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Lymphatic collection and cell isolation from mouse models for multiomic profiling

Nature Protocols volume 20, pages 884–901 (2025)Cite this article

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An Author Correction to this article was published on 10 February 2025

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Abstract

Premetastatic cancer cells often spread from the primary lesion through the lymphatic vasculature and, clinically, the presence or absence of lymph node metastases impacts treatment decisions. However, little is known about cancer progression via the lymphatic system or of the effect that the lymphatic environment has on cancer progression. This is due, in part, to the technical challenge of studying lymphatic vessels and collecting lymph fluid. Here we provide a step-by-step procedure to collect both lymph and tumor-draining lymph in mouse models of cancer metastasis. This protocol has been adapted from established methods of lymph collection and was developed specifically for the collection of lymph from tumors. The approach involves the use of mice bearing melanoma or breast cancer orthotopic tumors. After euthanasia, the cisterna chyli and the tumor are exposed and viewed using a stereo microscope. Then, a glass cannula connected to a 1 mL syringe is inserted directly into the cisterna chyli or the tumor-draining lymphatics for collection of pure lymph. These lymph samples can be used to analyze the lymph-derived cancer cells using highly sensitive multiomics approaches to investigate the impact of the lymph environment during cancer metastasis. The procedure requires 2 h per mouse to complete and is suitable for users with minimal expertise in small animal handling and use of microsurgical tools under a stereo microscope.

Key points

  • The elevated pressure in the lymphatics around the tumor is leveraged for the collection of lymph from each tumor and the subsequent metabolic and lipidomic characterization using mass spectrometry, as well as lymph-derived cell characterization by flow cytometry.

  • Lymph can be alternatively collected by cannulating the thoracic duct; however, this requires surgery and specialized equipment, and has not been applied to tumor-draining lymphatics.

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Fig. 1: Protocol overview for lymph collection in tumor-bearing mice.
Fig. 2: Experiment set up.
Fig. 3: Detailed stages of the lymph collection from cisterna chyli.
Fig. 4: Anatomical variation of mouse cisterna chyli.
Fig. 5: Detailed stages of the lymph collection from tumor-draining lymphatic vessel.
Fig. 6: Application and anticipated results obtained after lymph collection.

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Data availability

The authors declare that the main data discussed in this protocol are available in the supporting primary research paper as ‘source data’ (https://doi.org/10.1038/s41586-020-2623-z). All photographic images shown in this manuscript are the original, unaltered photographs. The deidentified metabolomics heat map contains no source data, as it is provided as a representative example of the protocol application.

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Acknowledgements

We thank T. P. Padera for insightful review and suggestions, which greatly improved the manuscript text. This work was supported by funding from the Breast Cancer Alliance Young Investigator Award, NIH/NCI 1R01CA282202, DF/HCC Incubator Award sponsored by the Ludwig Center at Harvard and the Melanoma Research Foundation Career Development Award.

Author information

Authors and Affiliations

  1. Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA, USA

    Marie Sabatier & Jessalyn M. Ubellacker

  2. Animal Resources Center, University of Chicago, Chicago, IL, USA

    Ani Solanki

  3. Legend Biotech, Somerset, NJ, USA

    Sangeetha Thangaswamy

  4. Edwin L. Steele Laboratories, Department of Radiation Oncology, Massachusetts General Hospital Cancer Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

    Pin-ji Lei, Hengbo Zhou, Meghan O’Melia & Lutz Menzel

  5. Breast Surgical Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA

    Samir Mitri

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Contributions

M.S. and J.M.U. conceived the protocol. M.S. developed the lymph collection from cisterna chyli procedure. M.S. and J.M.U. developed the isolation of cancer cells from lymph procedure. A.S. and S.T. provided expertise and technical support for lymph collection. P.L., H.Z., M.O., L.M., and S.M. provided conceptual support and input on the manuscript content. J.M.U. provided anticipated results. M.S. and J.M.U. wrote the manuscript. All authors read and edited the manuscript.

Corresponding author

Correspondence to Jessalyn M. Ubellacker.

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Ethics

All experiments were conducted in compliance with the approved Harvard T.H. Chan School of Public Health IACUC protocol IS00003460, and proper permission was granted for the publication of the included videos and images.

Competing interests

The authors declare no competing interests.

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Nature Protocols thanks Nathan Reticker-Flynn and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference

Ubellacker, J. M. et al. Lymph protects metastasizing melanoma cells from ferroptosis. Nature 585, 113–118 (2020): https://doi.org/10.1038/s41586-020-2623-z

Supplementary information

Reporting Summary

Supplementary Video 1

Lymph collection from the cisterna chyli. The cisterna chyli is dried using a gauze pad and a cotton swab. Then, a glass cannula (attached to the 1 mL syringe/Luer/tubing apparatus, Fig. 2b) is slowly inserted into cisterna chyli. Lymph fluid is collected within the glass cannula by capillary action and gentle aspiration. The collected lymph is transferred into a cold 0.2 mL microcentrifuge tube. The cannula is inserted twice again to recover the entire lymph fluid coming out from the cisterna chyli and the lymph is transferred into the same cold 0.2 mL microcentrifuge tube.

Supplementary Video 2

Visualization of the tumor-draining lymphatics. The focus is made on the cranial section of the subcutaneous B16 melanoma tumor from a C57BL/6J female mouse. The tumor-draining blood vessel that goes from the tumor to the axillary region is identified. The tumor-training lymph vessel is commonly located close to the tumor-draining blood vessel and can be differentiated by is white/translucent color. A snapshot highlighting in yellow both the tumor-draining blood and lymph vessels appears at t = 18 s and is represented in Fig. 5d.

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Sabatier, M., Solanki, A., Thangaswamy, S. et al. Lymphatic collection and cell isolation from mouse models for multiomic profiling. Nat Protoc 20, 884–901 (2025). https://doi.org/10.1038/s41596-024-01081-0

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