Abstract 2717: The role of lymphatic metastasis: Development of a lymphatic cannulation method for cell injection and lymph sampling in mice

The lymphatic network has a critical role in metastasis of certain cancers. Lymph directly originates from the interstitial fluid and thus can give first-hand access to by-products of cellular activity before molecules get diluted in blood. Therefore, the composition of lymphatic fluid provides valuable information about the immunologic, metabolic, and physiological status of tissues. However, due to inherent difficulties associated with studying lymphatic metastasis in animal models, our current understanding of the role of the lymphatics in the metastatic process is limited. Current lymph collection methods tend to have low success rates, yield low lymph volumes and are time-consuming, thus are not generally practical. In our novel approach, the mesenteric lymph duct (MLD) is cannulated by a needled-catheter enabling both the intra-lymphatic injection of cancer cells and also lymph collection with high success rates. To begin to investigate the role of lymphatics, we injected 2 groups of C57Bl/6 mice intra-lymphatically with either murine colon cancer cells or PBS. The lymph was collected 48hrs post-injection. Briefly, the tip of a 30G needle was inserted into a catheter which was introduced into the MLD. The catheter was connected to a micro-suction pump, creating a low negative pressure, that drew lymph into the catheter. The collection procedure took only 35mins per mouse and yielded ~30 μl of pure lymph from each mouse. The lymph samples were analyzed for protein composition by high-resolution mass spectrometry (LC-MS/MS). We identified 302 proteins with two or more unique peptides. Our results revealed exclusive expression of 14 proteins in lymph samples from cancer cell-injected mice, comprising proteins with a well-established link to cancer metastasis/tumor progression, such as tropomyosin alpha-1 chain, insulin-like growth factor-binding protein, and macrophage colony-stimulating factor-1. Biological pathway analysis showed that anti-oxidant pathways and proteins involved in inflammatory processes/immune responses were significantly downregulated in the cancer mice compared to the PBS group. Quantitative profiling of the proteins found in all samples revealed 36 proteins with significantly higher expression levels in the cancer mice. These proteins are involved in multiple processes, including MAPK cascade signaling, apoptosis, and B cell signaling. Our protocol enables both the delivery of cells of interest to the lymphatic system and also the investigation of the effects of injected material by the collection of lymph samples. The data demonstrate that mesenteric lymph serves as a sensitive fluid pool for investigating the role of lymphatics in cancer studies. The findings suggest that lymph analysis will illuminate potential mechanisms regarding the contribution of the lymphatic system to systemic metastasis, and may also suggest biomarkers of metastatic risk. Citation Format: Babak Banan, Jacob A. Beckstead, Lauren E. Dunavant, Barbara Fingleton. The role of lymphatic metastasis: Development of a lymphatic cannulation method for cell injection and lymph sampling in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2717.