<p>The study explores the development and characterization of lymph node stromal cell cultures (LNSCs) from patients with oral squamous cell carcinoma (OSCC), highlighting the importance of understanding tumor-node cross-talk for effective prognostic and therapeutic interventions. Herein, we describe the development and characterization of primary lymph node stromal cells (LNSCs, N = 14) from nodes of metastatic and non-metastatic OSCC patients. Primary cultures were established by the explant method from positive (N + ; N = 2), and negative nodes (N0_m; N = 4) of the metastatic patients (N = 3) as well as negative (N0_nm; N = 8) nodes from non-metastatic (N = 4) patients. STR profiling confirmed the purity and novelty, while characterization by immunocytochemistry/flow cytometry revealed heterogeneous cell populations consisting of fibroblastic reticular cells (CD31-Gp38 +) and double negative cells (CD31-Gp38-). Transcriptomic profiling indicated molecular alterations in the cells based on the non-metastatic, the pre-metastatic or metastatic status of the nodes, pro-inflammatory, matrix remodeling, and immune evasion being the primary pathways. Assessment of the protein levels for five selected markers (MX1, ISG15, CPM, ITGB4 and FOS) in the cell lines revealed that CPM levels were significantly reduced in the N + and N0_m nodes whereas ISG15 levels reduced in N0_m. Significantly, the profiling also provided insights into possible glycosylation of CPM (N0_nm) and ISGylation of ISG15 (N0_m). Cytokine profiling indicated release of chemokines/anti-proliferative cytokines from the negative nodes, while angiogenic/pro-metastatic cytokines were released from the nodes of metastatic patients. The lymph node stromal cell models established in the study with distinctive transcriptomic/cytokine characteristics will be invaluable in delineating the processes underlying nodal metastasis.</p>