Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis

Abstract

The 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 of CEMIP⁺ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling 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 exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

Figure 1 –. Exosomes from brain metastatic cells support brain metastatic colonization and are enriched in CEMIP protein.

a, Left, representative images of 231 BrT1-GFP⁺ cells growing on top of brain slices pre-treated with exosomes or PBS. Right, quantification of cancer cell number. b, Left, representative images of 231 BrT1-GFP⁺ cells invading brain slices pre-treated with exosomes or PBS. Brain slice sections were stained with DAPI (blue); dotted blue lines delineate the top and bottom limit of the brain slice. Right, quantification of invading cancer cell number. c, Heatmap of 20 differentially expressed exosomal proteins and β-Actin (ACTB) based on the quantitative mass spectrometry label-free quantification (LFQ) values (technical triplicates, *FDR - false discovery rate < 0.05 by ANOVA). Hierarchical clustering (one minus the sample Spearman’s rank of correlation between observations) was performed on protein expression levels. d, Top, CEMIP, ACTB (loading control), and CD81 (exosomal marker) immunoblot in cells and exosomes from organ-specific metastasis models. Bottom, densitometry quantification of CEMIP e, Top, CEMIP, ACTB (loading control), and CD81 (exosomal marker) immunoblot in cells and exosomes from human cancer cell brain metastasis models. Bottom, densitometry quantification of CEMIP. The number of cells per field of view (FOV) are averages ± SEM, from n = 9 individual brain slices (a), or n= 6, 7, 8 individual brain slices (b), scoring two fields per slice (a, b). Heatmap depicting differentially expressed proteins in BrT-derived exosomes displays average of three independent exosome sample replicates (c). Densitometry graphs show CEMIP expression normalized to CEMIP expression in BrT1 exosomes, and CEMIP expression was normalized to ACTB for each sample (d and e). A representative experiment of three (a, b and e) or four (d) independent biological replicates is shown. Scale bars, 100μm (a, b). Error bars depict mean ± standard error of the mean (SEM). P values were calculated by ANOVA (a, b). See Supplementary Figure 6 for unprocessed blots. See Supplementary Table 1 for statistics source data.

Figure 2 –. Exosomal CEMIP modulates the brain vascular niche to support vascular co-option and invasion.

a, Left, representative fluorescence microscopy images of vessel association of GFP-expressing BrT1 wild-type (WT, control cells with WT CEMIP expression) or GFP-expressing BrT1 CEMIP knockout (KO1 and KO2) cells growing on top of brain slices. Brain vasculature is shown by Col IV⁺ staining (red, all fluorescent images in Fig. 2a – 2d). Cells with spindle-like morphology and spread along vasculature (white arrows) were considered vessel-associated. Right, quantification of vessel-associated cancer cell number. b, Left, representative fluorescence microscopy images of BrT1 WT, BrT1 CEMIP-KO1, and CEMIP-KO2 GFP⁺ cells invading brain slices. Cancer cells were considered invasive when migrating inwards past the top cell layer of the brain slice (white arrows). Dotted blue lines delineate the top and bottom limits of the slice. Right, quantification of invading cancer cell number c, Left, representative fluorescence microscopy images of vessel association of BrT1 CEMIP-KO2 GFP⁺ cells growing on top of brain slices pre-treated with exosomes or PBS. White arrows indicate vasculature-associated cancer cells. Right, quantification of vessel-associated cancer cell number. d, Left, representative fluorescence microscopy images of BrT1 CEMIP-KO2 GFP⁺ cells invading (white arrows) brain slices pre-treated with exosomes or PBS. Dotted blue lines delineate the top and bottom limits of the brain slice. Right, quantification of invading cancer cell number. The number of cells per FOV are from n = 8, 9, 9 (a), n = 6 (b), n = 9, 7, 9, 9 (c), or n = 5 (d) individual brain slices, scoring two fields per slice. A representative experiment is shown from three (a - d) independent biological replicates. Brain slice sections are stained with DAPI, shown in blue (b, d). Scale bar, 100μm (a - d). Error bars depict mean ± SEM. P values were calculated by ANOVA (a - d). See Supplementary Table 1 for statistics source data.

Figure 3 –. Exosomal CEMIP supports brain metastasis in vivo.

a, Quantification of brain metastasis in mice intracardiacally injected with BrT1 WT or CEMIP-KO cells. Left, cranial bioluminescence signal (Total photon flux – photons/ second (p/s)) in mice over 4 weeks post-intracardiac injection of GFP-labelled BrT1 WT or BrT1 CEMIP-KO luciferase-positive cells and representative IVIS image of brain signal at week 4. Right, representative immunofluorescence images of whole brain sagittal sections from mice with brain metastatic lesions after 4 weeks (green, white arrows). Quantification of the number of lesions per brain (left graph) and total brain metastatic lesion area (μm², right graph) is shown below the immunofluorescence images. The number of lesions and total metastatic area per brain represent averages ± SEM, scored from lesions in two sagittal brain sections from different brain areas per mouse, and n = 4, 5, 5 mice per group b, Quantification of brain metastasis in mice pre-educated with exosomes or PBS. Left, cranial bioluminescence signal (Total photon flux – photons/ second (p/s)) of mice educated for 3 weeks with exosomes or PBS, followed by intracardiac injection of GFP-labelled BrT1 luciferase-positive cells, and representative IVIS image of brain signals at week 3 post-cell injection. Enlarged inset, middle, representative images of whole brain sagittal sections from mice showing GFP+ brain metastases (green, white arrows) 3 weeks post-cell injection. Right, quantification of total brain metastatic lesion area (μm², upper graph) and number of lesions per brain (lower graph), representing averages ± SEM scored from lesions in two sagittal brain sections representative of different brain areas per mouse, with n = 9, 9, 9, 7 mice per group. Scale bar, 1mm (a, b). Error bars depict mean ± SEM. P values were calculated by ANOVA (a, b). One representative experiment of two is shown (a, b). See Supplementary Table 1 for statistics source data.

Figure 4 –. Exosomal CEMIP uptake by BrECs and microglia induces vascular remodeling and inflammation in the brain vascular niche.

a, Representative images of fluorescently-labelled 231 BrT1 exosomes (green) and brain endothelial cells (BrECs, CD31⁺), microglia (Iba1⁺), astrocytes (GFAP⁺), or neurons (NeuN⁺) (all in red). White arrows indicate co-localization of exosomes and the indicated cell type. b, Representative confocal microscopy image of Glut1⁺ BrECs (blue, long arrows) and Iba1⁺ microglia (green, short arrows) interacting with fluorescently-labelled BrT1 exosomes (red). Double arrows depict joint interaction of BrECs and microglia with exosomes. c, Left, representative images of calcein AM-loaded BrEC vascular networks (green) formed in vitro upon pre-treatment with exosomes or PBS. Vascular tree general topology is depicted by identification of the tree’s master junctions (red) and master segments (yellow). Right, quantification of vascular network branch number (left graph) and length (right graph). d, Top, pathways affected by exosomal CEMIP in BrECs (left) and microglia (right) isolated from exosome-treated brain slices. Z-score indicates activation (orange) or inhibition (blue), and ratio indicates number of genes from the CEMIP list that map to a pathway divided by the total number of genes that map to that same pathway. Associated p-value of the Fisher’s exact test is displayed in black. Bottom, heatmap of differentially expressed genes involved in selected pathways. The number and length of branches per FOV are averages ± SEM, from n = 5, 7, 8, 7, 8, 8 individual μ-slide wells (c), scoring a representative field per well. One of three independent biological replicates is shown for a, b, c. The average of three independent biological replicates is displayed in (d). Scale bars, 100μm (a, c) and 50μm (b). Error bars depict mean ± SEM. P values were calculated by ANOVA (c) and Fisher’s exact test (chart) or two-sided Student’s t-test (heatmap) (d). See Supplementary Table 1 for statistics source data.

Figure 5 –. CEMIP is a prognostic biomarker of brain metastasis in patients.

a, Representative image of a lung cancer brain metastatic tumour patient sample analyzed by H&E (top) and CEMIP immunohistochemistry (bottom). The metastatic tumor is outlined by the black dashed line. b, Representative immunohistochemistry images illustrating CEMIP expression for each scoring category in patient tumour samples. Samples with no (0) or low (1 and 2) CEMIP staining were considered CEMIP^low (green). Samples displaying high expression (3 and 4) were considered CEMIP^high (red). c, Top, quantification of CEMIP expression by immunohistochemistry in primary tumour (left) and metastatic tumour (right) from patients with or without brain metastasis. Bottom, percentage of CEMIP^high cases and information on total number of samples evaluated in each group. PT (Minimum: 0.00, 0.00, 0.33; Maximum: 3.00, 2.67, 4.00; and Median: 1.25, 1.33, 1.83), and MET (Minimum: 0, 0; Maximum: 3, 4; and Median: 1.00, 1.83). d, Progression-free survival Kaplan-Meier curve for brain metastasis patients depicting time to brain metastasis based on primary tumour CEMIP expression, low (green) or high expression (red). Scale bars, 50μm (a), and 300μm (b). Human data consists of n=317 total unique tumour samples (213 primary and 104 metastatic) from 278 breast and lung cancer patients (a - c). Immunohistochemistry score represents the average intensity in tumour cores analyzed (1 – 3 per sample) on a scale from 0 to 4 (b). Dashed line across violin plots depicts quartiles and full line depicts median (c). P values were calculated by ANOVA and two-sided Student’s t-test (c), or Log-rank (Mantel-Cox) test (d). See Supplementary Table 1 for statistics source data.