Nano-scavengers for blood biomarker discovery in ovarian carcinoma

Highlights

• Clinical exploitation of protein corona fingerprinting as a novel tool for blood proteomics analysis and biomarker discovery.

• Proteomic analysis of the liposomal corona to amplify and uncover disease specific signatures in the blood of ovarian cancer patients.

• Discovery of previously unknown potential biomarker proteins in ovarian carcinoma with higher specificity and sensitivity than the clinically used biomarkers.

Abstract

The development and implementation of biomarker-based screening tools for ovarian cancer require novel analytical platforms to enable the discovery of biomarker panels that will overcome the limitations associated with the clinically used CA-125.The systematic discovery of protein biomarkers directly from human plasma using proteomics remains extremely challenging, due to the wide concentration range of plasma proteins. Here, we describe the use of lipid-based nanoparticles (NPs) as an ‘omics’ enrichment tool to amplify cancer signals in the blood and to uncover disease specific signatures. We aimed to exploit the spontaneous interaction of clinically-used liposomes (Caelyx®) with plasma proteins, also known as’ protein corona’ formation, in order to facilitate the discovery of previously unreported differentially abundant molecules. Caelyx® liposomes were incubated with plasma samples obtained from advanced ovarian carcinoma patients and healthy donors and corona-coated liposomes were subsequently recovered. Comprehensive comparison between ‘healthy’ and ‘diseased’ corona samples by label-free proteomics resulted in the identification of multiple differentially abundant proteins. Moreover, immunoassay-based validation of selected proteins demonstrated the potential of nanoparticle-platform proposed to discover novel molecules with great diagnostic potential. This study proposes a nanoparticle-enabled workflow for plasma proteomic analysis in healthy and diseased states and paves the way for further work needed to discover and validate panels of novel biomarkers for disease diagnosis and monitoring.

Introduction

Much effort is currently focused on the development of robust and high-throughput ‘omics’ platforms for the discovery of minimally invasive molecular biomarkers to aid early and accurate cancer diagnosis, monitor tumour growth and response to therapies. Despite significant investment by major stakeholders, few protein cancer biomarkers have been validated and received FDA approval, raising concerns regarding the efficiency of the biomarker-development pipeline. It is noteworthy that of the FDA-approved biomarkers, the majority are used to monitor the progression of cancer, rather than enabling its early diagnosis [1].

Proteins are the biological endpoints that govern most pathophysiological processes and they have therefore attracted most interest so far as biomarkers for cancer diagnostics [2]. Blood is frequently the biosample of choice for biomarker identification; however the discovery of tumour-derived protein signatures directly from blood is hindered by the wide concentration range of blood proteins, in addition to the preponderance of highly abundant proteins [3]. Despite significant improvement in the sensitivity of mass spectrometry-based proteomics, the issue of the high dynamic range of plasma protein abundances still remains unresolved and the diagnostic information blood can offer is partially inaccessible [4].

Nanotechnology-based platforms hold great promise in addressing the above issues associated with biomarker discovery [5]. It should be emphasised however, that the vast majority of nanoparticle-based technologies developed so far have been designed to capture and quantify already known cancer-specific analytes [[6], [7], [8], [9]], enabling the verification and validation phases of biomarker development. The NP-enabled discovery of new plasma buried biomarkers has only been recently attempted [10].

The fact that the surface of NPs is instantly covered by a wide range of adsorbed proteins and other biomolecules once in contact with blood, a self-assembly phenomenon known as ‘protein’ or ‘biomolecule’ corona formation [11,12], makes NPs ideal biomarker discovery platforms. Biomolecule corona formation has become a popular line of research in the last decade and ongoing research is mainly focused on the proteomic analysis of corona profiles after the ex vivo and more recently the in vivo interaction of NPs with biofluids (mainly plasma) [[13], [14], [15], [16], [17]]. Nanoparticle-protein interactions at the bio-nano interface not only can shed new light on the development of nanotechnologies but are now gradually being exploited as an engineering tool with therapeutic and diagnostic capabilities [10,11,[18], [19], [20]].

The surface-capture of a complex blood proteome by NPs as well as the recently proposed concept of ‘personalized corona’ has sparked interest for utilizing the biomolecule corona fingerprinting as a proteomic discovery platform [10,18,21,22]. We have recently demonstrated that the NP protein corona formed in the blood circulation of humans has the potential to be exploited as an enrichment and pre-fractionation tool that allows in depth coverage of the plasma proteome [18]. In a subsequent study, we employed two different tumour mouse models (a subcutaneous melanoma model and human lung carcinoma xenograft model) to demonstrate that intravenously injected lipid-based NP-scavengers (liposomes) surface-capture low MW, low abundant and disease-specific plasma proteins which cannot be detected by conventional plasma proteomic analysis [10]. Moreover, this study demonstrated that protein coronas, formed around intravenously injected NPs, differ both quantitatively and qualitatively in the presence and absence of a disease, allowing the uncovering of differentially abundant potential biomarker proteins [10].

When animal models are employed for biomarkers discovery, the exploitation of the molecularly richer in vivo protein corona is advantageous as opposed to its counterpart ex vivo corona [16]. However, hypothesis-free discovery proteomics often require the use of human clinical samples and therefore, in this study we aimed to explore the use of the ex vivo protein corona formed around the clinically used PEGylated liposomal doxorubicin formulation (Caelyx®), to identify disease-specific proteins directly from plasma samples, obtained from patients with recurrent ovarian carcinoma.

The work flow of this study is summarized in Fig. 1A and involved the incubation of Caelyx® liposomes with plasma samples from patients with recurrent ovarian cancer and from healthy donors and the comprehensive comparison of the resultant protein coronas by label-free mass spectrometry. The above analysis led to in the discovery of 413 differentially abundant proteins between ‘healthy’ and ‘diseased’ corona samples, of which nine were quantified by immunoassays to further validate the potential use of the nanoparticle-protein corona technology for plasma proteomic analysis and biomarkers discovery.