Deciphering the Premetastatic Lymphatic Niche of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most prevalent types of malignancies in the oral cavity, with a high incidence of lymph node (LN) metastasis. While previous studies have explored the mechanisms of lymphatic metastasis, little is known about the cellular architecture within the premetastatic niche of LNs. In this study, we established mouse models of premetastatic LNs, which demonstrated an immunosuppressive premetastatic environment in tumor-draining LNs prior to metastasis. We performed single-cell RNA sequencing on LNs from patients with OSCC, including premetastatic tumor-draining LNs and paired contralateral LNs. Our analysis identified a subset of CD4+ T cells that exclusively expressed <i>MIR155HG</i>, characterized by a preexhausted state and active immune exhaustion signaling with myeloid cells (henceforth, CD4+ Tex-pre cells). In silico analyses and in vivo experiments revealed a higher abundance of CD4+ Tex-pre cells in tumor-draining premetastatic LNs when compared with contralateral LNs, with their numbers increasing as LN metastasis progressed. Moreover, adoptive transfer of CD4+ Tex-pre cells aggravated immune suppression in tumor-draining premetastatic LNs and promoted LN metastasis. The presence of CD4+ Tex-pre cells was further validated by integrating external data sets and conducting in situ RNAscope staining. Finally, using bulk RNA sequencing data sets, we found that CD4+ Tex-pre infiltration was associated with lymphatic metastasis and that CD4+ Tex-pre scores could inform treatment decisions for low-grade cases without clinical nodal involvement. Overall, our study provides a comprehensive view of the single-cell landscape in the premetastatic niche of OSCC LNs and highlights the role of CD4+ Tex-pre cells in shaping the premetastatic niche.