Abstract 3022: Lymph node metastasis in solid tumors: A marker or driver of disease progression
Authors: Pereira Ethel R. , Kedrin Dmitriy , Seano Giorgio , Gautier Olivia , Meijer Eelco F. , Jones Dennis , Chin Shan-Min , Kitahara Shuji , Bouta Echoe M. , Chang Jonathan , Beech Elizabeth , Jeong Han-Sin , Carroll Michael C. , Taghian Alphonse G. , Padera Timothy P. 
Published: August 2018

Abstract
Background: The presence of lymph node metastasis in patients with solid tumors is associated with tumor aggressiveness, poorer prognosis and the recommendation for systemic therapy. However, whether tumor cells exit the lymph node and contribute to distant metastases remains controversial. Methods: In this study, we used syngeneic murine cell lines representing breast and melanoma cancer that spontaneously metastasize to the lymph node. We engineered these cells to express Dendra2, a photoconvertable protein. Dendra2 is a green-fluorescent protein that can be converted to emit red light by exposure to 405nm light. Once tumor cells spontaneously metastasized to the lymph node from the primary site, a 405nm laser diode was used on 5 consecutive days to convert Dendra2-cancer cells from green to red fluorescence, restricting the light exposure only to the metastatic lymph node. This technology allowed us to specifically trace the fate of cancer cells in the lymph node and beyond to other organs. Results: We show that spontaneous lymph node metastasis from breast cancer and melanoma mouse models can leave the lymph node and enter the blood circulation. We identified micrometastatic disease in the lung that originated from the lymph node in both models. We hypothesized that cancer cells escape the lymph node by directly invading lymph node blood vessels, as opposed to draining through the efferent lymphatic vessel. Immunohistochemical analysis of metastatic lymph nodes revealed isolated cancer cells in close association with CD31+ blood vessels, within high endothelial venules (HEVs) and breaching the vascular basement membrane. Quantitative analysis showed that 23±2% of isolated cancer cells were within 5μm of a blood vessel, compared to only 11±1% using a predictive model of randomly distributed cells in the lymph node (p&lt;0.05). Further, 6±2% of the cancer cells were inside blood vessels defined as cells within the lumen of blood vessels and having cell centroids more than 3μm from the blood vessel endothelium. To further confirm that metastatic cancer cells in a lymph node have affinity for lymph node blood vessels, we used multiphoton intravital microscopy to measure cancer cell migration in an optical lymph node window in mice. Dendra2 expressing metastatic cancer cells are first seen in the subcapsular sinus of the lymph node and later invade the cortex of the lymph node where they accumulate around rhodamine-dextran labeled blood vessels. Cancer cells can be observed in directed migration toward blood vessels as well as moving inside blood vessels. Finally, we analyzed lymph nodes with metastatic lesions from patients with head and neck cancer and identified cancer cells that were closely associated with and inside blood vessels. Conclusions: Together, our data show for the first time that in spontaneous breast and melanoma mouse models, tumor cells in the lymph node can invade blood vessels, exit the node and colonize the lung.
Citation Format: Ethel R. Pereira, Dmitriy Kedrin, Giorgio Seano, Olivia Gautier, Eelco F. Meijer, Dennis Jones, Shan-Min Chin, Shuji Kitahara, Echoe M. Bouta, Jonathan Chang, Elizabeth Beech, Han-Sin Jeong, Michael C. Carroll, Alphonse G. Taghian, Timothy P. Padera. Lymph node metastasis in solid tumors: A marker or driver of disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3022.