RNA-seq of lungs from SCID mice pre-treated with EVs from Tsc1-null or wild-type E15.5 neural progenitors prior to ELT3 cell injection

Pulmonary lymphangioleiomyomatosis (LAM) is a rare metastatic sarcoma characterized by the abnormal proliferation and dissemination of TSC1/TSC2-null smooth muscle-like cells. While extracellular vesicles (EVs) are known to regulate cancer metastasis, their roles in LAM have not been fully elucidated. Here, we identify distinct LAM-EV subtypes derived from primary tumor or metastasizing LAM cells that promote metastasis via ITGα6/β1–c-Src–FAK signaling. These EVs enhance LAM cell migration, invasion, and stemness by either shuttling ATP synthesis to cell pseudopodia or activating integrin adhesion complexes. In vivo, CD9⁺CD63⁺CD81⁺ EVs isolated from Tsc1-null or wild-type E15.5 mouse embryonic neural progenitors were labeled and injected intravenously into SCID mice, followed 48 hours later by intravenous injection of 0.5×10⁶ ELT3 cells, a well-characterized LAM model. Lungs were harvested 72 hours post-injection for RNA-seq analyses. Lungs from Tsc1-null EV-treated mice showed increased metastatic burden (rat DNA content) and differential expression of 815 genes (522 upregulated, 293 downregulated) compared to wild-type EV-injected mice. Reactome analysis revealed enrichment in extracellular matrix remodeling, collagen biosynthesis, and the assembly of multimeric structures. These findings establish EVs as key mediators of LAM metastasis and identify them as potential therapeutic targets. To investigate the role of extracellular vesicles (EVs) in pulmonary lymphangioleiomyomatosis (LAM) metastasis, we performed RNA sequencing of lung tissues from SCID mice treated with EVs derived from Tsc1-null or wild-type neural progenitor cells. CD9⁺CD63⁺CD81⁺ EVs were isolated from E15.5 mouse embryonic neural progenitors lacking Tsc1 or from wild-type controls. EVs were fluorescently labeled and injected into the tail vein of female SCID mice. After 48 hours, mice were intravenously injected with 0.5 × 10⁶ rat ELT3 cells, a well-characterized LAM model. Lung tissues were collected 72 hours after ELT3 cell injection for RNA isolation and sequencing. The experiment was designed to assess EV subtype-dependent modulation of the lung microenvironment and its contribution to the formation of a metastatic niche. RNA-seq was performed on lung tissues from Tsc1-null EV- and wild-type EV-treated mice (n = 3 per group). Differential gene expression and pathway analyses were conducted to identify molecular pathways influenced by Tsc1-null EV.