Exosome-mediated diagnosis of pancreatic cancer using lectin-conjugated nanoparticles bound to selective glycans

Highlights

• The use of specific glycan moieties allows the development of clinical diagnostics with high ensitivity and selectivity.

• Lectin-glycan interactions have the inherent potential to capture exosomes derived from cancer cells without antibodies.

• The relative fluorescence evaluation of exosomes presents an opportunity for evaluating cancer metastasis.

Abstract

The unique profile of upregulated glycosylation in metastatic cancer cells may form the basis for the development of new biomarkers for the targeting and diagnosis of specific cancers. This study introduces a pancreatic cancer cell-derived exosome detection technology, which is based on the specific binding of lectins to distinctive glycan profiles on the surface of exosomes. Lectins with a high and specific affinity for sialic acid or fucose were attached to bifunctional Janus nanoparticles (JNPs), which facilitated interactions with pancreatic cancer cell-derived exosomes in a microfluidic device. Here, we show that pancreatic cancer cell-derived exosomes from two cell lines and plasma samples collected from patients diagnosed with pancreatic cancer were successfully captured on the lectin-conjugated JNPs with affinities that were comparable to those of CA19-9, a conventional antibody. In addition, exosome detection using our platform could differentiate between metastatic and nonmetastatic pancreatic cancer cells. This study opens the possibility to achieve a new early diagnosis marker based on the glycan properties of pancreatic cancer cell-derived exosomes.

Introduction

Pancreatic cancer, ranked the 12th most common type of cancer among 27 types of cancer, is often asymptomatic in early stages (Siegel et al. 2019), and no methods for diagnosing early-stage cancers exist (Nie et al. 2014). Recent studies have focused on cancer cell-derived secretomes (i.e., proteins secreted by cancer cells) to identify biomarkers for early-stage pancreatic cancer (Wan et al. 2017). One such example is an exosome, which has received increasing attention for the diagnosis and evaluation of cancers (Liu et al. 2018; Srinivasan et al. 2019). Exosomes potentially serve as suitable clinical biomarkers owing to their stability and accumulation in the circulatory system (Liang et al. 2017). Furthermore, exosomes potentially offer intracellular and extracellular compartments for condensed packaging of biomarkers. When normal cells are transformed into cancer cells, membrane proteins or lipids and cytoplasmic proteins can be glycosylated via post-translational modification (PTM). Exosomes secreted by cells not only contain various proteins and genetic components but also have membranes consisting of glycosylated proteins. Intracellular glycosylation is a crucial phenomenon regulating the cell status, physiological functions, and pathophysiological information. Moreover, glycosylation in cancer is considered a potential pathway for predicting cancer prognosis, and exosomal glycan moieties are promising diagnostic markers for cancer detection (Williams et al. 2018; Yokose et al. 2020).

The selective detection of glycans, or evaluation of their expression levels, could be promising for the diagnosis and prognosis of various cancers (Park et al. 2013; Su et al. 2015; Winter et al. 2013). As an indicator of cancer, aberrant glycosylation reflects alterations in glycan synthesis pathways, including overexpression of sialyltransferases or fucosyltransferases (Wang et al. 2014). The overexpression of these enzymes leads to an increased presentation of sialic acid- or fucose-linked glycans in proteins secreted by cells or on cellular membranes (Rillahan et al. 2012). In particular, α2,6- and α2,3-sialylation are closely related to cancer cell characteristics, and sialylations associated with antibodies, such as sialyl-Lewis A (SLe^A) and sialyl-Lewis X (SLe^X), have previously been utilized as cancer-related glycan markers (Tang et al. 2015, 2016).

The glycans related to cancer also exist in vesicles that are secreted by the cells (Escrevente et al. 2013). Among the cellular vesicles, exosomes are enriched with biomolecular contents, such as cell genetic factors, proteins, and major histocompatibility complexes. Consequently, exosomes have the potential to be used as biomarkers (Chulpanova et al. 2018). However, there are many obstacles to using exosomes as biomarkers, such as the difficulties associated with isolating exosomes as well as those with the selective detection of normal and disease-related exosomes. Conventional exosome-mediated detection techniques are based on the use of immunocapture assays which use antibodies to selectively detect disease-specific markers for separate downstream analysis (Im et al. 2014; Liang et al. 2017; Melo et al. 2015; Zhang et al. 2019; Zong et al. 2016). Although antibody-based exosome detection has been widely used, its accuracy for cancer diagnosis could be compromised due to the heterogeneity of cancer cells and the subsequent variations in the concentration or type of genetic information and proteins associated with cancers (Chen et al. 2017; Lindstrom et al. 2012).

Approaches targeting cancer-derived exosomes using glycan-specific moieties might effectively overcome the current obstacles in cancer diagnosis and detection. Lectins, which are glycan-binding proteins, could be utilized to specifically target glycan characteristics in cancer cells for the diagnosis of diseases. Because lectins have multiple glycan-binding sites, there have been numerous attempts to investigate glycan-regulated biological processes to enable their use and application in clinical diagnoses (Wan et al. 2015). In particular, a recent study reported the use of lectins to quantify specific glycosylated proteins from the sera of cancer patients (Bertok et al. 2013). The glycan-binding affinity of lectins could be utilized to develop a selective and simple exosome-capture method based on the specific interactions of certain glycans with lectins. Moreover, unlike conventional biomolecular detection for cancer diagnosis, the use of the lectin–glycan interaction is likely to avoid non-specific competition from proteins or peptides in blood or the denaturation of the markers.

Therefore, lectin-based exosome detection was achieved using bifunctional nanoparticles in a microfluidics chip (Exo-chip) (Fig. 1). The bifunctional nanoparticle, Janus nanoparticle (JNP), was first prepared using polystyrene nanoparticles by depositing gold on the half surface of particles. The JNP offers two moieties: one for the binding of lectin on the polystyrene surface and the other for the immobilization of particles in a microfluidic device (i.e., Exo-chip). Exo-chip contains two separate areas: sample mixing and detection. Exo-chip can be used for capturing and monitoring exosomes, using JNPs. Selective exosome detection was evaluated using lectin-conjugated JNPs in Exo-chip. The performance of exosome detection mediated by glycan was compared with the results of that using CA19-9, a conventional pancreatic cancer marker (Fig. 1a). This proposed system may be particularly useful for clinical diagnosis because of its capability to detect specific exosomes in biological fluids containing a series of biomolecules.