Abstract 2703: Lymph node colonization promotes distant tumor metastasis through the induction of systemic immune tolerance
Authors: Reticker-Flynn Nathan E. , Martins Maria M. , Basto Pamela A. , Zhang Weiruo , Bejnood Alborz , Gentles Andrew J. , Sunwoo John B. , Plevritis Sylvia K. , Engleman Edgar G. 
Published: August 2019

Abstract
The majority of cancer-associated deaths are the result of distant organ metastasis, an event that is typically preceded by metastasis to regional or distant lymph nodes (LNs). LNs are education hubs of the adaptive immune system wherein antigens derived from pathogens or malignancies are presented to lymphocytes in a manner that facilitates elimination of the threat. Nonetheless, LN metastasis, which is typically attributed to passive drainage of tumor cells through lymphatics, frequently does not lead to the generation of an anti-tumor immune response, but instead correlates with poor prognosis and further disease progression. Here, we find that LN metastasis represents a critical step in tumor progression through the capacity of such metastases to induce systemic immune tolerance in a manner that promotes further dissemination of tumors to distant organs. Through serial in vivo passaging of a syngeneic melanoma in mice, we generate nearly 300 unique cell lines that exhibit an enhanced capacity to metastasize to LNs. Transcriptional profiling of these lines reveals increased expression of immune-related programs. We show that the presence of these LN metastases enables distant organ seeding of metastases in a manner that the parental tumor cannot, and this differential seeding is eliminated in mice that lack an adaptive immune response. To query the effects of the LN metastases on the systemic immune response, we perform organism-wide immune profiling by mass cytometry and identify a number of cellular mediators of tolerance. In particular, we find that LN metastases have the capacity to both resist NK cell cytotoxicity and induce regulatory T cells (Tregs) in vitro. Furthermore, depletion of NK cells in vivo enables non-metastatic tumors to disseminate to LNs, and ablation of Tregs using FoxP3-DTR mice eliminates the occurrence of lymphatic metastases. We further identify an interferon signaling axis that is constitutively activated within the LN metastases in the absence of exogenous interferon signaling. Through the use of ATAC-seq, we find that this program is conferred through epigenetic regulation of chromatin accessibility. Knockout of key interferon-induced genes using CRISPR/Cas9 in the LN-metastatic cells reveals that this program is required for enhanced LN metastatic seeding in vivo, and their overexpression increases LN metastasis of the non-metastatic cells. Using additional mouse models of pancreatic ductal adenocarcinoma and head and neck squamous cell carcinoma (HNSCC), we show that these findings are conserved across multiple malignancies. Additionally, we perform RNA-seq on sorted malignant populations from node-positive and node-negative HNSCC patients and confirm that these differences in transcriptional profiles extend to the human disease. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor immune tolerance.
Citation Format: Nathan E. Reticker-Flynn, Maria M. Martins, Pamela A. Basto, Weiruo Zhang, Alborz Bejnood, Andrew J. Gentles, John B. Sunwoo, Sylvia K. Plevritis, Edgar G. Engleman. Lymph node colonization promotes distant tumor metastasis through the induction of systemic immune tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2703.