Abstract
Tumor initiation remains one of the least understood events in cancer biology, largely due to the challenge of dissecting the intricacy of the tumorigenic process in laboratory settings. The insufficient biological complexity of conventional in vitro systems makes animal models the primary experimental approach to study tumorigenesis. Despite providing valuable insights, these in vivo models function as experimental black boxes with limited spatiotemporal resolution of cellular dynamics during oncogenesis. In addition, their use raises ethical concerns, further underscoring the need for alternative ex vivo systems. Here we provide a detailed protocol to integrate state-of-the-art microfabrication, tissue engineering and optogenetic approaches to generate topobiologically complex miniature colons (‘mini-colons’) capable of undergoing tumorigenesis in vitro. We describe the key methodology for the generation of blue light-inducible oncogenic cells, the establishment of hydrogel-based mini-colon scaffolds within microfluidic devices, the development of mini-colons and the induction of spatiotemporally controlled tumorigenesis. This protocol enables the formation and long-term culture of complex cancerous tissues that capture in vivo-like tumoral biology while offering real-time and single-cell resolution analyses. It can be implemented in 4–6 weeks by researchers with prior experience in 3D cell culture techniques. We anticipate that these methodological guidelines will have a broad impact on the cancer research community by opening new avenues for tumorigenesis studies.
Key points
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This protocol details the generation of topobiologically complex miniature colons capable of undergoing tumorigenesis in vitro. This approach uses key methodologies for the generation of blue light-inducible oncogenic cells, the establishment of hydrogel-based mini-colon scaffolds within microfluidic devices, the development of mini-colons and the induction of spatiotemporally controlled tumorigenesis.
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The spatiotemporal resolution of cellular dynamics revealed by this approach makes it a versatile in vitro alternative to the animal models.
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Data availability
The laser ablation template used for the generation of hydrogel mini-colon scaffolds is available at GitHub (https://github.com/LorenzoLF/Mini-colon_bioengineering) and Zenodo35. All other relevant data can be accessed through the original publication describing the development of mini-colons for tumorigenic studies7.
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Acknowledgements
We thank A. Bowler and F. Radtke for the inducible A, AK and AKP mouse colon organoids; M. Thomson for the original light-inducible plasmids; M. Wernig and G. Neumayer for the initial idea and work on the doxycycline- and light-inducible system; A. Aebi and B. Schneider for lentivirus production; C. Baur for discussing, designing and building the custom illumination device; A. Chrisnandy for assistance on photomask fabrication; O. Mitrofanova, B. Elçi and Y. Tinguely for assistance on microfluidic device fabrication; L. Tillard for technical support; and J. Prébandier for administrative assistance. We acknowledge support from the following EPFL core facilities: CMi, CPG, PTBTG, HCF, BIOP, FCCF, BSF and GECF.
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L.F.L.-M. conceived the study, designed experiments, carried out the experimental work, analyzed the data, performed artwork design and wrote the manuscript. T.H. generated the OptoCre module and blue light-associated systems, performed experimental work and analyzed data. J.L. produced the microfluidic devices and optimized hydrogel scaffolds. M.N. designed and developed the first mini-gut system. M.P.L. conceived the work, designed experiments and edited the manuscript.
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Competing interests
The EPFL has filed for patent protection (nos. EP16199677.2, PCT/EP2017/079651, US20190367872A1) on the scaffold-guided organoid technology used here, and M.P.L. and M.N. are named as inventors on those patents. M.P.L. and M.N. are employees of Roche. The other authors declare no competing interests.
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Nature Protocols thanks Mehdi Nikkhah, Xiling Shen and Yuan Wang for their contribution to the peer review of this work.
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Key references
Nikolaev, M. et al. Nature 585, 574–578 (2020): https://doi.org/10.1038/s41586-020-2724-8
Lorenzo-Martín, L. F. et al. Nature 629, 450–457 (2024): https://doi.org/10.1038/s41586-024-07330-2
Lorenzo-Martín, L. F. et al. Nat. Biotechnol. 43, 727–736 (2025): https://doi.org/10.1038/s41587-024-02301-4
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Lorenzo-Martín, L.F., Hübscher, T., Langer, J. et al. Bioengineering mini-colons for ex vivo colorectal cancer research. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01292-z
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DOI: https://doi.org/10.1038/s41596-025-01292-z
