Tumor draining lymph nodes connected to cold triple-negative breast cancers are characterized by Th2-associated...

Tumor draining lymph nodes (TDLN) represent a key component of the tumor-immunity cycle. There are few studies describing how TDLNs impact lymphocyte infiltration into tumors. Here we directly compare tumor-free TDLNs draining "cold" and "hot" human triple negative breast cancers (TDLN_Cold and TDLN_Hot). Using machine-learning-based self-correlation analysis of immune gene expression, we find unbalanced intranodal regulations within TDLN_Cold. Two gene pairs (TBX21/GATA3-CXCR1) with opposite correlations suggest preferential priming of T helper 2 (Th2) cells by mature dendritic cells (DC) within TDLN_Cold. This is validated by multiplex immunofluorescent staining, identifying more mature-DC-Th2 spatial clusters within TDLN_Cold versus TDLN_Hot. Associated with this Th2 priming preference, more IL4 producing mast cells (MC) are found within sinus regions of TDLN_Cold. Downstream, Th2-associated fibrotic TME is found in paired cold tumors with increased Th2/T-helper-1-cell (Th1) ratio, upregulated fibrosis growth factors, and stromal enrichment of cancer associated fibroblasts. These findings are further confirmed in a validation cohort and public genomic data. Our results reveal a potential role of IL4⁺ MCs within TDLNs, associated with Th2 polarization and reduced immune infiltration into tumors.