Spatio-temporal mapping of immune cell dynamics during human sequential lymph node metastasis 2309999

Abstract Introduction Regional lymph node (LN) metastasis critically influences distant metastatic progression, anti-tumour immunity, and patient prognosis. While tumour-induced immune modulation in tumour-draining LNs (TDLNs) has been extensively studied using murine models, the systematic reconstruction of the immune system from primary tumours through TDLNs and subsequent lymph nodes in human cancer progression remains understudied. Methods Here, we utilised integrated multi-omics approaches, including imaging mass cytometry, single-cell RNA sequencing, Visium and Xenium spatial transcriptomics, and multi-colour immunofluorescence to systematically characterise immune cell dynamics across 147 paired primary tumours, sentinel TDLNs (S-TDLNs), and secondary axillary LNs (ALNs) obtained from 50 treatment-naïve triple-negative breast cancer patients with different progression statuses. Results We observe depletion of cDC2 and naïve T cells, with expansion of immunosuppressive MARCO+ macrophages associated with survival, as identified by a newly developed single-cell transformer model. Spatial analysis and a unified cell—cell interaction framework reveal disrupted immune communication and impaired dendritic cell—T cell priming circuits within metastatic LNs and matched tumours. In an independent neoadjuvant immunotherapy cohort (36 patients, 52 LNs), preservation of CD1c+ cDC2 predicts pathological complete response and prolonged event-free survival, positioning cDC2 as a biomarker and rational therapeutic target in TNBC. Conclusion Collectively, this systematic mapping of immune landscape alterations during human sequential LN metastasis provides essential insights for understanding cancer metastasis mechanisms and paves the way for innovative immunotherapeutic strategies. Funding Source Royal Society Fund (IEC\NSFC\233577); CRUK Career Development Fellowship (RCCFEL\100095); National Natural Science Foundation of China (Grant No. 81672638, W2421095) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)