Cancer-Derived Exosomes: Their Role in Cancer Biology and Biomarker Development

Introduction

There exist six well-researched cancer hallmarks, and they are as follows: (1) sustaining proliferative signaling, (2) evading growth suppressors, (3) enabling replicative immortality, (4) activating invasion and metastasis, (5) inducing angiogenesis, and (6) resisting cell death. However, two emerging hallmarks were identified and added by Hanahan and Weinberg:^1,2 (7) deregulating cellular metabolism and (8) avoiding immune destruction. Cancer cells typically acquire these core capabilities through sustaining selective pressures and adopting alterations in specific and ubiquitous cellular function, defined as “enabling characteristics,” to permit cancer hallmark capabilities. Enabling characteristics, which foster cancer growth and spread, are typically adopted by cancer cells to survive, proliferate, and escape elimination by the host environment. In the next-generation review by Hanahan and Weinberg on cancer hallmarks’ molecular mechanisms, they proposed two enabling characteristics that are present in cancer cells across the board, which are genome instability/mutation and tumor-promoting inflammation.¹

In this review, we propose pathological trafficking of genetic materials carried by cancer-derived exosomes (CDE) cargo as a new enabling characteristic that fits the proposed criteria (Table 1). This CDE cargo phenomenon has been demonstrated to be present in virtually all kinds of cancer cell types, fostering the core hallmarks of cancer by effective homotypic or heterotypic intercellular (tumor-to-microenvironment) communications, which facilitate cancer invasion and metastasis as well as evasion from immune destruction. The second part of the review is dedicated to the updated status of CDE being evaluated as a valid diagnostic cancer biomarker in human studies.

Table 1 Criteria for Classification as a Hallmark of Cancer or an Enabling Characteristic, According to Hanahan and Weinberg1,2

This study aims to present a comprehensive overview of the roles of CDE in cancer progression and development, assimilated from the current literature and translational cancer research to stimulate cancer biologists, scientists, and oncologists who are interested in the involvement of CDE in cancer pathogenesis, cancer microenvironment, molecular mechanisms on cancer progression and who plan to apply the knowledge in developing more effective diagnostic strategies for various types of cancer. Ongoing research studies evaluating CDE as a cancer biomarker registered in the ClinicalTrials.gov were selected to enrich the discussion on the clinical application of CDE as a biological cancer marker and to indicate future opportunities for cross-disciplinary collaborations.

Literature Search

To ensure a comprehensive and unbiased literature review, we performed both electronic and manual literature search in the PubMed^® and Scopus database to retrieve relevant original articles. We leveraged the use of PubMed Advanced Search Builder, Medical Subject Headings (MeSH), and Boolean logic to add terms or combine search terms using connector words, such as AND, OR, or NOT, as well as truncate terms. We used a controlled vocabulary to produce highly relevant search results. The search terms included exosome, exosomal cargo, cancer-derived or tumor-derived, cancer biology, proteasome, and cancer biology. Subsequently, we surveyed the ClinicalTrials.gov registry for clinical studies conducted in the United States and around the world.

Cancer-Derived Exosomes in Cancer Biology

The tumor microenvironment (TME) surrounding cancer cells is identified to be comprised of cancer-associated fibroblasts, blood vessels, nerve fibers, immune cells, other stromal cells, and extracellular vesicles containing various kinds of genetic signals. Considering all are functional in anticancerous immunosuppressive cells, the TME is known to create a milieu that prevents the free spread of the malignant cells.³ The cancer cells, however, communicate with the neighboring stromal and immune cells, promoting immune evasion, and could also activate angiogenesis, tumor innervation, and epithelial-mesenchymal transition (EMT), in order to facilitate neoplastic growth. Recently, a phenomenon involving tumor-infiltrating innervation in the TME has also been proposed as a prerequisite for cancer cells of many types such as in prostate, gastric, pancreatic, and rectal cancers.^4–6 Tumors are capable of recruiting nerves via the release of neurotrophic factors and axonal guidance molecules, and, with the contribution of CDEs, induction of axonogenesis is initiated, whereby the communication between the tumor and potentially innervating nerves work in concert to promote tumor innervation.⁴ Thus, it has been proposed that tumor innervation with neurite outgrowth (axonogenesis), just like angiogenesis, might be considered a new emerging hallmark of cancer.^7–13 Although research has demonstrated that angiogenesis is frequently associated with axonogenesis, more studies are eagerly required to elucidate the roles of tumor-infiltrating innervation in tumor initiation, growth, and spread for this phenomenon to be widely accepted as a hallmark of cancer. Recent studies were able to confirm that the neurite recruitment/outgrowth and tumor innervation were promoted by the release of exosomes in the head and neck squamous cell carcinomas model⁵ and human papillomavirus-positive uterine cervical cancer cell lines.⁶

The name “tumor-derived exosomes” was coined in 1981, and this phenomenon has received extensive research attention over the past decade.¹⁴ Exosomes are extracellular, membranous, cup-shaped microvesicles 30 to 100 nm in size, which are produced by most types of cells.^15–17 They originate from intracellular multivesicular bodies and are released by exocytosis into the extracellular microenvironment.¹⁸ Exosome typically consists of a variety of genetic messengers such as DNA, mRNA, microRNA, cytosolic proteins, and lipids.^19–23 Exosomal markers such as tetraspanin proteins CD63, CD9, and CD81 allow sorting, selective recruitment, capturing, or profiling of CDEs.^24,25 Once the recipient cells internalize tumor-derived exosomes, the ensuing biological response is determined explicitly by the dedicated trafficking routes, the exosomal internalization pathway, and the complex surface molecules on the membrane of both the extracellular vesicle and the recipient cell. With the advent of theranostic nanotechnologies such as differential ultracentrifugation, nanofluidic technology, and the exosome total isolation chip (ExoTIC), a size-based extracellular vesicle isolation apparatus, researchers nowadays are now able to capture nano-sized CDE for further analyses.^26–28 The latest biosensing technologies, such as afterglow sensors with aptamer-based signal amplification, improve the limit of detection (LOD) that is nearly two orders of magnitude lower than that of fluorescence methods.²⁹ With the advent of these sensitive biosensors, the LOD can practically be improved to 10² exosomes per milliliter.

These exosomes, particularly those that are tumor-derived, act as signal transducers or messengers in the cell-cell communication.^5,30–33 The recipient cells respond to the exosomal contents (such as microRNA) by changing their phenotypes. microRNAs are considered an evolutionarily conserved family of molecules that bind to complementary sequences in the 3ʹ-untranslated region (3ʹUTR) of their target mRNAs, post-transcriptionally repressing gene expression.³⁴ It has been demonstrated that in high-grade bladder cancer cell line, TCC-SUP, for example, exosomes promoted angiogenesis and migration of both cancer and endothelial cells.³⁵ In another study in prostate cancer, the malicious CDEs induced differentiation of the stromal mesenchymal stem cells toward alpha-smooth muscle actin-positive myofibroblasts, which secreted high levels of proangiogenic VEGF-A, pro-invasive HGF, MMP-1, MMP−3, and MMP−13.³⁶

The role of CDEs as characteristic enablers of cancer hallmarks to facilitate organ-specific metastasis has been demonstrated by the proof-of-principle study conducted by Hoshino et al,³⁷ In their peripheral blood study of mouse and human cell lines, they claimed that during the metastatic cascade, organ-specific metastasis took place not by a random process but by somewhat predictable and trackable events. This happened through distinct integrin expression patterns contained in the CDEs, a phenomenon that now elucidates the mechanism of specific cancer organotropism adequately. The exosomal integrin αvβ5 was associated with hepatic metastasis, while exosomal integrins α6β4 and α6β1 were linked to lung metastasis.³⁷

In the following sections, we will use three different cancer types: hematopoietic, epithelial, and sarcomatous malignancies (leukemia/myeloma, breast cancer, soft tissue sarcoma, and osteosarcoma) to prove, using compelling evidence, that cancer cells across the board leverage the pathological trafficking of exosomes to promote neoplastic growth, facilitate cancer spread through tumor-stromal interaction, and evade destruction by the host (Figure 1).

Figure 1 Two established enabling characteristics (genome instability or mutation and tumor-promoting inflammation): one investigating feature (tumor innervation) and one hitherto proposed enabling characteristic, that is, pathological exosome trafficking. Enabling characteristics are defined as the capabilities possessed by most cancer types to foster and/or expedite the acquisition of one or some core hallmarks of cancer.

Exosomal Trafficking in Leukemia Pathogenesis

Although leukemia can reach every part of the host body through the ever-reaching blood vessels, recent research has reported that leukemic cells also employ paracrine exosome trafficking to achieve leukemogenesis, maintain leukemic persistence by shaping the leukemic niche and its progression, suppress hematopoiesis, modify anti-leukemic immunity, and evade destruction by chemotherapy. Table 2 illustrates these aspects taking acute myeloid leukemia (AML) as an example.

Table 2 Paracrine Exosome Trafficking Employed by Acute Myeloid Leukemia (AML) as an Example and Its Specific Functional Outcomes

Patients with chronic lymphocytic leukemia (CLL) have been identified to have decreased T-cell immunity. A recent study showed that CLL induced myeloid-derived suppressor cells (MDSCs), which, in turn, suppressed T-cell activation and induced suppressive regulatory T cells (Treg) through exosomal miR-155 transfer.^38,39 This exosome-mediated transfer of microRNAs to monocytes could significantly contribute to CLL-related immune escape via PD-L1 expression. Colleagues from the University of Liverpool verified that CLL-derived exosomes encapsulate small RNAs, and the encapsulated microRNA miR-202-3p enhanced the expression of a Hedgehog signaling intermediate.⁴⁰ An enriching body of evidence shows that the TME created by the bone marrow significantly favors the survival, growth, and proliferation of leukemic cells. For example, CLL leukemic cells can establish their favorable leukemic niche in the TME. Paggetti et al have discovered that CLL-derived exosomes could affect bone marrow stromal cells in adopting a cancer-associated fibroblast phenotype, which would contribute to a tumor-supportive microenvironment.⁴¹ In the chronic myeloid leukemia (CML) model, CML-derived exosomal paracrine trafficking stimulated bone marrow stromal cells to produce interleukin (IL)-8.^42,43 Further, another study demonstrated that exosomes released from CML cells affect the endothelium directly to modify the neovascularization process.⁴⁴

Exosomal Trafficking in Multiple Myeloma (MM) Pathogenesis

In a murine MM model, the myeloma exosomes were identified to have a proangiogenic function to enhance the viability of bone marrow endothelial cells; besides, an in vivo experiment demonstrated that these exosomes increased the presence of bone marrow MDSCs and changed their subsets to a more tumorigenic profile.⁵⁵ MM-derived exosomes could modify the bone marrow microenvironment to facilitate myeloma progression. Conversely, the bone marrow stromal cells could, reciprocally, also release certain exosomes to be taken up by MDSCs through the STAT3 and STAT1 pathways, which leads to increased immunosuppression, thereby inducing MM expansion.⁵⁶ Initially, investigators from the Dana-Farber Cancer Institute demonstrated that there were significant differences in microRNA profiling between normal and bone marrow mesenchymal stromal cell-derived exosomes in MM.⁵⁷ A recent study in patients with MM using small RNA sequencing of circulating exosomes from ten patient samples confirms that microRNAs are the most predominant small RNAs present in MM exosomes.⁵⁸ Meanwhile, investigators from the Karolinska Institute have examined the human bone marrow stromal cell line L88 and verified that caspase-3 is activated by the stroma cell–released exosomes, which can cleave the anti-apoptotic protein Bcl-xL, localized on the outer exosomal membrane. Through the cleavage of Bcl-xL, these exosomes could then be internalized using plasma cell myeloma, which led to their increased proliferation.⁵⁹

Finally, in another study performed at Tokyo Medical University, researchers established a hypoxia-resistant MM cell model to mimic the in vivo hypoxic microenvironment induced by the rapid proliferation of MM in the bone marrow. Their experiment showed that under normoxic or acute hypoxic settings, the hypoxia-resistant MM cells produced more exosomes than the parental cells, and the major functional protein in the exosomal cargo was identified to be miR-135b. This protein directly suppressed factor-inhibiting hypoxia-inducible factor 1 (FIH-1) in bone marrow endothelial cells.⁶⁰ Hence, further studies are needed to test if miR-135b could be used as a target for therapeutically avert angiogenesis in MM.

Exosomal Trafficking in Breast Cancer Pathogenesis

A study using plasma exosomal microRNAs as a diagnostic biomarker in breast cancer patients demonstrated that these molecules have outstanding power to distinguish breast cancer patients from normal counterparts. Zhai et al used a nucleic acid-functionalized Au nanoflare probe, which are known to have the ability to directly enter plasma exosomes and generate quantitative fluorescent signals for successful in situ detection of exosome-located microRNA-1246. At its best cutoff point, the in situ detection of the exosomal miRNA-1246 in the peripheral blood was able to distinguish 46 breast cancer patients from 28 healthy controls with 100% sensitivity and 93% specificity.⁶¹ Another clinical study on the exosomal microRNA signatures of 20 healthy women and 435 breast cancer patients discovered that 10 miRNAs in the entire breast cancer patient cohort, 13 in the HER2-positive subgroup (211 patients), and 17 in the triple-negative subgroup (224 patients) were significantly deregulated in comparison to those in healthy women, indicating different underlying aspects of cancer biology in different breast cancer types.⁶² These different exosomal microRNA signatures are associated with the clinicopathological features of each subgroup. In addition, exosomes that are released by breast cancer cells could modify TME through direct suppression of T-cell proliferation and inhibition of NK cell cytotoxicity, thus dampening the anticancer immune response in pre-metastatic organs.⁶³

Another hallmark of cancer is the transfer of chemoresistant or hormone-resistant propensity from breast cancer stem cells to the daughter cells, explored in the study of Santos et al, who demonstrated that miR-155 was upregulated in breast cancer stem cells and chemoresistant cells and was involved in the EMT. An enrichment in miR-155 was noted in exosomes isolated from stem-like breast cancer stem and chemoresistant cells. Moreover, the experiments demonstrated the capability of the horizontal transfer of miR-155 from the chemoresistant cells’ exosomal cargo to the recipient sensitive cells.⁶⁴ This study supports the presence of exosome-mediated chemoresistance and EMT in refractory cancer. Estrogen receptor (ER)-positive cancers are found to transition from an endocrine sensitive/dormant state to a resistant one, acquiring host mitochondrial DNA, which promoted oxidative phosphorylation (OXPHOS) and signaled the transition from metabolic quiescence toward hormonal therapy resistance.⁶⁵ Further, functional studies have identified cancer-associated fibroblast-derived extracellular vesicles containing whole genomic mitochondrial DNA in patients and xenograft models.

In a breast cancer cell line, recipient cells treated with exosomes from stemness-related breast cancer CXCR4-positive cells showed an increase in the same oncogenic abilities.⁶⁶ This experiment has also demonstrated that inoculating exosomes derived from CXCR4-positive cells into immunocompromised mice can stimulate primary tumor proliferation and metastatic potential. The same investigators also discovered a “stemness and metastatic” signature in the exosomes of patients with worse prognoses after comparing exosomal nucleic acid contents.⁶⁶

Exosomal Trafficking in the Pathogenesis of Soft Tissue Sarcoma

In 2013, for the first time, a study has showed that exosome-mediated pathogenesis, similar to epithelial carcinoma and hematopoietic malignancy, was also present in Ewing sarcoma.⁶⁷ Microarray analysis of exosomes shed by the Ewing sarcoma cell line revealed that their exosomal content shared a transcriptional signature potentially involved in the modification of the surrounding microenvironment via G-protein-coupled signaling, neurotransmitter signaling, and stemness.⁶⁷

A recently published study used both patient plasma samples and cell lines to demonstrate that liposarcoma cells secreted miR-25-3p and miR-92a-3p in exosomes. Subsequently, it stimulated the secretion of the proinflammatory cytokine, interleukin (IL)-6, in tumor-associated macrophages through a TLR7/8-dependent mechanism, which can ultimately cause liposarcoma progression.⁶⁸ In another study using patient-derived Ewing sarcoma cells, miR-34a, an inhibitor of Notch-NFκB signaling, was enriched and secreted through exosomes shed by CD99-silenced (by small interfering RNA) cells.⁶⁹ CD99 has been identified as a cell surface molecule involved in cell differentiation, migration, and death. In Ewing sarcoma cells, it is pro-oncogenic due to its effect on the prevention of NFκB-mediated neural differentiation and is continuously present at high levels. The horizontal transfer of miR-34a through exosomes to recipient cells enhanced neural differentiation in Ewing sarcoma.⁶⁹

Moreover, another study demonstrated that the membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP14) was released by exosomes of cultured human fibrosarcoma (HT-1080) cells.⁷⁰ MT1-MMP is a crucial metalloproteinase that facilitates tumor invasion by remodeling the extracellular matrix. Pathological sarcomatous exosomal trafficking carrying MT1-MMP could be detrimental to the host by providing a favorable microenvironment for sarcoma.

Exosomal Trafficking in Osteosarcoma Pathogenesis

Osteosarcomas are known as malignant mesenchymal-derived bone tumors and the most common bone cancers in children and adolescents. Emerging evidence has also shed light on the exosomal trafficking employed by osteosarcoma cells to shape its supporting TME and facilitate growth, as well as hematogenous spread. Among the specific exosomal contents, miR-148a and miR-21-5p are known to help shape the TME.⁷¹ The microRNA, miR-21, is a common oncological molecule taking part in the pathogenesis of various types of malignancies.^71–80 Take esophageal cancer as an example; it has been demonstrated in a human esophageal carcinomas cell line co-cultivation experiment that miR-21 in the CDE shuttled from donor cells significantly promoted the migration and invasion capability of recipient cells by activating c-Jun N-terminal kinase signaling pathway.⁷⁴ A recent multi-omics study observed that the progression from localized to metastatic osteosarcoma was accompanied by an elevation of the levels of urokinase plasminogen activator (uPA) and uPA receptor in the metastatic cells’ exosomal cargo.⁸¹ The impact of abundant miR-25-3p in the liposarcoma-derived exosomes on the surrounding microenvironment was similar to what was observed in osteosarcoma cases.⁸² Jerez et al conducted a gene ontology analysis of predicted targets for the miRNAs present in osteosarcoma-derived extracellular vesicles. Their bioinformatics analysis indicated that miRNAs derived from osteosarcoma cell lines might regulate metastatic potential by inhibiting a network of genes involved in apoptosis and cell adhesion.⁸³ Further research is needed to provide more evidence on the details and importance of exosomal trafficking in osteosarcoma pathogenesis and to determine it as a core hallmark of cancer.

Other Molecules (Proteins, Enzymes, Receptors, Ligands, or Signaling Molecules) That Can Exert Neoplastic Functional Activities Carried by Exosomes

Certainly, exosomal cargoes are not limited by miRNAs only but also by a lot of other candidate molecules such as proteins, lipids, enzymes, signaling molecules, which can exert their functional activities far from the exosome-producing cells.⁸⁴ Table 3 demonstrates that CDE cargoes could contain basically any purpose-built loaded nano-molecules for its ultimately release from the parental cells. It is evident that horizontal or paracrine transfer of these molecules, when received by the specific recipient cells in the TME or any distant metastatic niches, could facilitate the progression, invasion, and metastatic spread of cancer cells. Some of these exosomal molecules have the potential to serve as valid biomarkers, and, thus, there should be worthwhile testing for cancer detection and/or diagnosis.

Table 3 Representative Exosomal Cargoes Other Than miRNAs (Such as Proteins, Lipids, Signaling Molecules, DNA, Mitochondrial DNAs, circRNAs, lncRNAs, Integrin, and Enzyme), Which are Involved in Cancer Progression or in the Interplay in Anticancer Immunity or Served as a Biomarker in Various Cancer Types

In summary, it is evident that cancer cells from entirely different lineages, such as those from leukemia to osteosarcoma, rely on their exosomes to carry the genetic information for homotypic and heterotypic intercellular communications in the TME (Figure 2). This communication creates a favorable environment for cell proliferation and invasion and further establishes a pre-metastatic niche that is readily welcoming for the arrival of cancer cells when they carry the correct form of exosomal integrins. Therefore, these CDEs and their pathological trafficking capabilities should be considered as an emerging enabling characteristic for the well-established hallmarks of cancer.

Figure 2 Effects of tumor-derived exosomes and their horizontal paracrine trafficking to impact on the tumor microenvironment. For example, breast cancer-derived exosomes modify the TME through the suppression of T-cell proliferation and NK cell cytotoxicity. Also, the exosomal content (eg, miR-1246 or miR-155) might contribute to the chemoresistance or hormone-resistance in tumor cells. Exosomes secreted by liposarcoma cells containing miR-25-3p and miR-92a-3p have been found to stimulate IL-6 secretion in tumor-associated macrophages, leading to liposarcoma progression. miR-34a in the released exosomes enhances the neural differentiation of Ewing sarcoma. Myeloma-derived exosomes could modify the microenvironment, affecting various recipient cells such as bone marrow endothelial cells or myeloid‐derived suppressor cells. In the case of myeloma, these exosomal cargoes include miR-135b, miR18a, and let7b. After being internalized by recipient cells, miRs could bind to their target genes and trigger numerous pathways to facilitate tumor progression.

Cancer-Derived Exosomes in Biomarker Development

The first half of the review has exemplified the role of exosome in specific cancer types. Shifting gear to discuss the potential clinical application, this review will examine and discuss how we can develop cancer biomarkers based on characteristics such as exosomal cargo contents, detection methods, and localization of these exosomes in peripheral blood, pleural effusions, ascites, or urine. Further clinical studies are eagerly awaited to establish and validate the usefulness of specific CDE biomarkers in the different clinical setting during the cancer management. For example, in breast cancer, we can leverage the characteristics of exosomal microRNA signatures and exosomal nucleic acid contents in assisting in breast cancer subtyping or discovering a stemness and metastatic signature, as mentioned previously. Similarly, in the case of soft tissue sarcoma, in recognition of the role of exosomal cargo transcriptional signatures or microRNA profiling including miR-25-3p, miR-92a-3p, miR-34a, MT1-MMP, and MMP-14 playing in the promotion of sarcoma progression, remodeling extra-sarcomatous matrix to facilitate tumor invasion, and establishing a favorable TME for sarcoma growth, we can develop a practical analysis to investigate these soft tissue sarcoma-derived exosomes in assisting diagnosis or monitoring of disease along with the treatment milestones for a patient.

Although the technology for developing exosome-encapsulated therapeutics as targeted drug delivery is still in infancy, with the help of improving detection methods, rapid application of the analytic tests for specific exosomal cargoes for diagnostic purposes has become feasible, facilitating exosomal biomarker development. Several characteristics of CDEs, previously discussed in the cancer biology section, such as analyzable cancer-specific and stage-specific genetic contents in the cargo of CDEs, allow us to capture, profile and quantify using the current nanoanalytical technology. The phenomenon of pathological exosomal trafficking during cancer development and progression can be utilized in cancer diagnosis, prognostication, and treatment strategies. Studies have demonstrated that CDEs containing enriched genetic signals involved in cancer initiation and progression are shed by cancer cells into the blood, saliva, urine, ascites, and even cervicovaginal lavage. In clinical oncology, a cancer biomarker can be used for a diagnostic purpose, for example, in differentiating cancer from the non-cancer conditions. It can also be used for disease monitoring during antineoplastic therapy or follow-ups, for prognosticating a patient’s survival, and for predicting a tumor response after anticancer treatment.

Current sophisticated purification techniques offer an opportunity to utilize isolated exosomal cargoes to assist in differentiating the type of cancer and high tumor grade from low-grade cancer.^{61,75,100–102} In a recent study, using a urinary exosome 3-gene signature obtained from the ExoDx Prostate IntelliScore urine exosome assay, the investigators can differentiate high-grade (Gleason’s score > 7) vs low-grade prostate cancer and benign prostatic hyperplasia.¹⁰⁰ This noninvasive urine testing implies that many unnecessary invasive transrectal biopsies could be avoided. As was aforementioned in the review, in patients with multiple myeloma, serum exosomal miRNAs could add to the risk stratification in identifying newly diagnosed multiple myeloma with particularly poor outcomes.⁵⁸ Table 3 presents the select representations of exosomal cargo other than miRNAs, namely, proteins, lipids, signaling molecules, DNA, mitochondrial DNAs, circRNAs, lncRNAs, integrin, and enzyme, which potentially serve as a biomarker in various cancer types. Laboratory analytic methods to measure these contents in the research included triple SILAC quantitative proteomic analysis, mass spectrometry (MS)-based proteomic assays, lectin blotting, NP-HPLC analysis, ultrahigh-resolution Fourier transform MS, shotgun and targeted molecular quantitative lipidomic assays, capillary liquid chromatography-MS, MALDI MS, electrospray ionization MS, dsDNA-specific shrimp DNase and atomic force microscopy, RNA-seq analysis, transmission electron microscopy, nanoparticle tracking analysis, etc., depending on the study design (Table 3).

One of the advantages of investigating the CDEs as either a diagnostic, prognostic, or predictive biomarker is that physicians can obtain a specimen for CDE testing from a patient via relatively noninvasive methods. The shed CDEs into body secretion or discharges such as saliva, ascites, and cervicovaginal lavage can now be noninvasively or microinvasively assessed. In the past 5 years, clinical studies on exploiting CDEs as a clinical biomarker reported some promising results in various types of cancer (Table 4). The CDE cargo tested include lncRNAs, microRNAs, exosomal phosphatidylserine, urinary 3-gene expression profile, shuttle RNA pattern, RNA cargo, exosomal cancer stem cell-like marker CD133, exosomal EpCAM protein, and exosomal glutathione S-transferase P1 (Table 4).

Table 4 Published Clinical Studies on Exploiting Exosomes as Diagnostic, Prognostic, or Predictive Biomarkers in Various Types of Cancer

As of this writing, there have been several dozens of prospective observational studies being carried out to investigate the role of specific exosomal cargo as a cancer biomarker in various cancers and their diagnostic performance in a particular clinical setting (Table 5).

Table 5 Ongoing Human Studies Investigating Exosomes as a Biomarker in Various Types of Cancer, as Registered in ClinicalTrials.gov

Rapidly evolving nanotechnologies provide an opportunity to exploit and engineer exosomes for therapeutic purposes, which is gradually becoming a new class of cell-free nanomedicine. Therapeutic blockade of the exosome biogenesis to halt cancer progression at specific stages of the disease could be enticing in the development of cancer therapeutics.^80,114,115 The potential application of responsive exosome nano‐bioconjugates for cancer therapy has also been confirmed in a recent study; the nano‐bioconjugates can actively target tumors through the specific recognition on the surface of tumor cell and abolished signaling and improved phagocytosis of macrophages.¹¹⁶ There are growing interests in investigating engineered exosomes as potential therapeutic vehicles or an active drug delivery system.^117–123 Making use of the exosomal organotropic characteristics, exosomes loaded with therapeutic compounds could be employed to target a recipient cell to carry out gene therapy selectively.

The following examples shall illustrate how the application of exosomal engineering technology may enhance cancer therapeutics. Targeting the immune cells in the TME as an adjunct of anticancer treatment has been becoming a hot research area. In the application of nanomedicine, various forms of nanoparticles-bioconjugate exosomes have been synthesized and tested to target specific immune cells in the acidic TME. Recent research has demonstrated that anti-tumoral M1 macrophages-derived exosomes conjugated with CD47 and SIRPα antibodies effectively reprogrammed the macrophages from M2 to M1 phenotype in the TME.¹¹⁶ In another study in the living mice, cancer-associated fibroblasts in the TME can be specifically targeted by activated fibroblasts whose cell membrane was coated with semiconducting polymer nanoagents aiming to enhance multimodal cancer theranostics.¹²⁴

MicroRNA-21 is a well-known microRNA that overexpresses in almost all cancer types, where its upregulation promotes cell proliferation, invasion, and metastasis.^69–78 MiR-21 derived from the exosomes of MSCs regulates the death and differentiation of neurons in patients with spinal cord injury. Recent efforts involve utilizing an exosomal transfer of miRNAs or anti-miRNAs to tumor cells as a new approach for the therapeutic application of miRNAs to combat the most aggressive form of glioma, glioblastoma multiforme. Monfared and coworkers recently attempted to down-regulate miR-21 expression in glioma cell lines, U87-MG and C6, and rat glioblastoma models treated with miR-21-sponge exosomes and demonstrated a decline in tumor cell proliferation, a dramatic enhancement of apoptotic rate, and a significant reduction in tumor volume.¹²⁵

Conclusion

Cancer-derived exosomal trafficking is observed in almost all types of liquid or solid tumors, including leukemia, soft tissue sarcoma, and osteosarcoma, which supports its role as an enabling characteristic for cancer hallmarks. The cargoes carried by CDEs contain enriched genetic signals in the form of DNA, RNA, microRNA, lncRNA, protein, lipid composition, or specific gene expression profiles, which are shed into blood, saliva, urine, effusions, ascites, and cervicovaginal lavage. There are a growing number of studies that investigate CDE as either a diagnostic, prognostic, or predictive nano-biomarker in various kinds of cancer. Out of the published clinical studies on exploiting CDE as a cancer biomarker, 70% of them were looking at the CDE as a diagnostic biomarker. In contrast, the rest of the studies were testing the role of CDE as a prognostic or predictive biomarker. Not surprisingly, only a few of them have reached the state of validation trials. In the near future, we shall expect to see more prospective clinical trials to validate the performance of these nanoparticle biomarkers aiming to improve the overall cancer diagnostic capability in the clinic.