Exploring the potential of exosomes in diagnosis and drug delivery for pancreatic ductal adenocarcinoma

4.1 Representative isolation and detection techniques for PDAC-exosomes

Isolation of exosomes is the first step in the diagnosis based on PDAC-exosomes. Ultracentrifugation, ultrafiltration, size-exclusion chromatography (SEC) and immunoaffinity chromatography (IAC) are representative exosome separation techniques.^35-42 Electron microscopy (EM) and flow cytometry are classic exosome detection methods.^43-47 Microfluidic technology is a novel exosome detection technology (Table 1).

Ultracentrifugation is the most widely used exosome-separation technology. Ultracentrifugation is suitable for the initial purification of exosomes.³⁷ Ultrafiltration places a high demand on membranes with specific pore sizes. Before the discovery of exosomes, SEC was widely used in the high-resolution separation of macromolecules, such as proteins, polymers and various liposome particles.^{35, 38} However, SEC cannot effectively separate substances with similar molecular weights.^{35, 38} High-purity exosomes separated by IAC provide convenience for quantitative analysis. Only exosomes containing the target protein can be adsorbed by the corresponding antibodies.⁴⁰ Different separation and purification methods have different effects on exosomes. Ultracentrifugation may lead to the structural disruption of exosomes and co-precipitation of soluble protein impurities. Moreover, the yield of exosomes obtained by ultracentrifugation is low.⁴⁸ The efficiency and yield of ultrafiltration to isolate exosomes are higher than those of ultracentrifugation, but ultrafiltration may also lead to the deformation and rupture of exosomes, which may bias the results of downstream analysis.³⁷ Compared to ultracentrifugation and ultrafiltration, SEC not only protects the structure of exosomes, but also effectively removes the contamination of soluble proteins and improves the purity of exosomes to provide the basis for DDSs and biomarkers.⁴¹ High cost remains an important factor limiting the large-scale application of IAC.⁴¹

EM and flow cytometry are classic techniques used to characterize the nature and structure of exosomes.^43-47 Microfluidics technology uses microtubes to analyze trace amounts of samples. Microfluidic technology has the characteristics of high detection sensitivity and fast detection speed that give it good clinical application potential.^{50, 51} Immunoaffinity-based microfluidic technology uses antibodies immobilized on chips to specifically recognize and adsorb exosomes with corresponding ligands. For example, immunoaffinity-based microfluidic technology can efficiently isolate exosomes from trace serum and cell culture media using anti-CD63 antibodies.⁵² Microfluidic technology can integrate exosome isolation and analysis to change the paradigm of exosome-based diagnostics.

4.2 The feasibility of exosomes as indicators for PDAC detection

Current diagnostic tools for PDAC are computed tomography (CT), magnetic resonance imaging (MRI), endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) and blood tests (Table 2).^{53-55, 64}

Carbohydrate antigen 19-9 (CA19-9) is the most commonly used blood test marker for PDAC. In addition to PDAC, other intestinal diseases may also lead to the upregulation of CA19-9 markers in the blood.⁵⁶ CA19-9 does not have sufficient specificity for early PDAC. The development of new auxiliary detection markers will contribute to improving the early PDAC diagnosis. Detecting PDAC by PDAC-exosomes has enough feasibility as follows: (a) MRI requires patients not to carry any metal objects to ensure safety, which means that people with metal grafts in their bodies cannot be tested by MRI. Contrast agents used for CT may cause serious adverse effects. EUS-FNA can lead to pathogenic infections and severe discomfort. In contrast, body fluid detection based on exosomes has lower requirements and side effects for patients.^{53-55, 65, 66} (b) Real-time accurate tracking of tumor physiological status can be realized via PDAC-exosomes, providing a reliable basis for the diagnosis and PDAC treatment.^{51, 67, 68} (c) In some clinical trials, the diagnostic sensitivity and specificity of miRNA detection indicators in PDAC-exosomes extracted from plasma were better than those of CA19-9. Although more clinical data are needed for further verification, PDAC-exosomes can be reliable diagnostic markers for PDAC.⁶⁹ Compared to other solid tumors, the early symptoms of PDAC are not obvious. It is difficult to detect early PDAC by using conventional methods. PDAC-exosomes carry various biological information about tumor cells and the detection of PDAC-exosomes is helpful for the early diagnosis of PDAC. Therefore, exosome diagnosis is an attractive option for the detection of PDAC.

Proteins and RNAs are the main indicators of PDAC-exosomes. The proteins in PDAC-exosomes can be divided into two categories: (a) proteins that are ubiquitous in most exosomes from different cells, such as CD9, CD63, CD81 and heat shock proteins^{57, 58}; and (b) proteins whose contents are specifically upregulated in PDAC-exosomes.^{57, 58} These proteins can be used as reliable detection indicators. For example, Glypican-1 (GPC1) is specifically enriched in PDAC-exosomes. GPC1 detection accurately distinguished healthy individuals from patients with early or advanced PDAC.⁵⁹

Circular RNAs in exosomes can be stored for a long time at room temperature and remain stable.⁶⁰ The expression of circular RNA-IARS was upregulated in PDAC tumors and plasma exosomes of patients with metastatic disease.⁶⁸ Circular RNA-IARS accessed human microvascular vein endothelial cells via PDAC-exosomes, followed by increased endothelial monolayer permeability to promote cancer invasion and metastasis.⁶⁸ Circular RNA-IARS could be a reliable indicator for early diagnosis and prognostic prediction of PDAC.⁶⁸ In addition, miRNAs are another candidate detection indicator. A clinical trial showed that miRNA-200b and miRNA-200c were overexpressed in serum PDAC-exosomes. The combined diagnosis with miRNA-200b and miRNA-200c made the diagnostic accuracy of CA19-9 increase to 97%.⁶¹ In another clinical trial, miRNA-1246, miRNA-4644, miRNA-3976 and miRNA-4306 were significantly upregulated in 83% of PC-exosomes of serum. The levels of these miRNAs in exosome-depleted serum were also upregulated. Their levels in exosome-depleted serum were still much lower than those in exosomes. This clinical trial combined miRNA detection with flow cytometry to provide reliable references for the early diagnosis of PDAC.⁴⁹ A recent clinical trial showed that the combination of CA19-9 detection and miRNA sequencing analysis of exosomes in blood both increased the sensitivity and specificity of distinguishing PDAC and pancreatitis to approximately 90%.⁶² The serum-based exosome detection is expected to become an auxiliary detection method for PDAC screening in the future.

A recent clinical trial demonstrated the feasibility of screening for early-stage cancer by detecting tumor-specific substances in extracellular vesicles and particles (EVPs) in body fluids.⁶³ More than 400 samples of human tissues, blood and other bodily fluids were collected. These samples covered 18 different stages of cancer, such as breast, colon, lung, pancreatic and so forth.⁶³ The results showed that (a) there were large differences in EVPs proteins among humans and mice, which meant that we cannot rely solely on mouse models to simulate human liquid biopsy markers; (b) there was a group of multiple proteins in EVPs. The levels of these proteins were significantly higher in EVPs derived from patients with cancer. They could be used as tumor diagnostic markers, and (c) cancer types corresponding to EVPs could be analyzed and judged using reasonable statistical methods.

Compared to classical imaging methods such as CT, MRI and EUS-FNA, PDAC diagnostic methods based on exosome detection are better at providing case analysis based on biochemical information rather than imaging information. In fact, for small lesions in the early stage of solid tumors, classical imaging methods may not provide sufficient information. PDAC diagnostic methods based on exosome detection may be more important in the early stages of a cancer diagnosis.

The following points can help improve the early diagnosis of PDAC: (a) middle-aged and elderly people are at higher risk of developing PC. They should receive regular early screening and diagnosis. (b) The comprehensive application of different diagnostic methods can help promote the early diagnosis of PC. CT and ultrasonography are noninvasive imaging methods. The combined detection of CT and ultrasonography is suitable for diagnosing the underlying lesion structures. Serum exosome screening can be used to detect tumor-specific markers of PDAC. The combined detection of CA19-9 and exosomal diagnostic markers in serum can help to improve the accuracy of diagnosis; and (c) the development of more efficient automated detection systems can cover more populations and facilitate an early diagnosis of PDAC.

5.1 Tumor targeting of exosomes

Tumor targeting is an important DDSs indicator. Studies have shown that unmodified tumor-derived exosomes are poorly targeted to tumors.⁷⁰ Unmodified tumor-derived exosomes were quickly cleared after intravenous injection in mouse models, after which tumor-derived exosomes showed affinity for tumors, and it was related to the similar cell membrane composition of tumor-derived exosomes and intratumoral cells.⁷⁰ Therefore, it is important to enhance the tumor targeting of native exosomes.

Displaying molecules that enhance tumor targeting on the cell membrane of tumor-derived exosomes is an important way to enhance tumor targeting. For example, the expression level of the epidermal growth factor receptor (EGFR) is significantly upregulated in human breast cancer cells and exosomes. EGFR can serve as a receptor target for DDSs. Epidermal growth factors significantly promote mitosis and angiogenesis in cancer cells. Therefore, Ge11 peptides with higher biosafety were screened.⁷¹ Ge11 peptides were found to specifically bind to EGFR in breast cancer.⁷¹ Most importantly, Ge11 peptides did not significantly promote mitosis and neovascularization in breast cancer. The Ge11 peptide gene was then integrated into HEK293 cells (RRID: CVCL_0045). Exosomes secreted by HEK293 cells could efficiently target breast cancer cells in mouse models. Considering that the expression of EGFR is significantly increased in most PDACs, similar tumor-targeting enhancement strategies may also be applicable to PDACs in the future.⁷²

5.2 Advantages of exosomes as drug delivery systems for PDAC

Chemotherapies generally have defects such as a high circulation clearance rate and serious side effects. As novel DDSs, liposomes, nanomaterials, antibody-drug conjugates and exosomes are expected to improve the PDAC prognosis.^73-78

Exosomes have the following characteristics required for ideal DDSs: (a) they generally have low levels of immunogenicity and cytotoxicity⁷⁹; and (b) they can improve drug stability to reduce dosages and side effects. For example, CD47 is ubiquitous in most exosomes derived from human tissue. CD47 significantly delay the exosome clearance by monocytes and macrophages⁸⁰; and (c) exosomes efficiently penetrate biological membranes to improve drug delivery efficiency. Exosomes generally have good biocompatibility and affinity with corresponding donor cells or tissues.⁸¹ Exosomes, nanomaterials, liposomes and antibody-drug conjugates have been compared from the perspective of their properties as DDSs (Table 3).

Most studies have transformed exosomes derived from healthy cells into DDSs for the PDAC treatment. Exosomes rich in miRNA-1231 extracted from mesenchymal stem cells (MSCs) significantly inhibited the PDAC progression in mouse models.⁸⁸ The miRNA-3607-3p in exosomes derived from NK cells significantly inhibited PDAC metastasis by directly targeting interleukin-26 in PANC-1 cells (RRID: CVCL_0480) in mouse models.⁸⁹ In summary, some exosomes derived from healthy cells are not only good drug delivery vehicles, but also contain natural anticancer molecules.

The methods for loading therapeutic molecules into exosomes can generally be divided into endogenous and exogenous modes (Figure 4). The endogenous mode means that therapeutic molecules are first incubated with donor cells. When released from donor cells, the drug-loaded exosomes are separated and purified. The endogenous mode is simple to operate. However, it cannot precisely control drug loading efficiency and may destroy the natural physiological functions of membrane proteins.^82-84 Exosomes are first separated and purified from donor cells and then loaded with therapeutic molecules using methods such as electroporation. The exogenous mode may cause problems such as exosome aggregation and membrane damage.^82-84

5.3 Exosomes carry multiple types of anticancer drug molecules

Safe delivery of therapeutic molecules into PDAC tumors represents a bottleneck in clinical treatment. Studies have shown that exosomes have great potential to carry different therapeutic molecules, including proteins, cytokines, chemical drugs, RNAs, tumor antigens and immune adjuvants.

5.4 Key clinical applications of exosomes in PDAC

Diagnosis and treatment are two important applications of exosomes in the clinical treatment of PDAC.

Recently, two mRNAs (WASF2 and ARF6) and two small nucleolar RNAs (SNORA74A and SNORA25) of exosomes in serum have been proved to be effective in distinguishing patients with PDAC from control participants in clinical trials.¹⁰⁰ The sensitivity of this assay exceeds that of conventional CA19-9. The detection of tumor diagnostic markers in PDAC-exosomes is expected to become an important clinical auxiliary detection index in the future.

In terms of diagnosis, the isolation of high-purity PDAC-exosomes from clinical samples at a low cost is a key issue. Recently, microfluidic technology has become more effective in the separation and detection of exosomes in clinical samples. As early as 2010, a microfluidic immunoaffinity device was able to rapidly isolate exosomes using anti-CD63 antibodies.⁵² The device could effectively separate exosomes from the cell culture medium and serum for subsequent RNA detection and analysis. In the future, effective early screening of PDAC may be realized using trace amounts of blood extracted from fingertips.

In terms of treatment, the formulation of a scientific and effective manufacturing practice for medical exosomes remains controversial. The evaluation of the conditions under which patients are suitable for exosome-based treatment involves ethics and laws. In addition, it may not be realistic to extract sufficient amounts of exosomes from patients under poor conditions. In fact, PDAC-exosomes are at risk of promoting cancer development and metastasis. The potential risks of cancer-derived exosomes require further exploration. Recently, exosomes extracted from normal somatic cells, which are convenient for large-scale production, have been shown to be biologically safe. However, tumor-targeting of exosomes derived from healthy cells is usually insufficient. Modification of exosomes with materials such as nanoparticles and antibodies against cancer cells is an important method for enhancing the tumor targeting of exosomes. Modification and transformation based on tumor-targeting molecules and magnetic nanomaterials can effectively improve the tumor-targeting ability of exosomes derived from healthy cells.^101-104

Clinical trials on DDSs based on exosomes are ongoing. A phase I clinical trial of MSC-derived exosomes loaded with siRNA for the treatment of metastatic PDAC with KrasG12D mutations is currently underway (ClinicalTrials.gov identifier: NCT03608631). This phase I trial mainly studied the optimal dose and side effects of siRNA-loaded exosomes. Engineered exosomes may be effective in treating PC in related reports.

5.5 Engineered exosomes reverse drug resistance of PDAC

Colorectal cancer (CRC) is the third most common cancer in the world. The chemotherapy based on 5-FU is a typical treatment regimen for CRC. Long-term 5-FU treatment can lead to drug resistance. Deregulation of miRNAs in cells is an important factor in inducing CRC resistance to 5-FU. CRC upregulates intracellular miRNA-21 to inhibit the expression of human DNA MutS homolog 2 (hMSH2) and enhances drug resistance to 5-FU.¹⁰⁵

The resistance of colon cancer cells to 5-FU could be significantly reversed by exosomes loaded with the miRNA-21 inhibitor and 5-FU in mouse models.¹⁰⁶ First, engineered exosomes containing Her2 proteins were cultured and extracted from the genetically modified HEK293T cell line (RRID: CVCL_0063).¹⁰⁶ Her2 was a membrane protein and a specific tumor-homing polypeptide. Her2 enhanced the targeting of engineered exosomes to HCT 116 (RRID: CVCL_0291) colorectal cancer cells. The inhibitor of miRNA-21 and 5-FU were then co-loaded into engineered exosomes by electroporation.¹⁰⁶ After systemic administration, engineered exosomes significantly downregulated miRNA-21 levels and promoted 5-FU uptake in the 5-FU-resistant cell line HCT 116. The downregulation of miRNA-21 promoted the apoptosis of malignant cells and restored the expression of miRNA-21 regulatory targets PTEN and hMSH2. Compared to the administration of miRNA-21 inhibitor or 5-FU alone, the combination of miRNA-21 inhibitor and 5-FU via engineered exosomes effectively reversed the resistance of HCT 116 cells to 5-FU and significantly inhibited tumor progression in mouse models. In fact, the level of miRNA-21 was also significantly upregulated in malignant PDAC cells.¹⁰⁷ Therefore, the combined administration of miRNA-21 inhibitors and drug molecules via engineered exosomes could reverse PDAC resistance and improve prognosis.