Identification of novel therapeutic targets in Osteosarcoma for the development of Nanoparticle based drug delivery systems

Abstract

Osteosarcoma (OS) is the most common primary malignant tumour of the bone, with a high incidence rate in children and adolescents. Importantly, OS therapeutics has remained unimproved for the last 30 years and therefore, clinically unsatisfactory. It is understood that determining the mechanisms underlying OS tumorigenesis and progression, along with the identification of novel therapeutic targets can greatly aid in the clinical management of the disease. Thus, one important area in need of further exploration is the tumour microenvironment (TME), which consists of a plethora of extracellular matrix components, tumour associated fibroblasts and immune cells. To determine the underlying mechanism(s) and identify novel therapeutic targets, we searched an OS gene microarray dataset deposited within the Gene Expression Omnibus, which included 84 primary OS biopsies and 12 primary Mesenchymal stem cell control samples. An alternative to R coding (GEO2R) was utilised to identify differentially expressed genes (DEGs) in OS, with further R analysis conducted to quantify levels of genetic association to OS. 1161 DEGs in OS were identified, consisting of 535 upregulated and 626 downregulated genes at cut-offs of |log2FC|>1 and an adjusted P-value<0.01. Through functional annotations, we show that the DEGs are involved in immune system processes, including defence and general immune responses. This suggests that the immune system is strongly linked to the OS microenvironment with DEGs potentially contributing to OS development and metastasis. For the identification of immune based therapeutic targets in the OS TME, upregulated genes were additionally refined based on the gene ontology (GO) term “immune receptor activity”. Through GO based refinement, the immune receptor Formyl-Peptide Receptor 3 (FPR3) was found to be significantly upregulated in OS and to have a close genetic association. Through both in vitro and in vivo staining, we first show that FPR3 is specifically expressed on malignant bone cells of mesenchymal origin, with a lack of expression on normal bone cells. Thereafter, through functional in vitro cell migration assays, we determined its potential in becoming a therapeutic target in OS. Results showed that when targeted with a FPR3 specific peptide antagonist (WRW4), cell migration in two OS cell lines were significantly reduced. Observable differences in responsiveness to the peptide antagonist, between the two assayed OS cell lines, could be explained by differences in receptor expression levels as shown by flow cytometric analysis. An alternate role could relate to its use as a prognostic biomarker in OS. For the therapeutic targeting of FPR3 in OS, we propose the use of Nanoparticle based drug delivery systems (NP-DDS). Nanoparticles (NPs) are highly promising tools for both clinical and therapeutic purposes. The surfaces of NPs can be modified for targeted therapy and the NPs themselves are able to controllably release drugs when used as drug carriers in NP-DDS. However, NPs are known to exhibit varying levels of toxicity, therefore, their biosafety still remains a concern. Here, we put forward the Galleria Mellonella (GM) model organism as an efficient, low-cost, high-throughput tool for determining NP toxicity in vivo. We utilise, for the first time, techniques such as flow cytometry, immunohistochemistry and a 4HNE ELISA, on GM larvae for the determination of NP-induced toxicity, in vivo. Based on the various assays, NP toxicity was found to be heavily dependent on physicochemical properties such as surface composition and size. In addition, we investigate cellular ROS production, both in vitro and in vivo, to determine the mechanism’s likelihood in playing a major role in NP induced toxicity, observed in vivo. Results show that intracellular ROS production is the likely mechanism underlying NP toxicity in vivo, however, tissue aggregation is an important feature of CNTs that induces strong toxicity. Lastly, SPION induced immunotoxicity was observed in vivo with the use of GM larvae. Therefore, we aimed to determine if NP intracellular trafficking, intracellular fate and in particular, SPION related endosomal escape played a role in SPION induced immunotoxicity. Preliminary data is presented for this aspect in this study. Overall, certain NP variants that were identified to be biologically safe, can be put forward as potential drug delivery agents for the treatment of diseases such as OS. These NPs can be designed for tumour specific delivery of therapeutic compounds such as receptor peptide ligands, whereby, the tumour target can be a receptor of interest with therapeutic potential such as FPR3.