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. Overall design: This study was conducted in accordance with the Declaration of Helsinki. Study approval was granted by the institutional review boards of the Jesse Brown VA Medical Center and the University of Illinois at Chicago (UIC) Hospital and Health Sciences System. Briefly, subjects aged 18 years or older who were planned to undergo standard-of-care diagnostic bronchoscopy with EBUS to evaluate radiological abnormalities were screened for study enrollment. Subjects were included in the study upon providing informed consent if they did not meet exclusion criteria, which included the use of systemic corticosteroids or other immunosuppressants and/or a history of respiratory tract infections within 8 or 4 weeks prior to the diagnostic procedure, respectively. Study subjects were retrospectively divided into metastatic, or non-metastatic (control) groups based on histopathologic presence or absence of cancerous cells within sampled intrathoracic LNs. Since all subjects underwent clinically indicated EBUS sampling, additional criteria were established for inclusion into the control group. These included either no evidence of LN disease and no primary lung malignancy with at least six months of follow-up or confirmed negative tumor involvement from surgical LN dissection with no evidence of recurrence for at least one year of follow-up.