Abstract PR002: Lymph node colonization reprograms lymphocyte responses to generate systemic tolerance and promote distant metastasis
Authors: Breuer Cort B. , Gutierrez Norma A. , Xiong Zhewen , Kirane Amanda R. , Sunwoo John B. , Reticker-Flynn Nathan E. 
Published: November 2024

Abstract
The majority of cancer-associated deaths result from distant organ metastasis, yet the mechanisms that enable this process remain poorly understood. Colonization of locoregional or distant lymph nodes (LNs) typically precedes the formation of distant organ metastases, yet it has been unclear whether LN metastasis plays a functional role in disease progression. LNs are major sites of anti-tumor lymphocyte education, including in the context of immunotherapy, yet LN metastasis frequently correlates with further disease progression. By leveraging our mouse models of LN metastasis, we find that LN colonization represents a critical step in tumor progression by inducing tumor-specific immune tolerance in a manner that promotes further dissemination of tumors to distant organs. This promotion of distant seeding by LN metastases is reliant upon adaptive immunity and is tumor specific. Using flow cytometry and single-cell sequencing to perform comprehensive immune profiling, we identify multiple cellular mediators of tolerance. LN metastases evade NK cells, induce shifts in DC and B cell populations, and promote CD8 T cell exhaustion, but of particular importance, we find that regulatory T cells (Tregs) are enriched in the LNs of mice and patients with LN metastases. LN metastatic cells exhibit an enhanced capacity to induce Treg differentiation from naïve CD4 T cells, and ablation of Tregs using FoxP3-DTR mice eliminates the ability of LN metastases to promote distant metastasis. Furthermore, adoptive transfer of Tregs from the LNs of mice bearing LN metastases to naïve mice facilitates lung metastasis in a manner that Tregs from mice without LN metastases cannot. These Tregs are induced in a tumor antigen-specific manner and TCR recognition of tumors is required for the metastasis-promoting tolerance. Using photoconvertible mice, we track the trafficking dynamics of Tregs from tumor involved LNs and demonstrate their elevated trafficking to sites of distant metastasis prior to the formation of detectable metastases in those sites. Through bulk and single-cell TCR sequencing of the Treg repertoire we reveal how LN colonization alters the clonal architecture of the Treg compartment and demonstrate how the evolution of metastatic traits coincides with shifts in the Treg repertoire. Parallel analyses in patients with melanoma and head and neck cancer reveal similar CD4 T cell dynamics in the context of LN involvement. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor-specific immune tolerance and metastatic progression.
Citation Format: Cort B. Breuer, Norma A. Gutierrez, Zhewen Xiong, Amanda R. Kirane, John B. Sunwoo, Nathan E. Reticker-Flynn. Lymph node colonization reprograms lymphocyte responses to generate systemic tolerance and promote distant metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR002.