Extracellular vesicle proteomes of two transmissible cancers of Tasmanian devils reveal tenascin-C as a serum-based differential diagnostic biomarker

Abstract

The iconic Tasmanian devil (Sarcophilus harrisii) is endangered due to the transmissible cancer Devil Facial Tumour Disease (DFTD), of which there are two genetically independent subtypes (DFT1 and DFT2). While DFT1 and DFT2 can be differentially diagnosed using tumour biopsies, there is an urgent need to develop less-invasive biomarkers that can detect DFTD and distinguish between subtypes. Extracellular vesicles (EVs), the nano-sized membrane-enclosed vesicles present in most biofluids, represent a valuable resource for biomarker discovery. Here, we characterized the proteome of EVs from cultured DFTD cells using data-independent acquisition-mass spectrometry and an in-house spectral library of > 1500 proteins. EVs from both DFT1 and DFT2 cell lines expressed higher levels of proteins associated with focal adhesion functions. Furthermore, hallmark proteins of epithelial-mesenchymal transition were enriched in DFT2 EVs relative to DFT1 EVs. These findings were validated in EVs derived from serum samples, revealing that the mesenchymal marker tenascin-C was also enriched in EVs derived from the serum of devils infected with DFT2 relative to those infected with DFT1 and healthy controls. This first EV-based investigation of DFTD increases our understanding of the cancers' EVs and their possible involvement in DFTD progression, such as metastasis. Finally, we demonstrated the potential of EVs to differentiate between DFT1 and DFT2, highlighting their potential use as less-invasive liquid biopsies for the Tasmanian devil.

Keywords: Cancer diagnostics; Exosomes; Marsupials; Microvesicles; Proteomics; Size exclusion chromatography.

Fig. 1

Characterization of EVs from DFTD and devil fibroblast cells. Schematic representation of the isolation of EVs derived from cell cultures and the EV proteomic workflow analyses. Briefly, three biological replicates of each cell line (DFT1, DFT2, and devil fibroblast cells) were cultured in duplicates for 48 h at 35 °C (Tasmanian devil normal body temperature) in a fully humidified atmosphere of 5% CO2. After 48 h, the cultured medium of each cell line was collected, centrifuged, concentrated, and subjected to size exclusion chromatography to obtain extracellular vesicles. Additionally, lysate samples were prepared from each cell line. A pooled EV protein sample for EVs and cell lysates was prepared to generate specific-spectral libraries using data-dependent acquisition–mass spectrometry techniques (DDA–MS). Each fraction (16 in total) of the EV and lysate pooled samples were run in the mass spectrometer for a total of 90 min for each fraction. Individual EV and lysate protein samples (n = 36) were run using data-independent acquisition–mass spectrometry techniques (DIA–MS). Each DIA sample was run on the mass spectrometer machine for a total of 90 min

Fig. 2

Characterization of EVs from DFTD and devil fibroblast cells. A. Transmission electron microscopy images of isolated EVs from cell cultures. Red arrows indicate EV structures. B. Particle-size distributions of cell culture-derived EVs measured by nanoparticle tracking analysis (NTA), shaded areas represent 95% confidence intervals. C. EV concentration obtained by NTA. The letters “a” and “b” indicate significant pairwise differences between groups (i.e., groups denoted with the same letters are not significantly different; one-way ANOVA, Tukey post hoc test, p < 0.05); error bars represent 95% confidence intervals. D Venn diagram of overlapping genes identified in EVs derived from cell cultures with Vesiclepedia, and the top hundred exosomal genes reported in the Exocarta database. E EV and cell lysate Western blots of a purity (Golgi matrix protein ~ 130 kDa) and two cytosolic EV markers (Flotillin-1 ~ 48 kDa and Syntenin-1 ~ 32 kDa). F Heat map of expression patterns of mass spectrometry intensities of membrane and cytosolic EV markers presented in the cell culture-derived EV proteome

Fig. 3

EVs derived from DFTD cells represent their cell of origin. A Principal component analysis (PCA) biplots of cell lysate and cell culture-derived EV filtered proteomes. B Venn diagram of cell lysate and cell culture-derived EV protein overlap. D Venn diagram comparing significantly upregulated proteins of DFT2 EVs relative to devil fibroblasts EVs and significantly upregulated proteins of DFT2 lysates relative to fibroblast lysates. E Over-representation analysis (ORA) of cellular component gene ontology (GO) terms associated with proteins that are both significantly upregulated in DFT1 cells and their released EVs (relative to fibroblast cells and their released EVs; 129 proteins; FDR-corrected p ≤ 0.05). F ORA of cellular component GO terms associated with proteins that are both significantly upregulated in DFT2 cells and their released EVs (relative to fibroblast cells and their released EVs; 141 proteins; FDR-corrected p ≤ 0.05). For E and F, the total number of proteins included in each functional term is denoted by a number on the edge of each bar. G List of proteins that formed part of the enriched GO term myelin sheath in DFT1 cells and their EVs. H List of proteins that formed part of the enriched GO term myelin sheath in DFT2 cells and their EVs

Fig. 4

DFTD-derived EVs enriched cell and focal adhesion proteins relative to fibroblasts derived EVs. A. Over-representing analysis (ORA) for gene ontology (GO) terms, protein families, and pathways of the EV proteins upregulated in DFT1 (604) relative to fibroblast (569), and B. of the EV proteins significantly upregulated in DFT2 (517) compared to fibroblasts (634). Only functional terms with p ≤ 0.001 are illustrated in panels A and B (see Online resource 3 for all significant terms). The total number of proteins included in each functional term is denoted by a number on the edge of each bar. C. KEGG pathway map (shr04510) of the focal adhesion signalling pathway. Interconnected signalling pathways significantly upregulated in both DFT1 and DFT2 EVs are outlined in black borders. Proteins that are significantly upregulated in DFT1 EVs, DFT2 EVs or both are highlighted by red stars and labelled with gene names and with colours corresponding to the key shown. D. Heat map of expression patterns of mass spectrometry intensities of proteins belonging to the GTP binding GO term

Fig. 5

DFT2-derived EVs enriched the epithelial–mesenchymal transition hallmark relative to DFT1-derived EVs. A. Over-representing analysis (ORA) for gene ontology (GO) terms, protein families, and pathways of the EV proteins significantly upregulated in DFT1 (138) relative to DFT2 (294) (FDR-corrected p ≤ 0.05). The total number of proteins included in each functional term is denoted by a number on the edge of each bar. B. Enrichment plot contains enrichment score (ES), normalized enrichment score (NES), and FDR-corrected p value. The bottom portion of the plot shows the genes belonging to the hallmark, and they are ranked according to their differential expression. Higher and lower expressions are represented by red and blue colour, respectively. C Heat map showing the core list of proteins that contribute the most to the Epithelial–Mesenchymal Transition Hallmark

Fig. 6

Characterisation of extracellular vesicles (EVs) derived from Tasmanian devil serum. A. Transmission electron microscopy images for EVs isolated from serum of healthy control captive devils (n = 4), DFT1-infected devils (n = 4), and DFT2-infected devils (n = 5). Red arrows indicate EV structures. B. Size distribution profiles determined by nanoparticle tracking analysis (NTA) of EVs isolated from serum of captive healthy control devils (n = 4), DFT1-infected devils (n = 4), and DFT2-infected devils (n = 5). Shaded areas represent 95% confidence intervals. C EV concentrations of the same NTA groups. The letters “a” and “b” indicate significant pairwise differences among groups (i.e., groups denoted with the same letter are not significantly different; one-way ANOVA, Tukey post hoc test, p < 0.05). Error bars represent 95% confidence intervals. D. Heat map of intensity values of commonly recovered EV proteins, and E. serum contaminants found in EV samples derived from captive healthy controls devils (n = 10), DFT1-infected devils (n = 12), and devils infected with DFT2 (n = 5)

Fig. 7

The mesenchymal protein TNC as a potential biomarker for DFT2. A. Venn diagram of overlapping proteins identified as: (a) upregulated in EVs derived from devils infected with DFT2 relative to devils infected with DFT1; (b) upregulated in EVs derived from devils infected with DFT2 relative to healthy controls; (c) upregulated in EVs derived from DFT2 cultured cells relative to DFT1 cultured cells; and (d) the core list of mesenchymal proteins that contribute the most to the epithelial–mesenchymal transition hallmark enrichment in EVs derived from DFT2 cultured cells. Note that one protein in the core enrichment list was present, but not significantly upregulated in DFT2 cultured cell EVs. B. Dot plot showing the relative abundance of EV TNC detected in 10 healthy, 12 DFT1-infected devils, and 5 DFT2-infected devils, different letters “a” and “b” indicate significant pairwise differences between groups (i.e., groups denoted with the same letter are not significantly different; one-way ANOVA and Tukey post hoc test, p < 0.05). C. Receiver-operating characteristic (ROC) curve analysis for TNC EVs (5 DFT2-infected devils relative to 12 DFT1-infected devils). D. ROC curve analysis for TNC EVs (5 DFT2 infected devils relative to 10 healthy controls). For both C and D, the dashed red line indicates random performance. The cut-off values were determined using Youden’s index and are indicated in blue at the left top corner of the ROC curve, and specificity and sensitivity are indicated in brackets, respectively