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Secreted luciferases as a minimally invasive 3R-compliant tool for accurate monitoring of tumor burden

Nature Protocols (2026)Cite this article

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Abstract

Preclinical mouse models are indispensable in cancer research, providing insights into tumor biology and therapeutic responses. This protocol describes a minimally invasive blood-based tumor monitoring approach using secreted luciferases for longitudinal tracking of tumor burden in transplantable xenografts and genetically engineered mouse models. Unlike intracellular luciferases used in bioluminescence imaging, secreted luciferases are actively released into circulation, enabling precise quantification from microliter-scale blood samples. We describe a transplantable model, where tumor cells are labeled in vitro using lentiviral transduction before engraftment. Orthogonal secreted luciferases enable multiplexed analysis of distinct tumor populations within a single host, reducing animal numbers and enhancing data density. We also describe an autochthonous lung cancer model, where intratracheal adenoviral delivery of Cre recombinase and CRISPR nucleases induces tumorigenesis through somatic genome editing while activating a conditional secreted luciferase reporter transgene. Tumor-bearing mice undergo routine blood sampling, with luciferase activity measured ex vivo to quantify viable tumor burden. Compared to imaging techniques, this method eliminates anesthesia and contrast agents, minimizing animal stress and enabling frequent monitoring with superior temporal resolution and reduced logistical complexity. The protocol requires only standard molecular biology skills and basic mouse handling expertise. While tumor labeling and growth duration is model dependent, blood sampling requires ~5 min per animal, with all samples from one cohort processed and measured together within 2 h. This approach provides an accessible, cost-effective and scalable alternative to imaging-based tumor monitoring, that is aligned with the 3Rs principles, offering a powerful and ethically sound platform for preclinical cancer research.

Key points

  • Labeling tumor cells with secreted luciferases allows for precise, longitudinal monitoring of tumor growth through analyzing small blood samples. This protocol details the in vitro labeling for transplantable models, the in vivo labeling using conditional luciferase-transgenic mice and the analysis of luciferase blood levels with a plate reader.

  • Compared to elaborate imaging techniques, blood-based monitoring represents a simple, flexible and cost-effective tool that aligns with the 3Rs principles.

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Fig. 1: Principle of tumor monitoring with secreted luciferases.
Fig. 2: Overview of the protocol with applications.
Fig. 3: Monitoring transplanted tumors with secreted luciferases.
Fig. 4: Monitoring tumor drug response with a dual-luciferase approach.
Fig. 5: Monitoring autochthonous lung tumor growth.
Fig. 6: Construction and purification of recombinant adenoviral vectors for autochthonous mouse models.
Fig. 7: Equipment and workflow for intratracheal infections in mice.

Data availability

All data shown in this protocol were previously published. Data shown in Figs. 4 and 5 were extracted from ref. 31 and ref. 26, respectively, and are provided as Source data.

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Acknowledgements

The development of this protocol was supported by grants from von Behring-Röntgen Stiftung (71_0012), Deutsche Forschungsgemeinschaft (GRK 2573, STI 182/15-1), Wilhelm Sander-Stiftung (2022.129.1), Universitätsklinikum Giessen and Marburg (5/2025 MR), and State of Hesse (LOEWE, iCANx). Figures were created with BioRender.com.

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Author notes

  1. These authors contributed equally: Nastasja Merle, Imke Bullwinkel.

Authors and Affiliations

  1. Institute of Molecular Oncology and Genomics Core Facility, Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Philipps-University, Marburg, Germany

    Nastasja Merle, Imke Bullwinkel, Oleg Timofeev, Sabrina Elmshäuser & Thorsten Stiewe

  2. Institute for Lung Health (ILH), Justus-Liebig University, Gießen, Germany

    Nastasja Merle, Imke Bullwinkel, Oleg Timofeev, Sabrina Elmshäuser & Thorsten Stiewe

Authors
  1. Nastasja Merle
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  2. Imke Bullwinkel
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Contributions

N.M., I.B., O.T. and S.E. developed and refined the protocols. T.S. conceived the overall concept and designed the methodological framework. N.M. and I.B. drafted the manuscript, and all authors contributed to revising and refining the final version.

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Correspondence to Thorsten Stiewe.

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The authors declare no competing interests.

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Nature Protocols thanks Takahiro Kuchimaru, Scott Lyons and Laura Mezzanotte for their contribution to the peer review of this work.

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Key references

Charles, J. P. et al. Nat. Commun. 5, 3981 (2014): https://doi.org/10.1038/ncomms4981

Merle, N. et al. Mol. Cancer 21, 191 (2022): https://doi.org/10.1186/s12943-022-01661-2

Vogiatzi, F. et al. Proc. Natl Acad. Sci. USA 113, E8433–E8442 (2016): https://doi.org/10.1073/pnas.1612711114

Pavlakis, E. et al. J. Exp. Clin. Cancer Res. 42, 203 (2023): https://doi.org/10.1186/s13046-023-02785-z

Gremke, N. et al. Signal Transduct. Target. Ther. 10, 92 (2025): https://doi.org/10.1038/s41392-025-02180-4

Source data

Source Data Fig. 4, 5

Source data for graphs in Fig. 4a–f. Source data for graphs in Fig. 5b,c.

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Merle, N., Bullwinkel, I., Timofeev, O. et al. Secreted luciferases as a minimally invasive 3R-compliant tool for accurate monitoring of tumor burden. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01315-9

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