Identifying metabolic mechanisms of metastasis progression

The lung is one of the most common sites for metastasis, and various cell types within the lung microenvironment have been shown to facilitate and regulate metastatic outgrowth. Here, we aim to identify the metabolic mechanisms driving lung metastasis derived from MYC-driven mammary gland tumors. Using LC-MS, immunofluorescence (IF), and mass spectrometry imaging (DESI, OrbiSIMS), we observed several metabolic changes in metastatic lesions compared to metastasis-infiltrated lungs and normal healthy lungs. First, we detected an accumulation of lipids and fatty acids in the lung tissue surrounding metastatic lesions. IF staining for fatty acid synthase (FASN), a key enzyme in de novo lipogenesis, revealed its upregulation specifically in alveolar type II cells proximal to metastatic lesions. Second, we found increased levels of oxidized glutathione in metastasis-infiltrated lungs compared to normal lungs, along with higher levels of reduced glutathione in metastatic lesions relative to surrounding lung tissue. Finally, scRNA-seq data from normal and metastatic lungs allowed us to identify transcriptional differences (both general and specific to genes encoding metabolic enzymes) between cells in normal lung tissue and those in metastasis-infiltrated lungs. Overall design: MMTV-MYC mammary gland tumor-derived cells (LM1) were injected into the mammary fat pad (m.f.p.) of FVBN female mice (6–8 weeks old). Within a month, primary tumors (approximately 1.5 cm in diameter) developed, accompanied by the formation of metastatic lesions in the lung. Primary tumours and lung tissues were collected from 6 tumor-bearing mice and normal lungs were collected from 6 wildtype control mice. Tissues were then dissociated using a standard Liberase TM buffer dissociation protocol. The cells were filtered through 40 µm filters to ensure a single-cell suspension, and the samples were submitted for single-cell RNA sequencing. Cells from 3 primary tumours were used for the analysis. Cells from two lungs were pooled together for each out of 3 normal and 3 metastatic lungs. Additionally, original LM1 cells were also submitted for the sequencing.