Open access peer-reviewed chapter

Extracellular Vesicles for Cancer Immunotherapy: Biomarkers and Beyond

Written By

Baranya Murugan and Suresh Sagadevan

Submitted: 04 December 2021 Reviewed: 17 February 2022 Published: 20 July 2022

DOI: 10.5772/intechopen.103783

From the Edited Volume

Extracellular Vesicles - Role in Diseases, Pathogenesis and Therapy

Edited by Manash K. Paul

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Abstract

Extracellular vesicles (EVs), like exosomes and microvesicles, are membrane-bound vesicles released by most cell types in response to cellular stress as well as normal physiologic conditions. EV plays a vital part in cell communication and tumor immunology. Tumor-derived EVs carry a wide range of tumor neoantigens and have a distinct molecular signature that reflects the tumor’s genomic complexities. These tumor-derived EVs provide a glance into the immunological tumor microenvironment and have a perspective to be a novel, minimally invasive cancer immunotherapy biomarker. Antibodies against immune checkpoint inhibitors like anti-programmed death-1 (PD-1) and its ligand (PD-L1) have changed the treatment of broad diversity of solid tumors such as non-small cell lung cancer, head, and neck squamous cell carcinoma, urothelial carcinoma, melanoma, etc. Invasive tissue biopsy is necessary for both histologic diagnosis and next-generation sequencing efforts. The latter has become increasingly widespread in today’s healthcare. There is an unmet need for non-invasive or minimally invasive (e.g., plasma-based) biomarkers in both diagnosis and therapy monitoring. The selected investigation of EV in biospecimens, including plasma and saliva, can achieve this goal by potentially avoiding the need for tissue samples. In this chapter, we discuss the present challenges of biomarkers in cancer immunotherapy and the mechanistic role of tumor-derived EV in regulating the anti-tumor immune response.

Keywords

  • extracellular vesicles
  • cancer
  • immune response
  • biomarkers
  • immunotherapy

1. Introduction

Exosomes were initially discovered in 1981 as exfoliating vesicles from different normal and neoplastic cell lines. Exosomes correspond to the family of extracellular vesicles (EVs), composed of microvesicles, apoptotic bodies, and exosomes [1, 2, 3]. Exosomes arise from the endo-lysosomal pathway and originate from the endosomal compartment known as the multivesicular bodies [4, 5]. The microvesicles are developed by sprouting from the plasma membrane. The microvesicles size is around 100–1000 nm and originates from sprout and fusion of plasma membrane into extracellular space, and sharing out different models with the parental cells, involving membrane lipids, receptors, and different types of nucleic acids and proteins [6, 7]. Depending on their size and shape, exosomes can be divided into nine different subpopulations or categories, signifying that exosome that arises from a single cell line is different from morphologically functionally [8]. EVs play a potential role in many factors such as cellular homeostasis, physiology, and pathobiology [9, 10]. In the tumor milieu, EVs are isolated from cancer cells, immune cells, and non-immune host cells delivering as a critical component of the tumor microenvironment. Isolation of EV from various roots exhibits distinct roles in tumor immune cells, leading to tumor proliferation, metastasis, and drug resistance [11]. Examinations from the past years have shown a curious spike of exosomes initiating to fuse and communicate their vital functions. EV are linked with different cell types which also have required macromolecules comprising DNA, micro-RNA, messenger RNA, proteins, and lipids [12].

Immune checkpoint blockade therapy has modified an environment for the treatment of cancer [13]. Therefore, deeper knowledge on deciding the success and failure of this therapy is required. Tumor-derived EVs are linked in immunological crosstalk are likely to be an emerging biomarker for cancer immunotherapy [14, 15]. Tumor-derived EVs are notably interesting targets as they are discharged from cancer cells as to normal cells and it has been segregated from different biospecimens such as blood, urine, cerebrospinal fluid, and saliva [16, 17]. Therefore, this chapter focuses on the current availability of biomarkers for cancer immunotherapy.

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2. Exosomes contents

The contents of the exosomes not only reflect the composition of the donor cell but also consider the controlled sorting mechanism. Composites of different proteins involving receptors, transcription factors, enzymes, extracellular matrix proteins, lipids, nucleic acids (DNA, mRNA, and miRNA) interior and present on the surface of the exosomes [18]. As compared to the other cell membranes, Exosomes manifested a superior expression of sphingomyelin, cholesterol, phosphatidylserine, and saturated fatty acids as compared to whole cell membranes, according to earlier reports [19]. The proteins present in the exosomes consist of the endosome, plasma, and nuclear proteins. An exosome of various cell types consisting of TSG101, Alix, Rab GTPases, heat shock proteins (HSP70, HSP90), integrins, tetraspanins (CD9, CD63, CD81), and MHC class II proteins. Furthermore, exosomes also carry genetic material like mRNA, long non-coding RNA, micro-RNA, and double-stranded DNA [20]. Furthermore, the contents of exosomes may differ from the cells of their parent cells due to the precise distribution of cargo into exosomes. As a result, there is not enough information on their back-and-forth mechanisms.

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3. Extracellular vesicles as biomarkers

During the 1990s National Institute of Health (NIH) has explained biomarker as a biological molecule or gene that is found in body fluids estimated as a marker of a biological, pharmacological, pathogenic process for therapeutic intervention [21]. EVs are systematically involved in intercellular communication and exhibited as qualitatively and quantitatively in diseases like cancer, it also plays a significant role in drug resistance. EVs are unique and can serve as a valuable tool to aid in cancer detection, prognosis, and tracking therapeutic efficacy. EVs circulate in bodily fluids, making them an ideal candidate for non-invasive testing [22]. In addition, the lipid bilayer shields the biomacromolecules like RNA, proteins, and enzymatic activity, EVs constitute a safe vehicle for evaluating genetic sequences. Liquid biopsy is an emerging technique, that requires implying the circulating tumor cells (CTCs), cell-free DNA (cfDNA), and EVs, which has brought new understanding and aspects to the field of cancer therapeutic interventions [23]. CTCs are cancer cells that are slough off from the primary or metastatic sites through the circulation. According to a few recent studies, molecular profiling of CTCs can be used to monitor patients who are receiving therapies [24].

The foremost difficulty in examining CTCs such as rarity of the cells in circulation (one CTC is found in per billion blood cells). Up to date, cell search, targeting epithelial cell adhesion molecule, EpCAM to procure cell CTCs is the most prominent cell CTC enumeration assay. There are other biomarkers such as cfDNA known as short fragments of nucleic acids which are seen in body fluids, like blood or urine. It is demonstrated to be produced by the apoptotic degeneration of cellular DNA [25], and a fragment of cfDNA is procured from the tumor cells and is explained as circulating tumor DNA, which is known to imitate the genetic and epigenetic alterations of the initial tumor and its possible to be used as a diagnostic and prognostic biomarker for cancers. Also, NGS is the most common method for examination of the genetic information of the cfDNA [26]. As compared to NGS, digital PCR can only do the screening for the known variants and has the capability for the limited sample in one reaction. But NGS is a high cost and relatively time-consuming technique, which also necessitates bioinformatics skills for data analysis and interpretation.

EVs have great attention as like another kind of biopsy. EVs are mostly found in different kinds of body fluids such as blood, saliva, urine, bronchoalveolar fluid, breast milk, and semen. They reproduce the disease type by taking the molecules from benign cells, like miRNAs, proteins, long noncoding RNAs, and lipids. Out of the components of EVs, the associated proteins and RNA of EVs are demonstrated to be used as a tumor biomarker for detecting cancer and observing the progression of cancer [27]. Figure 1 and Table 1 show the development of novel exosome-based biomarkers that could benefit cancer patients in a variety of ways.

Figure 1.

Extracellular vesicles - body fluids as biomarkers and therapeutic clinical applications [28].

Exosomal cargosCancer typesClinical valueBiofluidsRefs
Glypican-1Pancreatic cancerPatients with early- and late-stage pancreatic cancer had higher levels of Glypican-1 positive exosomes as compared to healthy controls.Serum[29]
miRNA-375, 1290Prostate cancerPatients with castration-resistant prostate cancer who have high levels of both exosomal miRNA-375 and miRNA-1290 may have a worse prognosis.Serum[30]
miRNA-21HCCHCC cancer patients have increased levels of exosomal microRNA-21 than healthy normalSerum[31]
EML4-ALK fusionNSCLCExosomal RNA of NSCLC patients found to include EML4-ALK fusion transcriptsPlasma[32]
miRNA-19-3p,
21-5p, 221-3p
Lung adenocarcinomaLung adenocarcinoma patients have elevated levels of the miRNAs than healthy controlsPlasma[33]
PhosphatidylserineOvarian cancerOvarian cancer has elevated levels of phosphatidylserine positive exosomes patients than normal controlsPlasma[34]
LncRNA-p21Prostate cancerProstate cancer patients were found to have elevated levels of exosomal lncRNA-p21 than healthy controlsPlasma[35]
miRNA-21, 375Prostate cancerProstate cancer patients were found to have increased levels of urinary exosomal miRNA-21 and miRNA-375 as compared with normal controlsUrine[36]
Mesenchymal Stromal CellsProstate cancerPatients with SARS-Cov-2 infectionUrine[37]
Dendritic cellsNon-Small Cell Lung CancerImmunotherapyBlood[38]

Table 1.

Exosomes from different types of cancer patients—body fluids as biomarkers.

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4. Cancer immunotherapy biomarkers and challenges

Tumor-derived extracellular vesicles, which transfer immunosuppressive chemicals like PD-L1, TGF1, FasL, TRAIL, and NKG2D ligands, are the major transporters of tumor immune evasion and potential therapeutic targets. Anti-programmed death-1 (PD-1) and its ligand (PD-L1) antibodies are immune checkpoint inhibitors (ICI), stimulating an antitumor immune response over blocking inhibitory immune signaling. Immune checkpoint inhibitors therapy has become potential in determining effectiveness in different cancers such as non-small cell lung cancer, head, and neck squamous cell carcinoma, melanoma, etc. [39]. Un happily only a few patients have responded to the checkpoint inhibitors and there is a critical need to find out the reason for an adaptive immune response [40].

PD-1 and PD-L1 are located on the exterior of the tumor cells, and it acted as a second signal when it is attached to the PD-1 receptors on T-cells. There are a few blocking antibodies that target PD-1 and PD-L1, like pembrolizumab (Merck) and nivolumab (Bristol Meyer-Squibb) enhance the anti-tumor immune response by opposing this inhibitory signal [41]. The response rate percent is depending on the type of tumor and is a distinctive group of patients gained a very less response. PD-L1 as a biomarker and its expression levels on tumor cells were studied using immunohistochemistry has shown to act as a prognostic marker as well prognostic to anti-PD-1 therapy [42]. The tissue-based testing method needs a decent tissue biopsy. A tissue biopsy test method also has a few advantages like bleeding, infection, and other procedural complication like causing pneumothorax in the case of parenchymal lung biopsy. Tissue can be isolated either from the primary tumor or a metastatic tumor lesion upon the different factors that are considered. Hence the expression levels may differ based on the tumor tissues isolated. Researchers have also seen [43]. These results of PD-L1 expression levels by immunohistochemistry have influenced the practitioners in a way to treat patients. There are also limitations for immunohistochemistry staining, distinct antibodies have unique sensitizers. Additionally, the threshold value of PD-L1 staining is still a discussion between pathologists and oncologists. Hence there is a great challenge for researchers, clinicians, and patients, antibody clones have influenced the distinctive epitope of the PD-L1 molecule with unique scoring systems based on the assays [44].

In inclusion to PD-L1, a few other immunosuppressive molecules such as TGFB1 and NKG2D ligands were also augmented in TD-EVs and were capable to prompt T-cell suppression [45]. NKG2D is a receptor that is activated by NK cells and a few subgroups of T cells and acts as a prime recognition receptor for the detection and elimination of cancer cells. These are stress-influenced self-proteins, which are released as soluble molecules through protease-mediated cleavage. An excretion of NKG2D ligands is regarded to maintain their expression levels related to the immune evasion mechanism occupied by tumor cells to avoid NKG2D-mediated immune observation [46]. It has been observed that TD-EVs from ovarian cancer and melanoma expressed NKG2D ligands and intercept activation of cytotoxic NK cells [47]. FASL and TRAIL expression on TD-EVs persuades apoptosis in dendritic cells (DCs) leading to immunosuppression and stimulating the progression of tumors [48]. TD-EVs on FASL terminated an antigen-specific effector T cells. There are few other immunosuppressive proteins like COX2, CD39/CD73, PDL1, FASL, TGFβ, CTLA4, TRAIL, etc., are appeared to relate to the TD-EVs [49].

For a better immunotherapy effect, the immunosuppressive part of TD-EVs would be impeded, to initiate the immune system. For this case, DCs are the foremost step in the immunity cycle for terminating the cancer cells via T-cell activation [50]. The surface membranes are easily attracted with the immune cells, in which the dendritic cells procured extracellular vesicles can likely be manipulated to behave as anti-cancer vaccines thereby leading to novel immunotherapy to fight against cancer. The surface molecules of the TD-EVs would be guided to deliver cancer treatment. Hence, the tumor-associated antigens, immunogenic peptides, and heat shock proteins would bring about cancer treatment shortly [51].

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5. Extracellular vesicles towards cancer immunotherapy

An immunosuppressive molecule like PD-L1, TGF β1, FasL, TRAIL, and NKG2D ligands are carried by TD-EVs, these ligands are the most crucial mediators of tumor immune evasion and are also the feasible targets for immunotherapy [52]. PD-L1 is more expressed when the metastatic melanoma-derived exosomes are stimulated by interferon-γ, on EVs and impede the antitumor responses. When PD-L1 is expressed on the tumor cell surface, it enhances evasion of immune surveillance through interacting with the ligand, by curbing the T-cell function. EV released from metastatic melanoma brings a PD-L1 which terminates the cytotoxic function of CD8+ T cells. PD-L1 indicates EVs were released from human blood, which is not a soluble form of PD-L1 and is also connected with the head and neck cancer progression. Similarly, PD-L1 isolated from EVs from the supernatant of murine or human HNSCC cell lines inhibits the infiltration of CD4+ T and CD8+ T cells into the tumor microenvironment, therefore enhancing tumor progression [53]. Along with the PD-L1 ligand, the other immunosuppressive molecules like TGFβ1 and NKG2D were also augmented in TD-EVs thereby bringing out the suppression of T-cells. Another activating receptor NKG2D is activated by NK cells and by T-cells, thereby presenting a crucial receptor for recognizing and terminating the cancer cells [54]. The NKG2D ligands are stress-induced self-proteins that can be divided by proteases and released as soluble molecules. The release of NKG2D ligands in the extracellular environment is to fine-tune the surface expression levels, constituting the immune evasion mechanism employed by cancer cells to neglect the NKG2D-ligand interfered immune response [55].

Few reports demonstrated that ovarian and melanoma TD-EVs activate NKG2D ligands and inhibit the activation of cytotoxic NK cells [56]. Activation of FASL and TRAIL on TD-EVs causes apoptosis in DCs and peripheral blood mononuclear cells (PBMCs) thereby promoting tumor progression [57]. The other inhibitory immunosuppressive proteins like COX2, CD39/CD73, PDL1, CTLA4, FASL are related to TD-EVs. Apart from the immune-suppressive character of TD-EVs, it would obstruct for greater immunotherapy effect and trigger the immune system. For instance, DCs are the foremost process of the immunity cycle, it activates the T-cell and thereby eliminate tumor cells. The surface membrane components that communicate with other immune cells, DCs derived EVs likely to be used as cell-free antitumor vaccines thereby delivering a novel and potential immunotherapy towards cancer [58]. Therefore, with the above observation, TD-EVs showed a major part in tumor immune evasion and growth are presented in Table 2. Hence, peptides, antigens, and other small molecules such as heat shock proteins could be designed shortly for cancer therapy.

Treatment moleculesCells usedEffect of treatmentResearchResultsReference
Small moleculesMurine macrophages (RAW 264.7 cell line)Paclitaxel is loaded into exosomes through sonicationThe efficacy of paclitaxel for the treatment of multiple drug-resistant cancers was assessed when delivered via exosomesPaclitaxel with exosomes entered the tumour cells and inhibited the growth of pulmonary metastases[58]
Antigens and antibodiesHLA-DR15-positive human B cellsExosomes tagged with Hsp65 antigen or antigenic peptide.Evaluated the B cell-derived exosomes can activate T cells through MHC-mediated presentation of Hsp65 antigenExosomes tagged with Hsp65 could activate the T-Cells through MHC mediated presentation of Hsp65 antigen[59]
siRNA and RNAiPlasma cells of humansiRNA ElectroporationsiRNA is delivered towards MAPK1 to monocytes and T cellssiRNA delivered and downregulated the MAPK-1 transcription[60]
miRNAPanc-1DNA plasmids transfected for miR-155 and miR-125b2 into Panc-1 cellsUpon treatment with exosomes, murine macrophages overexpress miR-155 and miR-125b2After the treatment, transfected Panc-1 cells were reprogrammed from M2 to M1 macrophage phenotype[61]
Blocking exosomes biogenesis and secretion- GW4869All types of cellsExosome’s biogenesis was inhibitedCombination therapy of gemcitabine and an inhibitor of exosomesOn the release of exosomes by cancer-associated fibroblasts on exposure to gemcitabine, GW4869 prevented chemoresistance[62]

Table 2.

Exosomes—cancer therapeutic targets.

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6. Conclusions

Exosomes are endogenous tiny vesicles that enable communication between neighbor or far cells [63]. Exosomes originating from various origins can be initiated in circulation and target specific cells inside distant tissues [64]. Because of its stability and capacity to bypass natural barriers. Exosomes are a suitable and potential nanocarrier vehicle for cancer immunotherapy and chemotherapy because of their characteristics. However, exosomes are demonstrated to be involved in the triggering of immune responses towards cancer cells and stable of an immunosuppressive milieu. The qualitative and quantitative data of EVs molecular signatures are mandatory in extra cellular-based tumor diagnosis, monitoring, and therapeutic delivery to distinct cancer subtypes. As previously stated, researchers are most interested in EVs and therapeutic targets. However, much more research on these EVs for cancer immunotherapy and chemotherapy is needed to gain a better understanding. In the view of EVs have received great attention and would be a potential and promising biomarker and a better candidate for the treatment of deadly disease, cancer.

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Acknowledgments

The authors would like to acknowledge the financial support provided by a Research University grant from the University of Malaya (RU003-2021).

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Conflict of interest

The authors declare no potential conflict of interest regarding the publication of this work.

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Written By

Baranya Murugan and Suresh Sagadevan

Submitted: 04 December 2021 Reviewed: 17 February 2022 Published: 20 July 2022