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Development of a novel murine model of lymphatic metastasis


Current laboratory models of lymphatic metastasis generally require either genetically modified animals or are technically challenging. Herein, we have developed a robust protocol for the induction of intralymphatic metastasis in wild-type mice with reproducible outcomes. To determine an optimal injection quantity and timeline for tumorigenesis, C57Bl/6 mice were injected directly into the mesenteric lymph duct (MLD) with varying numbers of syngeneic murine colon cancer cells (MC38) or gastric cancer cells (YTN16) expressing GFP/luciferase and monitored over 2–4 weeks. Tumor growth was tracked via whole-animal in vivo bioluminescence imaging (IVIS). Our data indicate that the injection of tumor cells into the MLD is a viable model for lymphatic metastasis as necropsies revealed large tumor burdens and metastasis in regional lymph nodes. This protocol enables a closer study of the role of lymphatics in cancer metastasis and opens a window for the development of novel approaches for treatment of metastatic diseases.

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Dulbecco’s phosphate buffered saline


Inferior vena cava


Left renal vein


Mesenteric lymph duct


Polyvinyl chloride


Superior mesenteric artery


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This work was funded by an initial pilot project grant from the Vanderbilt Ingram Cancer Center/ Vanderbilt Institute for Infection, Inflammation and Immunology (VI4) and by the Office of the Assistant Secretary of Defense for Health Affairs, through the Peer Reviewed Cancer Research Program under award number W81XWH-18-1-0234 (to BF). Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense. BB was also supported by T32 CA106183 (awarded to JRG). The Vanderbilt Translational Pathology Shared Resource is supported by NCI/NIH Cancer Center Support Grant P30 CA068485 and the Vanderbilt Mouse Metabolic Phenotyping Center Grant U24DK059637. The Vanderbilt Center for Small Animal Imaging is supported by NCI/NIH Cancer Center Support Grant P30 CA068485 and S10 OD021804 for the bioluminescence imaging equipment.

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Correspondence to Barbara Fingleton.

Electronic supplementary material

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Supplementary Fig. 1. Example of luminescence signal and region of interest definition. (a) Luminescent signal overlaid on white light photographs of a mouse that had been injected with 3 million GFP + Luciferase + MC38 cells taken at days 1, 5, 8,12,15, 17 and 19 post-injection. (b) The region of interest was defined by the largest area of luminescence detected at any time point and then applied to all images from all time points. The luminescent signal from this region was graphed against time post-injection. (TIF 535 kb)

Supplementary Fig. 2. Evidence of lung and liver metastases in some, but not all, injected mice. (A) in situ lungs displaying numerous metastatic foci (arrows point to two examples).These lungs are from one of the mice shown in Fig. 5. (B) Hematoxylin and eosin-stained histological sections of lungs from two different mice showing metastatic lesions. Both images taken at 2X magnfication. (C) Hematoxylin and eosin-stained histological section of liver from an MLD-injected mouse showing metastatic lesions (4X magnification). (D) Lung section from another MLD-injected mouse showing absence of any metastatic foci (2X magnfication). (TIF 3063 kb)

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Banan, B., Beckstead, J.A., Dunavant, L.E. et al. Development of a novel murine model of lymphatic metastasis. Clin Exp Metastasis (2020).

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  • Mesenteric lymph duct
  • Colon cancer
  • Gastric cancer
  • Luciferase
  • Intralymphatic