Neutrophil Extracellular Trap Deposition Establishes an Immunosuppressive Environment in Non-metastatic Lymph Nodes and Facilitates Heterologous Metastatic Outgrowth

Abstract

Metastasis accounts for most cancer-related mortality, and regional lymph nodes (LNs) are among the earliest sites of dissemination for many solid tumours. Yet tumour-draining lymph nodes (TDLNs) are also sites of adaptive immune activation, raising the question of how these immune organs become permissive to metastatic growth. We investigated whether neutrophil extracellular traps (NETs), which accumulate in TDLNs before detectable tumour-cell arrival, contribute to immune conditioning of histologically tumour-negative nodes and whether the consequences of this conditioning extend beyond the initiating tumour. Using multiplex immunofluorescence and spatial transcriptomic profiling of TDLNs from 19 patients with gastroesophageal adenocarcinoma, we found that NET positivity in non-metastatic TDLNs was associated with inferior overall survival and a distinct immunoregulatory state characterized by closer NET-Treg spatial relationships, coordinated increases in Treg-associated transcriptional programs, altered immune composition and redistribution of CD8 T cells away from Tregs. NET-positive nodes also showed reduced CD8 naive/stem-like transcription, while greater peri-Treg CD8 redistribution was associated with lower cytotoxic-effector transcription. In vitro, direct NET exposure shifted human T cells toward regulatory and dysfunction-associated phenotypes and impaired tumour-cell control. NET-conditioned Tregs further suppressed CD8 T cell proliferation and granzyme B expression, while depletion of Tregs attenuated NET-associated CD8 apoptosis. In tumour-bearing mice, neutrophil elastase inhibition altered activated Treg distribution across tumour-draining and contralateral LNs. Importantly, prior H59 tumour conditioning increased susceptibility to a subsequent heterologous MC38 metastatic challenge after removal of the initiating tumour; this phenotype was attenuated by TDLN resection and PAD4 deficiency. Adoptive transfer of contralateral-LN T cells from conditioned donors further identified a transferable component of this metastasis-permissive state that was sensitive to NET-directed intervention. These findings support a model in which NET-associated nodal immune conditioning alters Treg and CD8 T cell organization and function before overt nodal metastasis. The consequences of this conditioning extend beyond the draining node and can shape host susceptibility to a subsequent heterologous tumour challenge, broadening the premetastatic-niche concept toward conditioning of the host immune state.