Abstract PR007: Lymph node colonization promotes distant tumor metastasis through the induction of systemic tumor-specific immunosuppression
Authors: Reticker-Flynn Nathan E. , Zhang Weiruo , Chang Serena , Gentles Andrew J. , Sunwoo John B. , Kong Christina S. , Plevritis Sylvia K. , Engleman Edgar G. 
Published: March 2021

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 anti-tumor immunity, but instead correlates with further disease progression. Here, 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. Using an in vivo passaging approach of a non-metastatic syngeneic melanoma, we generated 300 unique cell lines exhibiting varying degrees of LN metastatic capacity. 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 effect is eliminated in mice lacking an adaptive immune response. Furthermore, this promotion of distant seeding by LN metastases is tumor specific. Using flow cytometry and single-cell sequencing 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 non-metastatic tumors to disseminate to LNs, and ablation of Tregs using FoxP3-DTR mice eliminates the occurrence of lymphatic metastases. The immunosuppressive effects of LN metastases can be transferred to tumor-naïve recipients through specific lymphocyte populations in a manner that promotes distant seeding. Through the use of whole exome sequencing, we show that neither the metastatic proclivity nor immunosuppression evolve through the acquisition of driver mutations, loss of neoantigens, loss of MHC class I presentation, or decreases in melanoma antigen expression. Rather, by RNA-seq and ATAC-seq, we show that a conserved interferon signaling axis is upregulated in LN metastases and is rendered stable through epigenetic regulation of chromatin accessibility. Furthermore, using CRISPR/Cas9, we find that these pathways are required for LN metastatic seeding, and validate their conserved significance in 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. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor-specific immunosuppression.
Citation Format: Nathan E. Reticker-Flynn, Weiruo Zhang, Serena Chang, Andrew J. Gentles, John B. Sunwoo, Christina S. Kong, Sylvia K. Plevritis, Edgar G. Engleman. Lymph node colonization promotes distant tumor metastasis through the induction of systemic tumor-specific immunosuppression [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PR007.