Proteomic Assessment of Extracellular Vesicles from Canine Tissue Explants as a Pipeline to Identify Molecular Targets in Osteosarcoma: PSMD14/Rpn11 as a Proof of Principle

Abstract

Osteosarcoma (OS) is a highly malignant bone tumour that has seen little improvement in treatment modalities in the past 30 years. Understanding what molecules contribute to OS biology could aid in the discovery of novel therapies. Extracellular vesicles (EVs) serve as a mode of cell-to-cell communication and have the potential to uncover novel protein signatures. In our research, we developed a novel pipeline to isolate, characterize, and profile EVs from normal bone and osteosarcoma tissue explants from canine OS patients. Proteomic analysis of vesicle preparations revealed a protein signature related to protein metabolism. One molecule of interest, PSMD14/Rpn11, was explored further given its prognostic potential in human and canine OS, and its targetability with the drug capzimin. In vitro experiments demonstrated that capzimin induces apoptosis and reduces clonogenic survival, proliferation, and migration in two metastatic canine OS cell lines. Capzimin also reduces the viability of metastatic human OS cells cultured under 3D conditions that mimic the growth of OS cells at secondary sites. This unique pipeline can improve our understanding of OS biology and identify new prognostic markers and molecular targets for both canine and human OS patients.

Keywords: biomarker discovery; extracellular vesicles; molecular targets; osteosarcoma; tissue explants.

Figure 1

Isolation and characterization of extracellular vesicles from OS and non-malignant bone tissue explants. (A) General workflow for sample acquisition and vesicle characterization from canine appendicular OS patients. (B) Representative immunoblot for extracellular vesicle markers, CD63 and flotillin-1 (Flot-1) for patient 6 (left) and table summarizing immunoblot results for all patients (right). Green filled boxes represent positivity for the marker, empty boxes represent no signal detected for the marker, grey filled boxes indicate the sample was not obtained. Bold outlines represent the samples that were included in the UHPLC-MS/MS analysis as they were positive for CD63 and/or flotillin and had a total protein concentration of 2.9 μg (n = 12). (C) Representative size distribution analysis for pooled fractions that were CD63 and/or flotillin-1-positive for patient 6 (left) and summary graph of vesicle size distribution for all patients (right). (D) Representative transmission electron microscopy images of a vesicle preparation obtained with our protocol; scale bar = 50 nm (left) or 20 nm (right). (E) Immunoblots of OS samples from patients 3, 6, 7 for novel vesicle markers fibronectin, filamin A, stomatin and gelsolin.

Figure 2

Proteins identified in UHPLC-MS/MS analysis including all samples. (A) Pie charts showing the number of statistically significantly proteins when comparing non-malignant bone (NB) and OS samples using a t-test. (B) Table of significantly elevated proteins found in the non-malignant bone samples (top) and OS samples (bottom) when using a minimum peptide = 2 as a cutoff and a t-test. (C) The molecular functions and biological processes of the significantly elevated proteins identified in the OS samples. (D) Table of significantly elevated proteins in OS samples when using a minimum peptide = 1 as a cutoff and a t-test.

Figure 3

PSMD14 is a clinically relevant and viable target in OS. (A) Significant association between PSMD14 mRNA levels in treatment-naïve biopsies and the development of metastasis within 5 years (GSE21257, unpaired t-test). Significant difference in survival between canine OS patients with high versus low PSMD14 and PSMA7 mRNA, alone and combined (GSE27217, log-rank (Mantel-Cox) test). (B) Levels of PSMD14 in primary tumour-derived OS cell lines: HOS, Abrams (Ab), OVC-cOSA-75 (75), OVC-cOSA-78 (78), secondary tumour-derived OS cell lines: OVC-cOSA-31 (31), D17, K7M2, and non-malignant mesenchymal stromal cells (MSC) and Madin-Darby canine kidney (MDCK) cells. (C) MDCK, MSC, D17 and OVC-cOSA-31 cells were treated with capzimin at 0.001, 0.01, 0.1, 1, 10 μM for 24 or 48 h and incubated with resazurin for 8 h before reading the absorbance at 570/600 nm (five technical replicates per treatment). IC₅₀ curves depict normalized % viability, mean ± SD from n = 3 independent experiments. The IC₅₀ was determined using a four-parameter variable slope model using Prism 9 software. (D) Capzimin (CZM) treatment resulted in a slight increase in HIF1α levels with treatment. Representative HIF1α immunoblot after D17 and OVC-cOSA-31 (31) cells were subjected to DMSO (−) or their respective CZM IC₅₀ treatment (+) for 24 h (D17 = 205 nM, 31 = 140 nM) or 48 h (D17 = 400 nM, 31 = 280 nM). (E) CZM treatment led to an increase in ubiquitinated proteins 6 h post treatment. D17 and OVC-cOSA cells were treated with DMSO or CZM (D17 = 400 nM, 31 = 280 nM) for 6 h before lysis and adsorption with ubiquilin 1 tandem UBA (TUBE2) beads. Enriched ubiquitinated proteins were detected using an anti-ubiquitin antibody.

Figure 4

Capzimin induces apoptosis in a dose-dependent manner in canine OS cells. (A) CZM exposure at the calculated IC₅₀ dose leads to a reduction in full length PARP (fPARP), but an associated increase in the cleaved version of PARP (cPARP) was not observed at either 24 or 48 h (left). However, when cells were treated with the 4 μM dose of CZM, the associated increase in cPARP was more evident (right). Immunoblots are from one representative experiment. fPARP levels were normalized to β actin (loading control) and compared to the DMSO control group to determine relative differences. cPARP levels were first normalized to β actin and are depicted as changes relative to normalized fPARP levels. Densitometry graph shows mean ± SD from n = 3 independent experiments; * indicates p < 0.05 as determined by an unpaired, two-tailed t-test. (B) Annexin V/Propidium Iodide flow cytometry analysis of OS cells treated with a low and high dose of CZM. A high dose of CZM significantly increases the percentage of early and late apoptotic D17 cells. Increases were seen in both cell lines when treated with the 400 nM of CZM, but these differences were not statistically significant. Graphs depict mean ± SD from n = 3 independent experiments; * indicates p < 0.05 as determined by a one-way ANOVA and post-hoc Tukey’s multiple comparison’s test.

Figure 5

Capzimin reduces clonogenic survival, cell growth, and migration. (A) CZM reduces the clonogenic survival in both D17 and OVC-cOSA-31 cell lines at their respective IC₅₀ dose (left) and the 4 μM dose (right). Colony area is defined as the area of the well occupied by cells, while colony intensity is defined as the pixel intensity of the colony. Graph depicts mean ± SD from n = 3 independent experiments, each experiment was seeded in 3 technical triplicates; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 as determined by the Mann-Whitney test. (B) CZM reduces the percentage of mitotically active cells. Immunolabelling and quantification of DMSO or CZM-treated cells show differences in the nuclear levels of mitotic marker phosphorylated histone-H3 (pHH3). Graphs depict the relative nuclear positivity of pHH3 from n = 4 experiments, 5 images were evaluated per experiment; * indicates p < 0.05, **** indicates p < 0.0001. (C) CZM induces a G2/M arrest in D17 cells upon 4 μM treatment for 24 h. Graph depicts mean ± SD from n = 3 independent experiments (D) CZM decreases migration (left) and the expression of migration-associated markers (right). Images of a transwell insert stained with crystal violet 24 h post CZM or DMSO treatment at 10X magnification. Graphs show the percentage of migrated cells as determined after extracting stained transwell inserts with 10% acetic acid and measuring the absorbance at 590nm. The extracted dye was blank corrected to a media only stained insert and normalized to the DMSO control from n = 4 experiments, 2 technical replicates per treatment group; *** indicates p < 0.001. Representative immunoblots showing the impact of CZM treatment on NF-κB and fibronectin expression from one representative experiment. Protein levels were normalized to β actin (loading control) and compared to the DMSO control group to determine relative differences. Densitometry graph shows mean ± SD from n = 2 independent experiments.

Figure 6

Capzimin sensitizes OS cells to doxorubicin (Dox) treatment. D17 or OVC-cOSA-31 cells were treated with subjected to three different conditions to evaluate CZM’s efficacy combined with Dox. (A) In the CZM and then Dox conditions, cells were treated with DMSO or their respective CZM for 24 h and then subjected to their IC₅₀ dose of doxorubicin (30 μM for D17 and 26 μM for OVC-cOSA-31) or 10-fold or 100-fold lower concentrations for an additional 24 h. (B) In the Dox and then CZM condition, cells were treated with Dox for 24 h, after which, the media was removed and media with DMSO or CZM was replaced for 48 h. (C) In the Dox & CZM condition, cells were subjected to co-treatment of both compounds for 48 h. After each experimental time course, viability was measured by incubating with resazurin for 8 h and measuring absorbance at 570/600 nm (six technical replicates per treatment). Graphs depict normalized % viability, mean ± SD from n = 3 independent experiments, except for part C, where n = 2 independent experiments. Chart depicts select statistically significant differences as determined by a two-way ANOVA with a post-hoc Tukey’s multiple comparison test. Non-filled squares indicate no significant differences were observed.

Figure 7

Human OS cells are more sensitive to capzimin when cultured under 3D conditions. (A) CZM was unable to blunt proliferation up to a 2 μM dose in MG63.3 cells treated in 2D conditions for 6 days. Graph depicts the relative absorbance values at 490 nm and normalized to the DMSO (vehicle) control and represents the mean ± SD from n = 3 independent experiments. (B) CZM significantly decreased growth under 3D conditions. MG63.3 cells were cultured in a 3D Basement Membrane Extract (BME) system and subjected to various doses of CZM treatment for 6 days. Growth was significantly decreased by CZM at 300, 400, 500 and 1 μM concentrations (* p < 0.05 and **** p < 0.0001). Graph depicts corrected absorbance values at 490 nm with BME + media alone used to subtract background. The results are representative of one of 2 experiments, each with 4 technical replicates.