Hypoxia-Enhanced Podoplanin-Defined Tumour Plasticity Drives CCR7-Mediated Lymphatic Metastasis in Breast Cancer

Background: Lymphatic metastasis is strongly associated with poor prognosis. Although the chemokine receptor CCR7 is a well-established promoter of lymphatic dissemination, its prognostic relevance remains weak. We show that tumour cell plasticity, defined by upregulation of podoplanin (PDPN) and promoted by hypoxia, intersects with CCR7 function in triple-negative breast cancer (TNBC). Methods: In vivo and in vitro studies using a CCR7-expressing TNBC mouse model were combined with transcriptomic profiling. Human relevance was assessed using scRNA-seq datasets from cell lines and primary tumours, as well as the METABRIC breast cancer cohorts (n = 1784). Results: A PDPN-defined epithelial–mesenchymal transition (EMT) state—promoted by hypoxia—was required for efficient CCR7-driven lymphatic metastasis and tumour progression. PDPN-expression was linked to elevated tumour cell collagen-expression and suppression of interferon-signalling, features associated with an immune-cold microenvironment. PDPN-expression with effects on interferon and collagen programs were observed across murine and human TNBC/basal-like cell lines. In primary TNBC, PDPN-expression correlated with EMT, collagen, and hypoxia signatures, mirroring murine findings. In METABRIC, high CCR7–PDPN co-expression predicted poor survival in lymph node–positive patients, whereas either marker alone lacked prognostic value. Conclusions: PDPN is a tumour-cell-associated biomarker of plasticity in TNBC, revealing synergy between specific hypoxia-induced EMT-states and CCR7 in promoting lymphatic dissemination and poor prognosis. Overall design: Total RNA was extracted from four different subclones of EO771 mouse mammmary tumor cells (4 technical replicates/subclone), using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Libraries were generated using the TruSeq Stranded mRNA kit (Illumina) and sequenced on an Illumina NextSeq 550 platform in single-end mode, producing 75 bp reads mapped to the mouse reference genome (GRCm38/mm10). Sequencing was run in two batches: the first batch included X2 (EO771 cells expressing vector control), CCR7 (EO771 cells overexpressing CCR7), CCR7-PDPN (CCR7-overexpressing EO771 cells sorted for high expression of PDPN); and the second batch included X2, X2-PDPN (vector control cells sorted for high PDPN expression), and CCR7-PDPN. All the data was analysed using Python (v.3.11.5). Normalization was performed using Transcripts Per Million (TPM) by rnanorm (v.1.5.1). TPM values below 1 were considered as noise.