Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Spontaneous and experimental models of lymph node metastasis

Nature Protocols volume 20, pages 3170–3187 (2025)Cite this article

Subjects

Abstract

Lymph node (LN) metastasis is a conserved feature across most solid organ malignancies and portends worse prognoses. Functionally, LN metastases induce systemic tumor-specific immune tolerance and may serve as a reservoir for distant metastases. Nonetheless, there are relatively few preclinical models for interrogating the biology of LN metastasis and its systemic effects at various stages of metastatic progression. We describe a method for modeling LN metastasis of melanoma tumors in mice that enables assessment of tumor and immune cell phenotypes and the functional roles of nodal involvement on distant metastasis. Our model comprises a family of transplantable syngeneic melanoma tumor cell lines evolved to exhibit enhanced LN metastatic potential, which can be used to probe cancer–immune interactions and test new therapeutics. We present both (i) a spontaneous LN metastasis model involving primary tumor implantation and assessment of LN colonization 21–28 d later and (ii) an experimental metastasis model involving implantation of primary tumors followed by direct intra-LN injections of tumor cells. Both models can be extended to assess the impact of LN metastasis on the development of distant metastases through asynchronous intravenous injections of tumors. Finally, we discuss experimental design considerations including when to use spontaneous or experimental models and troubleshooting consistent LN metastasis, making this model accessible for researchers with basic mouse survival-surgery skills. We highlight how LN metastasis models can be used to profile metastatic immune reprogramming, measure the impact of nodal metastases on distant metastases and assess novel anti-metastatic therapeutics.

Key points

  • The procedure covers two models of LN metastasis. The spontaneous model recapitulates the metastatic cascade and can be used for mechanistic studies of the metastatic processes via the subcutaneous implantation of LN metastatic cells in mice.

  • The experimental model of LN metastasis is instead preferred for studying systemic alterations in the host during the metastatic process via the administration of a subcutaneous parental non-metastatic tumor followed by intra-LN implantation of metastatic cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Development of a syngeneic mouse model of LN metastasis.
Fig. 2: Schematic of LN metastasis models.
Fig. 3: Step-by-step description of subcutaneous and intra-LN tumor implant procedure.
Fig. 4: Acquisition of ISG signatures in LN metastatic cell lines.
Fig. 5: Characterization of the immune phenotypes of spontaneous and experimental LN metastases.
Fig. 6: Evaluation of distant metastasis formation and anti-metastasis therapies.

Similar content being viewed by others

Data availability

Cell line bulk RNA-seq data have been deposited at the Gene Expression Omnibus with accession code GSE117529.

References

  1. Gerstberger, S., Jiang, Q. & Ganesh, K. Metastasis. Cell 186, 1564–1579 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Amin, M. B. et al. The Eighth Edition AJCC Cancer Staging Manual: continuing to build a bridge from a population‐based to a more “personalized” approach to cancer staging. CA Cancer J. Clin. 67, 93–99 (2017).

    PubMed  Google Scholar 

  3. Leong, S. P. L. et al. Impact of nodal status and tumor burden in sentinel lymph nodes on the clinical outcomes of cancer patients. J. Surg. Oncol. 103, 518–530 (2011).

    Article  PubMed  Google Scholar 

  4. von Andrian, U. H. & Mempel, T. R. Homing and cellular traffic in lymph nodes. Nat. Rev. Immunol. 3, 867–878 (2003).

    Article  Google Scholar 

  5. Qi, H., Kastenmüller, W. & Germain, R. N. Spatiotemporal basis of innate and adaptive immunity in secondary lymphoid tissue. Annu. Rev. Cell Dev. Biol. 30, 1–27 (2014).

    Article  Google Scholar 

  6. Spitzer, M. H. et al. Systemic immunity is required for effective cancer immunotherapy. Cell 168, 487–502.e15 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Prokhnevska, N. et al. CD8+ T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor. Immunity 56, 107–124.e5 (2023).

    Article  CAS  PubMed  Google Scholar 

  8. Connolly, K. A. et al. A reservoir of stem-like CD8+ T cells in the tumor-draining lymph node preserves the ongoing anti-tumor immune response. Sci. Immunol. 6, eabg7836 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Fransen, M. F. et al. Tumor-draining lymph nodes are pivotal in PD-1/PD-L1 checkpoint therapy. JCI Insight 3, e124507 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Dammeijer, F. et al. The PD-1/PD-L1-checkpoint restrains T cell immunity in tumor-draining lymph nodes. Cancer Cell 38, 685–700.e8 (2020).

    Article  CAS  PubMed  Google Scholar 

  11. Huang, Q. et al. The primordial differentiation of tumor-specific memory CD8+ T cells as bona fide responders to PD-1/PD-L1 blockade in draining lymph nodes. Cell 185, 4049–4066.e25 (2022).

    Article  CAS  PubMed  Google Scholar 

  12. Francia, G., Cruz-Munoz, W., Man, S., Xu, P. & Kerbel, R. S. Mouse models of advanced spontaneous metastasis for experimental therapeutics. Nat. Rev. Cancer 11, 135–141 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hebert, J. D., Neal, J. W. & Winslow, M. M. Dissecting metastasis using preclinical models and methods. Nat. Rev. Cancer 23, 391–407 (2023).

    Article  CAS  PubMed  Google Scholar 

  14. Speak, A. O. et al. A high-throughput in vivo screening method in the mouse for identifying regulators of metastatic colonization. Nat. Protoc. 12, 2465–2477 (2017).

    Article  CAS  PubMed  Google Scholar 

  15. Gupta, G. P. & Massagué, J. Cancer metastasis: building a framework. Cell 127, 679–695 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Pereira, E. R. et al. Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice. Science 359, 1403–1407 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Aslakson, C. J. & Miller, F. R. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res. 52, 1399–1405 (1992).

    CAS  PubMed  Google Scholar 

  18. Green, E. L. (ed.) Handbook of Genetically Standardized JAX Mice (The Jackson Laboratory, 1962).

  19. Bertalanffy, F. D. & McAskill, C. Rate of cell division of malignant mouse melanoma B16. J. Natl Cancer Inst. 32, 535–545 (1964).

    CAS  PubMed  Google Scholar 

  20. Fidler, I. J. Selection of successive tumour lines for metastasis. Nat. N. Biol. 242, 148–149 (1973).

    Article  CAS  Google Scholar 

  21. Fidler, I. J. Biological behavior of malignant melanoma cells correlated to their survival in vivo. Cancer Res. 35, 218–224 (1975).

    CAS  PubMed  Google Scholar 

  22. Gengenbacher, N., Singhal, M. & Augustin, H. G. Preclinical mouse solid tumour models: status quo, challenges and perspectives. Nat. Rev. Cancer 17, 751–765 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Westcott, P. M. K. et al. The mutational landscapes of genetic and chemical models of Kras-driven lung cancer. Nature 517, 489–492 (2015).

    Article  CAS  PubMed  Google Scholar 

  24. McFadden, D. G. et al. Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma. Proc. Natl Acad. Sci. USA 113, E6409–E6417 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Reticker-Flynn, N. E. et al. Lymph node colonization induces tumor-immune tolerance to promote distant metastasis. Cell 185, 1924–1942.e23 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zhang, W. et al. Identification of cell types in multiplexed in situ images by combining protein expression and spatial information using CELESTA. Nat. Methods 19, 759–769 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Valastyan, S. & Weinberg, R. A. Tumor metastasis: molecular insights and evolving paradigms. Cell 147, 275–292 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Clark, E. A., Golub, T. R., Lander, E. S. & Hynes, R. O. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 406, 532–535 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Kang, Y. et al. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 3, 537–549 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Bos, P. D. et al. Genes that mediate breast cancer metastasis to the brain. Nature 459, 1005–1009 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Minn, A. J. et al. Genes that mediate breast cancer metastasis to lung. Nature 436, 518–524 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Nguyen, D. X., Bos, P. D. & Massagué, J. Metastasis: from dissemination to organ-specific colonization. Nat. Rev. Cancer 9, 274–284 (2009).

    Article  CAS  PubMed  Google Scholar 

  33. Tamada, Y., Aoki, D., Nozawa, S. & Irimura, T. Model for paraaortic lymph node metastasis produced by orthotopic implantation of ovarian carcinoma cells in athymic nude mice. Eur. J. Cancer 40, 158–163 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Takahashi, K. et al. Development of a mouse model for lymph node metastasis with endometrial cancer. Cancer Sci. 102, 2272–2277 (2011).

    Article  CAS  PubMed  Google Scholar 

  35. Lund, A. W. et al. VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics. Cell Rep. 1, 191–199 (2012).

    Article  CAS  PubMed  Google Scholar 

  36. Carr, I. & Carr, J. in Tumor Invasion and Metastasis. Developments in Oncology Vol. 7 (eds. Liotta, L. A. & Hart, I. R.) 189–205 (Springer, 1982).

  37. Li, L., Mori, S., Sakamoto, M., Takahashi, S. & Kodama, T. Mouse model of lymph node metastasis via afferent lymphatic vessels for development of imaging modalities. PLoS ONE 8, e55797 (2013).

  38. Pulaski, B. A. & Ostrand‐Rosenberg, S. Mouse 4T1 breast tumor model. Curr. Protoc. Immunol. 39, 20.2.1–20.2.16 (2000).

    Google Scholar 

  39. Banan, B. et al. Development of a novel murine model of lymphatic metastasis. Clin. Exp. Metastasis 37, 247–255 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Zawieja, D. C. et al. Lymphatic cannulation for lymph sampling and molecular delivery. J. Immunol. 203, 2339–2350 (2019).

    Article  CAS  PubMed  Google Scholar 

  41. Brown, M. et al. Lymph node blood vessels provide exit routes for metastatic tumor cell dissemination in mice. Science 359, 1408–1411 (2018).

    Article  CAS  PubMed  Google Scholar 

  42. Ubellacker, J. M. et al. Lymph protects metastasizing melanoma cells from ferroptosis. Nature 585, 113–118 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Smith, A. J., Clutton, R. E., Lilley, E., Hansen, K. E. A. & Brattelid, T. PREPARE: guidelines for planning animal research and testing. Lab. Anim. 52, 135–141 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Vleeschauwer, S. I. D. et al. OBSERVE: guidelines for the refinement of rodent cancer models. Nat. Protoc. 19, 2571–2596 (2024).

    Article  PubMed  Google Scholar 

  45. Sert, N. Pdu et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. PLoS Biol. 18, e3000410 (2020).

    Article  Google Scholar 

  46. Liu, S., Shen, M., Le, K., Hartono, A. B. & Stoyanova, T. Protocol for establishing spontaneous metastasis in mice using a subcutaneous tumor model. STAR Protoc. 5, 103239 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Duyverman, A. M. M. J., Steller, E. J. A., Fukumura, D., Jain, R. K. & Duda, D. G. Studying primary tumor–associated fibroblast involvement in cancer metastasis in mice. Nat. Protoc. 7, 756–762 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. McLane, L. M., Abdel-Hakeem, M. S. & Wherry, E. J. CD8 T cell exhaustion during chronic viral infection and cancer. Annu. Rev. Immunol. 37, 1–39 (2015).

    Google Scholar 

Download references

Acknowledgements

This work was supported by NIH grant DP2 AI177915 (to N.E.R.-F.), ARPA-H contract 1AY1AX000003 (to N.E.R.-F.). K.C.G. is an Investigator of the Howard Hughes Medical Institute. C.B.B. was supported by an Arc Institute Fellowship (Breuer). G.E.R. was supported by an NSF Graduate Research Fellowship and a Stanford Graduate Fellowship in Science & Engineering. Cell sorting/flow cytometry analysis for this project was performed on instruments in the Stanford Shared FACS Facility.

Author information

Authors and Affiliations

  1. Immunology Program, Stanford University, Stanford, CA, USA

    Cort B. Breuer, Grayson E. Rodriguez & Gita C. Abhiraman

  2. Department of Otolaryngology – Head & Neck Surgery, Stanford University, Stanford, CA, USA

    Cort B. Breuer, Zhewen Xiong, Alice Wang & Nathan E. Reticker-Flynn

  3. Stanford Cancer Institute, Stanford University, Stanford, CA, USA

    Cort B. Breuer & Nathan E. Reticker-Flynn

  4. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA

    Grayson E. Rodriguez, Gita C. Abhiraman & K. Christopher Garcia

  5. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA

    K. Christopher Garcia

  6. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA

    K. Christopher Garcia

Authors
  1. Cort B. Breuer
    View author publications

    Search author on:PubMed Google Scholar

  2. Zhewen Xiong
    View author publications

    Search author on:PubMed Google Scholar

  3. Alice Wang
    View author publications

    Search author on:PubMed Google Scholar

  4. Grayson E. Rodriguez
    View author publications

    Search author on:PubMed Google Scholar

  5. Gita C. Abhiraman
    View author publications

    Search author on:PubMed Google Scholar

  6. K. Christopher Garcia
    View author publications

    Search author on:PubMed Google Scholar

  7. Nathan E. Reticker-Flynn
    View author publications

    Search author on:PubMed Google Scholar

Contributions

C.B.B. and N.E.R.-F. conceived the study and wrote the manuscript. All authors reviewed and edited the manuscript. N.E.R.-F. derived the cell lines and established the protocol. C.B.B. refined the protocol and performed all experiments for anticipated results. Z.X. and A.W. assisted with flow cytometry experiments. G.E.R. and G.C.A. assisted with therapeutic testing of anticipated results. K.C.G. and N.E.R.-F. supervised the study.

Corresponding author

Correspondence to Nathan E. Reticker-Flynn.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Key reference

Reticker-Flynn, N. E. et al. Cell 185, 1924–1942.e23 (2022): https://doi.org/10.1016/j.cell.2022.04.019

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Breuer, C.B., Xiong, Z., Wang, A. et al. Spontaneous and experimental models of lymph node metastasis. Nat Protoc 20, 3170–3187 (2025). https://doi.org/10.1038/s41596-025-01200-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41596-025-01200-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer