Abstract PR04: Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immunosuppression
Authors: Reticker-Flynn Nathan E. , Basto Pamela A. , Zhang Weiruo , Bejnood Alborz , Kenkel Justin A. , Martins Maria M. , Chang Serena , Gentles Andrew J. , Sunwoo John B. , Plevritis Sylvia K. , Engleman Edgar G. 
Published: June 2020

Abstract
The majority of cancer-associated deaths result from distant organ metastasis rather than the primary tumor, yet the mechanisms that enable this process remain poorly understood. For most solid tumors, colonization of regional or distant lymph nodes (LNs) typically precedes the formation of distant organ metastases, yet it remains unclear whether LN metastasis plays a functional role in disease progression. LNs are education hubs of the adaptive immune system wherein antigens derived from pathogens or malignancies are presented to lymphocytes to elicit an adaptive immune response. Nonetheless, LN metastasis, which is typically attributed to passive drainage of tumor cells through lymphatics, frequently does not lead to the generation of an antitumor immune response but instead correlates with further disease progression. Here, using a novel mouse model, we find that LN metastasis represents a critical step in tumor progression through the capacity of such metastases to induce tumor-specific immunosuppression in a manner that promotes further dissemination of tumors to distant organs. We use an in vivo passaging approach of a nonmetastatic syngeneic melanoma to generate 300 unique cell lines of known phylogenetic relationships that exhibit varying degrees of LN metastatic capacity. Transcriptional profiling of the lines reveals a conserved enrichment for immune-related programs and constitutive activation of an interferon signaling axis, and using ATAC-seq, we find that these programs are rendered stable through alterations in chromatin accessibility. 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 lacking an adaptive immune response. Using mass cytometry to perform organism-wide immune profiling, we identify multiple 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 nonmetastatic tumors to disseminate to LNs, and ablation of Tregs using FoxP3-DTR mice eliminates the occurrence of lymphatic metastases. Through the use of whole-exome sequencing, we show that neither the metastatic proclivity nor immunosuppression evolves through the acquisition of driver mutations, loss of neoantigens, loss of MHC class I presentation, or decreases in melanoma antigen expression. Rather, using CRISPR/Cas9, we find that key interferon-induced molecules are required for LN metastatic seeding, and using additional mouse models of pancreatic ductal adenocarcinoma and head and neck squamous cell carcinoma (HNSCC), along with RNA-seq analysis of malignant populations sorted from HNSCC patients, we find that these effects are conserved across multiple malignancies and in humans. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor immune tolerance.
This abstract is also being presented as Poster B25.
Citation Format: Nathan E. Reticker-Flynn, Pamela A. Basto, Weiruo Zhang, Alborz Bejnood, Justin A. Kenkel, Maria M. Martins, Serena Chang, Andrew J. Gentles, John B. Sunwoo, Sylvia K. Plevritis, Edgar G. Engleman. Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immunosuppression [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR04.