Immune Landscape of Intrathoracic Lymph Nodes with Metastasis in Primary Lung Malignancy using EBUS-TBNA samples

Malignant cells modulate immune cell phenotype and function to generate a favorable microenvironment for tumor survival and progression. The draining lymph node (LN) is the most frequent and often first site of cancer metastasis. Notwithstanding, our understanding of how cancer, metastatic to LNs, alters the immune cells therein remains limited. Although LN biopsy is frequently performed as a standard practice in cancer diagnosis and staging, immunologic studies employing these samples are rare. The present study investigates the immune cell alterations in metastatic intrathoracic LNs. With the advent of single-cell technologies, we comprehensively analyzed immune cells in a series of bronchoscopic LN biopsies from human subjects undergoing diagnostic workup of cancer. Using single-cell RNA sequencing and mass cytometry analyses, we compared intrathoracic LN samples from 18 subjects with pathologically confirmed metastasis and four controls without evidence of metastasis. We found that immune cell composition and gene expression patterns differed markedly between metastatic and control LNs. In particular, metastatic LNs contained relatively more neutrophils and APOE-high myeloid cells, with the latter exhibiting significant transcriptional derangement and a powerful intercellular interaction signature. Additionally, CD8 T cells in metastatic LNs demonstrated a unique exhausted phenotype. Notably, our findings are corroborated in a publicly available lung adenocarcinoma cohort derived from surgical LN biopsies. Immune cell phenotypes and gene expression patterns from bronchoscopic LN biopsies can be leveraged to advance understanding of cancer immunology and may have independent diagnostic value when malignant cells fail to be identified on histopathology.