMID: 23070010; PubMed Central PMCID: PMCPMC3476471.
8. Cen H, Mao F, Aronchik I, Fuentes RJ, Firestone GL. DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells. FASEB J. 2008;22(7):2243-52. PubMed PMID: 18263700.
9. Komoriya A, Packard BZ, Brown MJ, Wu ML, Henkart PA. Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J Exp Med. 2000;191(11):1819-28. PubMed PMID: 10839799; PubMed Central PMCID: PMCPMC2213522.
10. Liu L, Chahroudi A, Silvestri G, Wernett ME, Kaiser WJ, Safrit JT, et al. Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates. Nat Med. 2002;8(2):185-9. PubMed PMID: 11821904.
11. Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M, et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem. 1997;272(29):17907-11. PubMed PMID: 9218414.
12. Poreba M, Szalek A, Kasperkiewicz P, Rut W, Salvesen GS, Drag M. Small Molecule Active Site Directed Tools for Studying Human Caspases. Chem Rev. 2015;115(22):12546-629. PubMed PMID: 26551511; PubMed Central PMCID: PMCPMC5610424.
13. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991;19(14):4008. PubMed PMID: 1861999; PubMed Central PMCID: PMCPMC328507.
14. Teschendorf C, Warrington KH, Jr., Siemann DW, Muzyczka N. Comparison of the EF-1 alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-associated virus. Anticancer Res. 2002;22(6A):3325-30. PubMed PMID: 12530082.
15. Qin JY, Zhang L, Clift KL, Hulur I, Xiang AP, Ren BZ, et al. Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One. 2010;5(5):e10611. PubMed PMID: 20485554; PubMed Central PMCID: PMCPMC2868906.
16. Yue X, Zhao Y, Xu Y, Zheng M, Feng Z, Hu W. Mutant p53 in Cancer: Accumulation, Gain-of-Function, and Therapy. J Mol Biol. 2017;429(11):1595-606. PubMed PMID: 28390900; PubMed Central PMCID: PMCPMC5663274.
17. Hobbs GA, Der CJ, Rossman KL. RAS isoforms and mutations in cancer at a glance. J Cell Sci. 2016;129(7):1287-92. PubMed PMID: 26985062; PubMed Central PMCID: PMCPMC4869631.
18. Neubert NJ, Soneson C, Barras D, Baumgaertner P, Rimoldi D, Delorenzi M, et al. A Well-Controlled Experimental System to Study Interactions of Cytotoxic T Lymphocytes with Tumor Cells. Front Immunol. 2016;7:326. PubMed PMID: 27625650; PubMed Central PMCID: PMCPMC5003846.
19. Nelson N, Lopez-Pelaez M, Palazon A, Poon E, De La Roche M, Barry S, et al. A cell-engineered system to assess tumor cell sensitivity to CD8(+) T cell-mediated cytotoxicity. Oncoimmunology. 2019;8(8):1599635. PubMed PMID: 31413906; PubMed Central PMCID: PMCPMC6682348.
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Published
Jun 12, 2020
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1.
Lanigan TM, Rasmussen SM, Weber DP, Athukorala KS, Campbell PL, Fox DA, Ruth JH. Real time visualization of cancer cell death, survival and proliferation using fluorochrome-transfected cells in an IncuCyte® imaging system. J Biol Methods [Internet]. 2020 Jun. 12 [cited 2024 Apr. 23];7(2):e133. Available from: https://jbmethods.org/index.php/jbm/article/view/323
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