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

Zhao, Q.; Lu, Y.; Shi, Z.; Zhang, H.; Li, C. S.; Zhao, R.; Ling, Y.; Gao, Y.; Zhang, Z.; Sun, X.; Qian, Y.; Wang, X.; Wang, C.; Cong, B.; Ni, X.; Liu, Y.; Zhao, M.; Wang, Y.; Mahata, B.; Qiu, P.

doi:10.64898/2026.03.17.712495

bioRxiv · 10.64898/2026.03.17.712495

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

Zhao, Q.·Lu, Y.·Shi, Z.·Zhang, H.·Li, C. S.·Zhao, R.·Ling, Y.·Gao, Y.·Zhang, Z.·Sun, X.·Qian, Y.·Wang, X.·Wang, C.·Cong, B.·Ni, X.·Liu, Y.·Zhao, M.·Wang, Y.·Mahata, B.·Qiu, P.

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Abstract

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. 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-naive triple-negative breast cancer patients with different progression statuses. Our comprehensive profiling revealed critical immune alterations, such as decreased type-2 conventional dendritic cells (cDC2), naive T cells, and B cells, along with an increase in immunosuppressive macrophages. Developing a novel single-cell transformer model, we identified substantial alterations in various immune cell populations, notably MARCO+ macrophages, which strongly correlated with breast cancer patient survival outcomes. Spatial analysis combined with our newly integrated cell-cell interaction platform revealed diminished immune cell communication and impaired priming interactions among dendritic cells, B cells, and T cells within metastatic lymph nodes and primary tumour sites. In an independent neoadjuvant immunotherapy cohort of 36 TNBC patients with 52 lymph node samples, we found preservation of CD1c cDC2 in lymph nodes predicted pathological complete response and longer event-free survival, highlighting cDC2 as a potential biomarker and therapeutic target. 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.

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Zhao, Q., Lu, Y., Shi, Z., Zhang, H., Li, C. S., Zhao, R., Ling, Y., Gao, Y., Zhang, Z., Sun, X., Qian, Y., Wang, X., Wang, C., Cong, B., Ni, X., Liu, Y., Zhao, M., Wang, Y., Mahata, B., Qiu, P.. 2026-03-19. Spatio-temporal mapping of immune cell dynamics during human sequential lymph node metastasis. https://doi.org/10.64898/2026.03.17.712495

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