Supplementary Components1604880_summary. pieces Cgp 52432 treated with PBS, 231 BrT1 WT, 231 BrT1 CEMIP KO1 and KO2 exosomes can be obtainable as Supplementary Table 3. The patient processed data from Fig. 5 and Supplementary Fig. 5 is available as Supplementary Table 8. Unprocessed scans and replicates for all immunoblots presented in the manuscript are available as Supplementary Figure 6. Abstract Development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic, but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake Rabbit Polyclonal to OPRD1 of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating cytokines, known to promote brain vascular remodeling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting of exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis. organotypic brain slice culture system (Supplementary Fig. 1a)14. We pre-treated brain slices with 5 g of exosomes from brain-tropic 231-BR (231 BrT1), lung-tropic 4175 (231 LuT1), bone-tropic 1833 (231 BoT1), or parental MDA-MB-231 (231 Parental) human breast cancer metastatic cells6,15 (Supplementary Fig. 1a), for two consecutive days, then added fluorescently-labelled 231 BrT1 cancer cells, measuring tumour cell colonization Cgp 52432 three days later (Supplementary Fig. 1b C cancer cell number). Pre-treatment of brain slices with 231 BrT1-derived exosomes increased colonizing 231 BrT1 cell number four-fold compared to PBS, and two-fold or more compared to pre-treatment with 231 parental and lung- or bone-metastatic exosomes Cgp 52432 (Fig. 1a), respectively. Pre-treatment with non-brain tropic exosomes did not induce significant cancer cell growth compared to PBS (Fig. 1a and Supplementary Fig. 1c). Open in a separate window Figure 1 C Exosomes from brain metastatic cells support brain metastatic colonization and are enriched in CEMIP protein.a, Left, representative images of 231 BrT1-GFP+ cells developing together with brain slices pre-treated with PBS or exosomes. Best, quantification of tumor cellular number. b, Still left, representative images of 231 BrT1-GFP+ cells invading brain slices pre-treated with PBS or exosomes. Brain slice areas had been stained with DAPI (blue); dotted blue lines delineate the very best and bottom level limit of the Cgp 52432 mind slice. Best, quantification of invading tumor cellular number. c, Heatmap of 20 differentially portrayed exosomal protein and -Actin (ACTB) predicated on the quantitative mass spectrometry label-free quantification (LFQ) beliefs (specialized triplicates, *FDR – fake discovery price 0.05 by ANOVA). Hierarchical clustering (one without the test Spearmans rank of relationship between observations) was performed on proteins expression amounts. d, Best, CEMIP, ACTB (launching control), and Compact disc81 (exosomal marker) immunoblot in cells and exosomes from organ-specific metastasis versions. Bottom level, densitometry quantification of CEMIP e, Best, CEMIP, ACTB (launching control), and Compact disc81 (exosomal marker) immunoblot in cells and exosomes from individual cancer cell human brain metastasis models. Bottom level, densitometry quantification of CEMIP. The amount of cells per field of watch (FOV) are averages SEM, from = 9 specific human brain pieces (a), or beliefs were computed by ANOVA (a, b). Discover Supplementary Body 6 for unprocessed blots. Discover Supplementary Desk 1 for figures supply data. Next, we asked if pre-conditioning with human brain metastatic tumour-derived exosomes impacted human brain metastatic cell invasiveness. Three times after tumour cell addition, we quantified invading 231 BrT1 cells in transversal parts of human brain pieces pre-treated with 231 BrT1 or 231 parental-derived exosomes.