Cell free RNA in cerebral spinal fluid identifies mechanisms of disease progression in leptomeningeal disease
Melanie Hayden Gephart
Introduction: Leptomeningeal brain metastases (LMD) are not biopsied and the number of cancer cells in the cerebral spinal fluid (CSF) are few, severely limiting the tissue samples available to research and develop effective treatments.
Objective: To overcome this critical hurdle, we developed innovative techniques including single cell and cell free gene expression analysis on primary human CSF specimens.
Methods: Through an IRB-approved study protocol we collected CSF specimens from patients with non-small cell lung cancer leptomeningeal disease or normal controls. Single cells or cell free RNA were isolated, sequenced, and analyzed.
Results: We successfully isolated and performed single cell RNA-sequencing on leptomeningeal cells directly from fresh human CSF samples. Prior methods were not from human specimens, nor could be applied to the extremely low numbers of cells present in CSF. We also successfully isolated and sequenced cell free messenger RNA in human CSF. Prior methods required high concentrations of RNA, RNA rapidly degraded, or had not been reliably applied to human CSF. This identified key genes validated in vitro to enable LMD disease progression.
Conclusions: We applied our novel techniques to extract and sequence single tumor cells or tumor-associated cfRNA from patient CSF samples. Gene expression analysis identified LMD-specific survival and migration mechanisms, which we are now validating in an LMD, human-in-mouse, intracerebral, xenograft model. Disruption of LMD-intrinsic mechanisms of disease progression will identify potential therapeutic targets for the prevention and treatment of LMD.