Identification of the lymphangioleiomyomatosis cell and its uterine origin

Lymphangioleiomyomatosis (LAM) is a metastasizing neoplasm of reproductive age women which causes cystic lung remodeling and progressive respiratory failure. While LAM lesions are known to contain abnormal smooth muscle-like cells which harbor mTOR activating mutations in TSC1 or TSC2, the tissue origins of the mutant “LAM cells” that invade the lung remain unclear. By employing single cell and single nuclear RNA sequencing on explanted LAM lungs, we identified a unique population of cells and associated signature genes and gene networks which were readily distinguished from those of endogenous lung cells. These unique LAMCORE cells shared closest transcriptomic similarity to normal uterus and share transcriptomic features with neural crest, as identified in uterine LAM lesions by single nuclear RNA-seq. Immunofluorescence microscopy demonstrated the expression of LAMCORE cell signature genes within LAM lesions in both lung and uterus. Serum aptamer proteomics and ELISA identified biomarkers consistent with the signature genes expressed and predicted to be secreted by LAMCORE cells. Single cell transcriptomics strongly supports a uterine neural crest origin of LAMCORE cells; providing insights into disease pathogenesis and informing future treatment strategies for LAM. scRNA-seq and snRNA-seq was performed using the 10x Chromium platform on four lung explants from LAM patients undergoing lung transplantation. We also performed scRNA-seq on a renal AML resected from a patient with a sporadic AML (S-AML), and snRNA-seq on uterine tissue obtained at hysterectomy from an S-LAM patient. As controls, we conducted scRNA-seq on a 31 years old female explanted lung, brain dead, beating-heart, organ donor and snRNA-seq on one normal uterine tissue at hysterectomy from a 29 years old female patient with cervical cancer, and obtained scRNA-seq data of six additional female donor lungs from Gene Expression Omnibus (GSE122960) and scRNA-seq data from normal mouse uterus (GSE118180). qRT-PCR, immunofluorescence and immunochemical stains to validate the spatial relationships of LAM cells and LAMCORE signature genes.Serum aptamer proteomics and ELISA assays screening were used for validation of secretome predictions.