AbstractNon-small cell lung cancer (NSCLC) metastatic to the brain leptomeninges is rapidly fatal, cannot be biopsied, and cancer cells in the cerebrospinal fluid (CSF) are few; therefore, available tissue samples to develop effective treatments are severely limited. This study aimed to converge single-cell RNA-seq and cell-free RNA (cfRNA) analyses to both diagnose NSCLC leptomeningeal metastases (LM), and to use gene expression profiles to understand progression mechanisms of NSCLC in the brain leptomeninges. NSCLC patients with suspected LM underwent withdrawal of CSF via lumbar puncture. Four cytology-positive CSF samples underwent single-cell capture (n = 197 cells) by microfluidic chip. Using robust principal component analyses, NSCLC LM cell gene expression was compared to immune cells. Massively parallel qPCR (9216 simultaneous reactions) on human CSF cfRNA samples compared the relative gene expression of patients with NSCLC LM (n = 14) to non-tumor controls (n = 7). The NSCLC-associated gene, CEACAM6, underwent in vitro validation in NSCLC cell lines for involvement in pathologic behaviors characteristic of LM. NSCLC LM gene expression revealed by single-cell RNA-seq was also reflected in CSF cfRNA of cytology-positive patients. Tumor-associated cfRNA (e.g., CEACAM6, MUC1) was present in NSCLC LM patients’ CSF, but not in controls (CEACAM6 detection sensitivity 88.24% and specificity 100%). Cell migration in NSCLC cell lines was directly proportional to CEACAM6 expression, suggesting a role in disease progression. NSCLC-associated cfRNA is detectable in the CSF of patients with LM, and corresponds to the gene expression profile of NSCLC LM cells. CEACAM6 contributes significantly to NSCLC migration, a hallmark of LM pathophysiology.