Mechanisms of tumor dormancy and awakening in the lymph node

Abstract

Mechanisms of tumor dormancy are being systematically uncovered through the major organs. Yet little is known about lymph nodes (LNs) as harbors of residual disease, despite their well-established prognostic significance for metastasis and direct contribution to metastasis in visceral organs. Further, few mechanisms of awakening from dormancy have been reported. To investigate tumor dormancy in the LN, the niche for dormant disseminated tumor cells (DTCs) was characterized by spatial analysis of LN tissues from a model of spontaneous murine mammary carcinoma dissemination to the lymph node. Ki-67- DTCs were significantly enriched near blood endothelia but not lymphatic endothelia versus a null distribution control, revealing that the lymph node blood vasculature is the primary niche for quiescent DTCs. An organotypic co-culture model of the LN vascular niche was developed to study the mechanisms underlying niche regulation of tumor quiescence. Human breast cancer cell line HMT-3522-T4-2 were growth suppressed in the presence of endothelial cells, recapitulating the effect of the vascular niche in vivo. RNA sequencing revealed that TGFβ signaling was among the most differentially upregulated pathways in the niches containing blood endothelial cells, which was validated through ELISA. Addition of TGF-β1 to tumor growth-permissive LN stromal cultures suppressed tumor cell growth as seen in vascular niches, providing evidence that TGF-β1 promotes DTC quiescence in the LN vascular niche. To investigate tumor awakening in the LN, the stable vascular niche was disrupted through sustained inflammation via bacterial lipopolysaccharide (LPS), which resulted in endothelial remodeling and LN expansion. LPS treatment of mice with LN DTCs resulted in over ten-fold increase in LN micrometastases, supporting the emerging theme that tissue dysregulation and loss of homeostasis triggers metastatic outgrowth. The inflamed stroma was transcriptionally profiled using single-cell RNA sequencing, which revealed that expression of the TGFβ pathway decreased upon inflammation. Knocking down requisite TGFβ receptor 2 in tumor cells enhanced micrometastatic outgrowth within the LN, supporting the hypothesis that blood endothelial-derived TGF-β1 is required for DTC quiescence. These studies reveal that the LN supports DTC dormancy and provide evidence that endothelial TGF-β1 promotes quiescence in the vascular niche. Further, inflammation is sufficient to induce micrometastatic outgrowth in the LN, concomitant with stromal remodeling and loss of endothelial TGFβ expression.