Surface proteomic analysis of osteosarcoma identifies EPHA2 as receptor for targeted drug delivery

Abstract

Background: Osteosarcoma (OS) is the most common bone tumour in children and adolescents. Despite aggressive therapy regimens, treatment outcomes are unsatisfactory. Targeted delivery of drugs can provide higher effective doses at the site of the tumour, ultimately improving the efficacy of existing therapy. Identification of suitable receptors for drug targeting is an essential step in the design of targeted therapy for OS.

Methods: We conducted a comparative analysis of the surface proteome of human OS cells and osteoblasts using cell surface biotinylation combined with nano-liquid chromatography - tandem mass spectrometry-based proteomics to identify surface proteins specifically upregulated on OS cells. This approach generated an extensive data set from which we selected a candidate to study for its suitability as receptor for targeted treatment delivery to OS. First, surface expression of the ephrin type-A receptor 2 (EPHA2) receptor was confirmed using FACS analysis. Ephrin type-A receptor 2 expression in human tumour tissue was tested using immunohistochemistry. Receptor targeting and internalisation studies were conducted to assess intracellular uptake of targeted modalities via EPHA2. Finally, tissue micro arrays containing cores of human OS tissue were stained using immunohistochemistry and EPHA2 staining was correlated to clinical outcome measures.

Results: Using mass spectrometry, a total of 2841 proteins were identified of which 156 were surface proteins significantly upregulated on OS cells compared with human primary osteoblasts. Ephrin type-A receptor 2 was highly upregulated and the most abundant surface protein on OS cells. In addition, EPHA2 was expressed in a vast majority of human OS samples. Ephrin type-A receptor 2 effectively mediates internalisation of targeted adenoviral vectors into OS cells. Patients with EPHA2-positive tumours showed a trend toward inferior overall survival.

Conclusion: The results presented here suggest that the EPHA2 receptor can be considered an attractive candidate receptor for targeted delivery of therapeutics to OS.

Figure 1

Cell surface protein isolation, visualisation of the proteomic data and EPHA2 expression levels. (A) General workflow of cell surface protein isolation. Cells were cultured in 75 cm² culture flasks and incubated with Sulfo-NHS-SS-Biotin that covalently binds to primary amino groups of extracellular proteins. The cells were lysed and lysates were incubated with Neutravidin Agarose beads to capture the biotinylated proteins. The biotinylated (cell surface) proteins were separated from the unbound (intracellular) proteins by centrifugation on a column and then eluted from the biotin-Neutravidin Agarose beads using DDT. (B) A heat map of a supervised cluster analysis of our obtained data set, visualising the differentially expressed proteins (cutoff: P<0.05) between the three human primary OBs (Hum31, Hum54 and ORT-1) and the five OS cell lines (SaOS-2, MG-63, U2OS, Cal-72 and LM7). (C) EPHA2 expression data acquired by nanoLC-MS/MS. Differential expression of EPHA2 between the OS cell lines (black bars) and hp-OBs (grey bars) is evident, approximately 12-fold across the two types of cell, a difference that is highly significant (beta-binominal test; ***P=0.0005). (D) EPHA2 expression data acquired by flow cytometry in OS cell lines SaOS-2, LM7, MG-63 and U2OS and hp-OBs Hum63 and Hum65, expressed as median (signal-to-background ratio) fluorescence intensities per cell line. Bars represent experiments performed in triplicate; error bars indicate s.d. The OS cell lines (black bars) show a convincingly higher EPHA2 expression than the hp-OBs (grey bars). On average, the OS cells were found to express four-fold higher levels of EPHA2 than the healthy bone cells and this difference is significant (Student's t-test; **P<0.01). (E) Histograms of EPHA2 expression in OS cell lines; visualisation of flow cytometry results. Staining positivity for EPHA2 is indicated by an increase in fluorescent signal and a concomitant right shift of the histograms.

Figure 2

EPHA2 receptor specifically mediates targeted adenoviral vector internalisation into OS cells. (A) Representative fluorescence images of SaOS-2 and MG-63 OS cells and Hum63 hp-OBs subjected to transduction with AdYSA at the indicated MOIs, acquired using the Acumen ^eX3 microplate cytometer. Composite images show Hoechst-stained cell nuclei (blue) and GFP-expression (green). (B) Transduction of OS cell lines (green bars) and hp-OBs (blue bars) with EPHA2-targeted adenoviral vector AdYSA, as measured by GFP expression 48 h after subjecting the cells to virus at MOI-100. Bars represent mean results of an experiment performed in triplicate; error bars indicate s.d. The observed difference between OS cells and hp-OBs is three- to six-fold (Student's t-test; *P<0.05). (C) Competition by pre-incubation of the cells with synthetic peptides before incubation with AdYSA. Bars represent mean results of an experiment performed in triplicate; error bars indicate s.d. Green bars represent the control condition in which the cells were incubated with AdYSA alone, grey bars represent the control condition in which cells were pre-incubated with Cys.S peptide, followed by incubation with AdYSA and blue bars represent the competition condition in which cells were pre-incubated with YSA peptide followed by incubation with AdYSA. Receptor blocking with YSA peptide results in significant reduction of transduction efficiency in the OS cell lines (****P<0.001 for SaOS-2; ***P<0.005 for LM7 and U2OS; **P<0.01 for MG-63), but not in the already poorly transduced healthy bone cells (NS).

Figure 3

EPHA2 is expressed on human OS tissue. (A) Immunohistochemical staining results for EPHA2 on human OS and normal bone tissue sections. Per category, two examples are shown. (B) Tissue staining per group. Staining was scored based on the percentage of positive cells and the intensity of the staining of the cells and allotted to the categories: negative, weak, moderate or positive. One bone sample could not be scored because of limited sample quality (NA). The staining intensity was significantly higher in the OS samples compared with healthy bone tissue, both in the primary and metastatic lesions (χ²; ***P<0.005). Clinical details of all samples are provided in Supplementary Table S5.

Figure 4

Kaplan–Meier curves showing overall survival of OS patients with EPHA2-positive or EPHA2-negative tumours. Kaplan–Meier survival plot showing the cumulative survival of patients suffering from localised OS. Patients were divided into two groups of patients with EPHA2-positive tumours (57 samples) and those with EPHA2-negative tumours (11 samples). The observed difference in the probability of survival between the two groups shows a trend towards inferior survival for patients with positive EPHA2 staining (log rank; P=0.065).