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Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins

A Corrigendum to this article was published on 01 April 2004

Abstract

We have shown that bacteria injected intravenously into live animals entered and replicated in solid tumors and metastases. The tumor-specific amplification process was visualized in real time using luciferase-catalyzed luminescence and green fluorescent protein fluorescence, which revealed the locations of the tumors and metastases. Escherichia coli and three attenuated pathogens (Vibrio cholerae, Salmonella typhimurium, and Listeria monocytogenes) all entered tumors and replicated. Similarly, the cytosolic vaccinia virus also showed tumor-specific replication, as visualized by real-time imaging. These findings indicate that neither auxotrophic mutations, nor vaccinia virus deficient for the thymidine kinase gene, nor anaerobic growth conditions were required for tumor specificity and intratumoral replication. We observed localization of tumors by light-emitting microorganisms in immunocompetent and in immunocompromised rodents with syngeneic and allogeneic tumors. Based on their 'tumor-finding' nature, bacteria and viruses may be designed to carry multiple genes for detection and treatment of cancer.

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Figure 1: Visualization with the low light imager of the distribution of light-emitting bacteria injected intravenously in nude mice.
Figure 2: Intravenously injected bacteria and viruses accumulate and replicate in subcutaneous C6 rat glioma tumors in nude mice as visualized by light emission.
Figure 3: Localization of bacterial colonization and viral infection in subcutaneous C6 glioma tumors.
Figure 4: Bacterial and vaccinia virus show tumor-specific localization in different tumorous mice models.
Figure 5: Intravenously delivered light-emitting bacteria and recombinant vaccinia virus mark the location of primary breast tumors and their metastases in nude mice.

References

  1. 1

    Abelmann, H.W. in Cancer as I See It (Philosophical Library, Inc., New York, 1951).

    Google Scholar 

  2. 2

    Llinares, P. et al. Infected atrial myxoma simulating infective endocarditis. Enferm. Infecc. Microbiol. Clin. 11, 378–381 (1993).

    CAS  PubMed  Google Scholar 

  3. 3

    Liao, W.Y. et al. Bacteriology of infected cavitating lung tumor. Am. J. Respir. Crit. Care Med. 161, 1750–1753 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Di Virgilio, G., Lavenda, N. & Siegel, D. Viral particles in human breast cancer. Oncologia 19, 341–348 (1965).

    CAS  Article  Google Scholar 

  5. 5

    Williamson, A.L., Jaskiesicz, K. & Gunning, A. The detection of human papillomavirus in oesophageal lesions. Anticancer Res. 11, 263–265 (1991).

    CAS  PubMed  Google Scholar 

  6. 6

    Liu, B. et al. Identification of a proviral structure in human breast cancer. Cancer Res. 61, 1754–1759 (2001).

    CAS  PubMed  Google Scholar 

  7. 7

    Chang, F., Syrjanen, S., Shen, Q., Wang, L. & Syrjanen, K. Screening for human papillomavirus infections in esophageal squamous cell carcinomas by in situ hybridization. Cancer 72, 2525–2530 (1993).

    CAS  Article  Google Scholar 

  8. 8

    Lemmon, M.J. et al. Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment. Gene Ther. 4, 791–796 (1997).

    CAS  Article  Google Scholar 

  9. 9

    Pawelek, J.M., Low, K.B. & Bermudes, D. Tumor-targeted Salmonella as a novel anti-cancer vector. Cancer Res. 57, 4537–4544 (1997).

    CAS  PubMed  Google Scholar 

  10. 10

    Sauter, B.V., Martinet, O., Zhang, W., Mandeli, J. & Woo, S.L.C. Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases. Proc. Natl. Acad. Sci. USA 97, 4802–4807 (2000).

    CAS  Article  Google Scholar 

  11. 11

    Gordon, E.M. et al. Systemic administration of a matrix-targeted retroviral vector is efficacious for cancer gene therapy in mice. Hum. Gene Ther. 12, 193–204 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Block, A. et al. Gene therapy of metastatic colon carcinoma: regression of multiple hepatic metastases by adenoviral expression of bacterial cytosine deaminase. Cancer Gene Ther. 7, 438–445 (2000).

    CAS  Article  Google Scholar 

  13. 13

    Chen, C.T. et al. Antiangiogenic gene therapy for cancer via systemic administration of adenoviral vectors expressing secretable endostatin. Hum. Gene Ther. 11, 1983–1996 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Gnant, M.F.X. et al. Tumor-specific gene delivery using recombinant vaccinia virus in a rabbit model of liver metastases. J. Natl. Cancer Inst. 91, 1744–1750 (1999).

    CAS  Article  Google Scholar 

  15. 15

    Gnant, M.F., Puhlmann, M., Bartlett, D.L. & Alexander, H.R. Jr. Regional versus systemic delivery of recombinant vaccinia virus as suicide gene therapy for murine liver metastases. Ann. Surg. 230, 352–360 (1999).

    CAS  Article  Google Scholar 

  16. 16

    McCart, J.A. et al. Complex interactions between the replicating oncolytic effect and the enzyme/prodrug effect of vaccinia-mediated tumor regression. Gene Ther. 7, 1217–1223 (2000).

    CAS  Article  Google Scholar 

  17. 17

    Puhlmann, M. et al. Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant. Cancer Gene Ther. 7, 66–73 (2000).

    CAS  Article  Google Scholar 

  18. 18

    Belas, R. et al. Bacterial bioluminescence: isolation and expression of the luciferase genes from Vibrio harveyi. Science 218, 791–793 (1982).

    CAS  Article  Google Scholar 

  19. 19

    De Wet, J.R., Wood, K.V., Deluca, M., Helinski, D.R. & Subramani, S. Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7, 725–737 (1987).

    CAS  Article  Google Scholar 

  20. 20

    Prasher, D.C., McCann, R.O., Longiaru, M. & Cormier, M.J. Sequence comparisons of complementary DNAs encoding aequorin isotypes. Biochem. 26, 1326–1332 (1987).

    CAS  Article  Google Scholar 

  21. 21

    Foran, D.R. & Brown, W.M. Nucleotide sequence of the LuxA and LuxB genes of the bioluminescent marine bacterium Vibrio fischeri. Nucleic Acids Res. 16, 777 (1988).

    CAS  Article  Google Scholar 

  22. 22

    Escher, A., O'Kane, D.J., Lee, J. & Szalay, A.A. Bacterial luciferase αβ fusion protein is fully active as a monomer and highly sensitive in vivo to elevated temperature. Proc. Natl. Acad. Sci. USA 86, 6528–6532 (1989).

    CAS  Article  Google Scholar 

  23. 23

    Lorenz, W.W., McCann, R.O., Longiaru, M. & Cormier, M.J. Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc. Natl. Acad. Sci. USA 88, 4438–4442 (1991).

    CAS  Article  Google Scholar 

  24. 24

    Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G. & Cormier, M.J. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233 (1992).

    CAS  Article  Google Scholar 

  25. 25

    Engebrecht, J., Simon, M. & Silverman, M. Measuring gene expression with light. Science 227, 1345–1347 (1985).

    CAS  Article  Google Scholar 

  26. 26

    Legocki, R.P., Legocki, M., Baldwin, T.O. & Szalay, A.A. Bioluminescence in soybean root nodules: demonstration of a general approach to assay gene expression in vivo using bacterial luciferase. Proc. Natl. Acad. Sci. USA 83, 9080–9084 (1986).

    CAS  Article  Google Scholar 

  27. 27

    Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. & Prasher, D.C. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994).

    CAS  Article  Google Scholar 

  28. 28

    Langridge, W.H.R. et al. A luciferase marker gene system to monitor gene expression in bacteria, plant and virus infected animal cells. Proceedings of the VIIth Int. Symp. on Bio & Chemiluminescence, Banff, Canada, March 14–18, 1993 (eds. Szalay, A.A. et al.) 222–226 (Wiley, Chichester, UK, 1993).

    Google Scholar 

  29. 29

    O'Kane, D.J. et al. Visualization of bioluminescence as a marker of gene expression in Rhizobium-infected soybean root nodules. J. Plant Mol. Biol. 10, 387–399 (1988).

    CAS  Article  Google Scholar 

  30. 30

    Wang, G., Mayerhofer, R., Langridge, W.H.R. & Szalay, A.A. in Proceedings of the VIIth Int. Symp. on Bio & Chemiluminescence, Banff, Canada, March 14–18, 1993 (eds. Szalay, A.A. et al.) 232–238 (Wiley, Chichester, UK, 1993).

    Google Scholar 

  31. 31

    Giacomin, L.T. & Szalay, A.A. Expression of a PALI promoter luciferase gene fusion in Arabidopsis thaliana in response to infection by phytopathogenic bacteria. Plant Sci. 116, 59–72 (1996).

    CAS  Article  Google Scholar 

  32. 32

    Lee, C.Y., Szittner, R. & Meighen, E.A. The lux genes of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish. Nucleotide sequence, difference in gene organization and high expression in mutant Escherichia coli. Eur. J. Biochem. 201, 161–167 (1991).

    CAS  Article  Google Scholar 

  33. 33

    Meighen, E.A. & Szittner, R. Multiple repetitive elements organization of the lux operons of luminescent terrestrial bacteria. J. Bacteriol. 174, 5371–5381 (1992).

    CAS  Article  Google Scholar 

  34. 34

    Fernandez-Pinas, F. & Wolk, C.P. Expression of luxCD-E in Anabaena sp. can replace the use of exogenous aldehyde for in vivo localization of transcription by luxAB. Gene 150, 169–174 (1994).

    CAS  Article  Google Scholar 

  35. 35

    Contag, C.H. et al. Photonic detection of bacterial pathogens in living hosts. Mol. Microbiol. 18, 593–603 (1995).

    CAS  Article  Google Scholar 

  36. 36

    Shabahang, S. & Szalay, A.A. in Proceedings of the 11th International Symposium on Bioluminescence and Chemiluminescence, Pacific Grove, CA, September 6–10, 2000 (eds. Case, J.F. et al.) 449–452 (World Scientific, Singapore, 2001).

    Google Scholar 

  37. 37

    Wang, Y., Wang, G., O'Kane, D.J. & Szalay, A.A. in Proceedings of the 9th International Symposium on Bioluminescence and Chemiluminescence, Woods Hole, MA, October 4–8, 1996 (eds. Hastings, J.W. et al.) 419–422 (Wiley, Chichester, UK, 1996).

    Google Scholar 

  38. 38

    Wang, Y., Wang, G., O'Kane D.J. & Szalay A.A. Chemiluminescence energy transfer to study protein-protein interaction in living cells. Mol. Gen. Genet. 264, 578–587 (2001).

    CAS  Article  Google Scholar 

  39. 39

    Wang, Y, Yu Y., Shabahang, S., Wang, G. & Szalay, A.A. Functional Renilla luciferase–Aequorea GFP (RUC-GFP) fusion protein as a novel dual reporter for imaging of gene expression in cell cultures and in live animals. Mol. Gen. Genet. 268, 160–168 (2002).

    CAS  Article  Google Scholar 

  40. 40

    Timiryasova, T., Yu, Y.A., Shabahang, S., Fodor, I. & Szalay, A.A. in Proceedings of the 11th International Symposium on Bioluminescence and Chemiluminescence, Pacific Grove, CA, September 6–10, 2000 (eds. Case, J.F. et al.) 457–460 (World Scientific, Singapore, 2001).

    Google Scholar 

  41. 41

    Gebauer, G., Jager, W. & Lang, N. mRNA expression of components of the insulin-like growth factor system in breast cancer cell lines, tissues, and metastatic breast cancer cells. Anticancer Res. 18, 1191–1195 (1998).

    CAS  PubMed  Google Scholar 

  42. 42

    Padera, T.P. et al. Lymphatic metastasis in the absence of functional intratumor lymphatics. Science 296, 1883–1886 (2002).

    CAS  Article  Google Scholar 

  43. 43

    Mose, J.R. & Mose, G. Oncolysis by Clostridia. I. Activity of Clostridium butyricum (M-55) and other nonpathogenic Clostridia against the Ehrlich carcinoma. Cancer Res. 24, 212–216 (1964).

    Google Scholar 

  44. 44

    Dang, L.H., Bettegowda, C., Huso, D.L., Kinzler, K.W. & Vogelstein, B. Combination bacteriolytic therapy for the treatment of experimental tumors. Proc. Natl. Acad. Sci. USA 98, 15155–15160 (2001).

    CAS  Article  Google Scholar 

  45. 45

    Yazawa, K., Fujimori, M., Amano, J., Kano, Y. & Taniguchi, S. Bifidobacterium longum as a delivery system for cancer gene therapy: selective localization and growth in hypoxic tumors. Cancer Gene Ther. 7, 269–274 (2000).

    CAS  Article  Google Scholar 

  46. 46

    Sznol, M., Lin, S.L., Bermudes, D., Zheng, L.M. & King, I. Use of preferentially replicating bacteria for the treatment of cancer. J. Clin. Invest. 105, 1027–1030 (2000).

    CAS  Article  Google Scholar 

  47. 47

    Ramshaw, I., Ruby, J., Ramsay, A., Ada, G. & Karupiah, G. Expression of cytokines by recombinant vaccinia virus: a model for studying cytokines in virus infections in vivo. Immun. Rev. 127, 157–182 (1992).

    CAS  Article  Google Scholar 

  48. 48

    Gnant, M.F.X., Puhlmann, M., Alexander, H.R. & Bartlett, D.L. Systemic administration of a recombinant vaccinia virus expressing the cytosine deaminase gene and subsequent treatment with 5-fluorocytosine leads to tumor-specific gene expression and prolongation of survival in mice. Cancer Res. 59, 3396–3403 (1999).

    CAS  PubMed  Google Scholar 

  49. 49

    Voisey, C.R. & Marincs, F. Elimination of internal restriction enzyme sites from a bacterial luminescence (luxCDABE) operon. Biotechniques 24, 56–58 (1998).

    CAS  Article  Google Scholar 

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Acknowledgements

The authors thank M. Lilly for generating mice with bladder tumors, I. Fodor for help with prostate tumor mice and for access to the rVV-RUC-GFP virus, D. Gridley for developing intracranial glioma tumors, D. deLeon and J. Tian for mice with MCF-7 implants and K. Oberg for access to the stereo fluorescence microscope. We would like to acknowledge the help and scientific criticisms of C. Slattery and F. Grummt during the preparation of this manuscript. Y.A.Y. was a recipient of a graduate fellowship from LLU. The research was supported in part by LLU, by Genelux, by an SFB travel award to A.A.S., by the research prize from A.V. Humboldt Foundation, Germany, awarded to A.A.S., and by an SFB award to W.G.

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Correspondence to Aladar A Szalay.

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A.A.S. is a cofounder and shareholder of Genelux. Y.A.Y., T.M.T. and Q.Z. are employed by Genelux.

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Yu, Y., Shabahang, S., Timiryasova, T. et al. Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins. Nat Biotechnol 22, 313–320 (2004). https://doi.org/10.1038/nbt937

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