Exosomes â Small Players, Big Sound - Bioconjugate Chemistry (ACS

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Exosomes – Small Players, Big Sound Diana Gulei, Alexandra Iulia Irimie, Roxana CojocneanuPetric, Joachim L Schultze, and Ioana Berindan-Neagoe Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00003 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 26, 2018

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Bioconjugate Chemistry

Exosomes – Small Players, Big Sound Diana Gulei1*, Alexandra Iulia Irimie2*, Roxana Cojocneanu-Petric3, Joachim L. Schultze4,5, Ioana Berindan-Neagoe1,3,6

1. MEDFUTURE-Research Center for Advanced Medicine, “Iuliu-Hatieganu” University of Medicine and Pharmacy, Marinescu 23 Street, 400337 Cluj-Napoca, Romania. Electronic address: [email protected]. 2. Department of Prosthetic Dentistry and Dental Materials, Division Dental Propaedeutics, Aesthetics, Faculty of Dentistry, "Iuliu Hatieganu" University of Medicine and Pharmacy, Cluj-Napoca, Romania 2. Research Center for Functional Genomics, Biomedicine and Translational Medicine, "Iuliu Hatieganu" University of Medicine and Pharmacy, Marinescu 23 Street, 400337 Cluj-Napoca, Romania. Electronic address: [email protected]. 3. Genomics and Immunoregulation, LIMES-Institute, University of Bonn, 53115 Bonn, Germany 4. Platform for Single Cell Genomics and Epigenomics, German Center for Neurodegenerative Diseases, 53175 Bonn, Germany 5. Department of Functional Genomics and Experimental Pathology, The Oncology Institute "Prof. Dr. Ion Chiricuta", Republicii 34-36 Street, Cluj-Napoca, Romania. Electronic address: [email protected].

Correspondence: [email protected], [email protected] (I. BerindanNeagoe)

Keywords Exosomes, cancer, biomarker, miRNAs, therapy, immunity

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Abstract Incipiently named extracellular vesicles, exosomes are forming now a separate class of cellular mediators with important functions in physiological and pathological states. Their ability to transfer information between cells through encapsulation of proteins, nucleic acids and lipids for the preservation of the homeostatic equilibrium is translated also in pathological conditions. The recipient cells react to the reception of foreign molecules adjusting their molecular state according to the enclosed message. Cancer cells, in order to influence the microenvironment and facilitate the malignant expanding, exploit this inter-cellular trafficking. Immune cells are also producing exosomes that ensure the transportation of immune mediators and signaling between cells. Current experimental attempts are concentrated on the adjustment of exosomes level for therapeutic purposes, enrolment of these vesicles as diagnosis or prognosis tools and also exosomes’ use as drug delivery vehicles or immune stimulatory agents.

1. Introduction The role of the immune surveillance in eliminating cancer cells has been strongly studied in the oncology area, where numerous immune-related therapies are now developed or tested in order to abolish the tumor development and to control the immune response

1, 2

. This natural

capacity of the immune cells to eliminate the malignant ones from the organism is sometimes bypassed by the tumor formation through several mechanisms: (1) expression of lower levels of surface antigens; (2) secretion of surface proteins able to inhibit the activity of the immune system; (3) release of immune inhibitory microvesicles/substances in the surrounding microenvironment. Every type of these mechanisms and also combined ones are able to tilt the balance in the favor of the malignant cells, allowing the development of carcinogenesis and metastasis

3-5

. Exosomes secreted by the tumor cells are able to influence the immune cells en-

countered in circulation or in the malignant environment through delivery of inhibitory signals that impair their function against the cancer mass 6-8. Nowadays, immunotherapies counteract this unbalanced regulation between cancer and immune cells in order to increase the potential of the immune system or to decrease the capacity of the cancer cells to escape from the immune surveillance. Recently, the role of exosomes has been also highlighted in the scientific area with the potential of therapeutic molecules as they can mediate communication between cells and also immune related mechanisms. More precisely, these nanovehicles can intervene in the opsonization process, antigen presentation and also inhibition or activation of the immune response 8-12. Exosomes could be explored in 2 ACS Paragon Plus Environment

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novel therapeutic strategies, where their aberrant function in cancer could be experimentally modulated in order to impair the malignant phenotype through activation of the immune system or suppression of the malignant inhibitory immune mechanisms. Despite the fact that exosomes were initially considered the “garbage bins” of cells, advances in the field of cellular communication demonstrated that these small vesicles (40-100 nm) are actually crucial players in the process of molecular trafficking, being able to perform intercellular transport and information exchange with different regulatory cargo molecules 12-15. The double lipid membrane structures are small vesicle-like bodies secreted by the cells and are ubiquitously present in all body fluids 16. The secretion of these transfer vehicles is a process which is naturally present in physiological conditions, facilitating the communication between cells in order to preserve homeostasis. The same process is replicated under pathological contexts, including cancer, promoting the establishment of a malignant environment with effects on tumor cell growth, proliferation, invasion and metastasis and immune system aberrant control

13, 17

. The content of exosomes, consisting primarily of various proteins and nu-

cleic acids – DNA, mRNAs, but also short and long non-coding RNAs – provides information about the state of the cell of origin, and, at the same time, offers clues concerning their possible effects on the recipient cells

18, 19

. Studies have shown that exosomes are in-

volved in many cellular activities, from physiological ones such as carrying immunogenic molecules (e.g. CD86, CD40, MHC-I, MHC-II, Hsp70 and galectin-5) or conveying biomessages to neighboring or distant cells – thus mimicking paracrine and endocrine communication - to pathological ones, for instance cancer growth and priming of the pre-metastatic site in various tumors

18, 20, 21

. The continuous expanding record of exosome implications in

malignant scenarios has also stimulated potential therapeutic applications of these small vehicles in the context of cancer management. At the moment, there are several ongoing clinical trials comprising the use of exosomes for anti-cancer therapy with an increased interest in lung malignancies, melanoma, colon and prostate cancer 22. The main approaches are (1) depletion of exosomes from biological fluids, (2) targeting of key molecules involved in the biogenesis of these small microvesicles or even (3) conversion of the secreted vesicles into delivery vehicles for anti-cancer therapeutics. The therapeutic direction as delivery vehicles seems to be the most promising one due to the “self” profile of the exosomes and implicit lack of immune recognition against exosomes by the host

23-25

. Despite all these advances

there are still numerous unclarified aspects that need to be revealed in order to efficiently target cancer cells and immune cells serving tumor microenvironment via exosomes.



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2. Exosome biogenesis – from donor to target cell The beginnings of 1980s marked an important milestone in extracellular vesicles (EVs) research when the concept that EVs are simply formed and released through outside budding of the plasma membrane was replaced by a more refined and realistic theory. Two independent studies described the formation and release pathways of these types of communication vesicles that actually implies an intercellular formation mediated by multi-vesicular bodies (MVBs) that is only afterwards followed by the discharge into the extracellular medium 26, 27. Several years later, Rose Johnstone suggested the term “exosomes” for the vehicles of endo28

somal origins to the scientific community

. Now, exosomes and microvesicles (MVs) are

clearly distinguished in the scientific nomenclature and their biogenesis is clearly separated in terms of mechanisms 29. Exosomes became increasingly popular being now at the center of interest for cancer diagnosis, prognosis and even therapy. The formation and release of exosomes is a finely tuned process that starts with the formation of an early endosome that will maturate into late endosomes under the control of endocytosisassociated proteins and lipid complexes

30

. The transformed vesicles are encapsulated into

multivesicular bodies (MVBs), which also harbor specific proteins and nucleic acids originating from processing steps within the donor cell. At the final stage, the MVBs that do not undergo lysosomal degradation are expulsed from the cell via RabGTPases, followed by the release of exosomes into the extracellular milieu (Figure 1)

31

. By these mechanisms, exo-

somes are able to target recipient cells by their engulfment into the recipient cell and the modulation of the recipient cell’s activity in a paracrine or endocrine manner, depending on their location 30.


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Figure 1. Exosomes biogenesis. Next to endocytosis, early endosomes are formed followed by maturation, which results in their budding into MVBs (Multivesicular Body) as ILVs (Intraluminal Vesicles). The MVBs that are not subjected to lysosomal degradation are merging with the plasma membrane allowing the exosomes to enter into the extracellular space. The interaction between exosomes and target cells can be mediated by different mechanisms including: (1) receptor binding, (2) phagocytosis or (3) membrane fusion as shown on the recipient cell. Once entered into the target cells, exosomes can modulate their state in a context dependent manner, depending on the state of the donor cell. The modulation is mediated by the exosomal cargo, mainly composed of DNA and RNA sequences – mRNA, miRNA, lipids and proteins, including those involved in antigen presentation (integrins, tetraspanins - CD9, CD63, CD81 and CD82 and MHC I and II). ER – Endoplasmic Reticulum; MVB – Multivesicular Body; ILV - Intraluminal Vesicles MHC - Major Histocompatibility Complex; miRNA – microRNAs; mRNA – Messenger RNA

As mentioned earlier, the biogenesis of exosomes is a strictly regulated process, every step within the formation of the EVs being controlled and directed by specific pathway components. MVBs appear inside the cells as units that hold vesicles in their lumen (ILVs). These vesicles are actually the future exosomes that will be released outside of the cell or will go for 5 ACS Paragon Plus Environment


degradation inside of the lysosome

32

. ILVs and exosome biogenesis represents a well-

documented research niche with two proposed controlling mechanisms that revolve around the Endosomal Sorting Complex Required for Transport (ESCRT) machinery 13, 29, 30. ESCRT is divided into four main groups (ESCRT-0, -I, -II and -III) that associate with different proteins in order to accomplish their functions (VPS4, VTA1, ALIX named also PDCD6IP) 33. Every type of complex has a designated role, where ESCRT-0 is responsible for the recognition and retreatment of transmembrane proteins that have been previously marked through ubiquitination, ESCRT-I and -II are involved in the deformation and invagination of the membrane that holds specific molecules (cargo) and ESCRT-III has been assigned a function as a vesicle scissor 33. The associated proteins, mainly VPS4 ATPase have the task to detach and recycle the entire machinery. The flow of vesicle formation via the ESCRT machinery consists of a continuous interaction between specific molecules that end up with the emergence of vesicles with cell-derived cargo inside MVBs. Even if the recirculation via exosome release or degradation through the lysosome pathway of the cytosolic proteins is still an incompletely deciphered mechanism, it is assumed that these molecules are recognized by the HSC70 chaperone that forms a bridge between MVBs and target proteins towards their integration into the ILVs 34. Studies involving the experimental depletion of cells from key ESCRT members revealed that ILVs and exosome biogenesis could take place even in the absence of these complexes, their role being replaced by heat shock proteins, lipids or tetraspanins 35. ESCRT-dependent and ESCRT-independent pathways are reviewed by Colombo and colleagues together with the implications for exosome formation 34. Once exosomes are formed inside of the MVBs, these final vehicles fuse with the cell membrane in order to release their content into the extracellular space. The secretion of the nanovesicles is strictly controlled by Rab guanosine triphosphatases (GTPases) together with other parameters 13. High concentration of Ca2+ inside the cell elevates the secretion of exosomes 36

. pH levels outside and within the cells are also critical factors for the expulsion of the vesi-

cles, where low pH status determines the acceleration of exosome release and also uptake by target cells 37. Experimental evidence shows that certain genes are also involved in the process of exosomes trafficking, the attention being now shifted towards oncogenes that are able to raise the level of exosomes from malignant cells targeted towards healthy cells with the purpose of malignant transformations 13. Concerning the mechanism by which recipient cells collect the released nanovesicles, it has been proposed that there are three types of interactions: (I) receptor-ligand cooperation; (II) 6 ACS Paragon Plus Environment

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direct fusion with the membrane of the target cell; (III) endocytosis in case of phagocytosis related pathways 38.

3. Exosome content Exosome content is usually specific to the cell of origin, and may vary according to the physiological and pathological condition. In this regard, the exosomal cargo can reveal the state of the donor cell and also can influence in a pathological manner the fate of the recipient cell 13. Moreover, the specificity of the molecular content can be exploited for the development of non-invasive diagnosis and prognosis tools, as well as targeted therapeutic strategies

39, 40

.

The reliability of intercellular communication is maintained and translated by specific components within the exosomes. These components are generally made of proteins, lipids, DNA fragments, mRNA and small RNA species. However, the cargo is not randomly distributed into exosomes, where the incorporation of these molecules is based on strictly regulated mechanisms referred as the term of “sorting”. Even if the pathways responsible for the sorting of exosomes content are not completely elucidated, the fact that some molecules are constantly incorporated and others are absent and also the fluctuating content that mirrors the state of the cell (homeostatic, pathological, stress conditions) are standing as a proof for the controlled loading of exosomes 41. Regarding the protein composition, exosomes share a common panel of molecules, but also contain other differential proteins that vary according to the type and state of the donor cell. The common list includes proteins from the endosome, plasma membrane of the cell and also from the cytosol. Another unifying aspect consists in the lack of proteins derived from the mitochondria, Golgi complex, nucleus and also endoplasmic reticulum, revealing the specificity of exosomes that are not just disposal units for random cell debris 34. The incorporation of proteins is thought to be controlled by lipid-dependent mechanisms, ESCRT and tetraspanins. As shown in the previous chapter, ESCRT mechanism is deeply involved in the biogenesis of exosomes 34. This complex is responsible for the identification and selection of ubiquitinated proteins, molecules that will be further processed into ILVs and then degraded upon the fusion of MVBs with the lysosome 42. In the case of exosomes, where the proteins escape from the degradation step encountered in the lysosomal media and are shipped extracellularly via these nanovehicles, the role of ESCRT is still debated. Experimental inhibition of different elements within the ESCRT complex, showed that the protein content of exosomes is affected in different manners depending on the targeted ESCRT member

43

. Impairment of

tsg101 (ESCRT-0/I), Hrs and STAM1 affected the level of exosomes with CD63, CD81 and 7 ACS Paragon Plus Environment


MHC-II

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43

. Hrs expression was also associated with a vital role in exosomes biogenesis in

dendritic cells 44. In the case of Alix or Vps4B (ESCRT-III) inhibition, the situation was reversed, where the amount of exosomes increased (measured by exosomal MHC II)

43

. Be-

sides ESCRT mechanisms, lipids also play a role in exosomal sorting, where the secretion of exosomes that are proteolipid (PLP)-positive is not modified upon the inhibition of Hrs, Alix or Tsg101 45. Lipids are also included in the exosomal cargo, although the studies regarding these types of molecules are more limited

34, 45-47

. The main lipid structures that were ob-

served as being enriched in exosomes are cholesterol, saturated fatty acids and sphingomyelin. More importantly, researchers reveled that exosomes contain reminiscent structures from the lipid rafts, but also lipid raft–associated proteins, GPI-anchored proteins and flotillins, present on the surface of the plasma membrane of the cells 48, 49. In light of these findings, it is still unclear if the ILVs form inside of the MVBs following mechanisms known for lipid raft formation, or whether domains present naturally in the membrane of the cell are enclosed together with the vesicles from the MVBs. The third mechanisms for protein sorting consist in the action of tetraspanins, membrane proteins found in high levels in exosomes 50. CD63, among others, is one of the tetraspanins that regulates the biogenesis but also the loading process of exosomes, where LMP1 protein integration into the nanovehicles is dependent on CD63

51

. A similar dependence mechanism takes place in the case of PMEL incorporation

into ILVs within melanogenesis 41. The presence of nucleic acids inside the exosomes had the biggest impact, especially in oncology, due to the specificity of the genetic material in accordance with the physiopathological state of the donor cell. Within the nucleic acids spectrum, miRNA sequences are the most studies ones, once it was discovered that the secreted non-coding cargo could affect the expression of genes within the recipient cell and even induce pathological modifications in order to augment the process of malignant development

52-55

. The fact that some miRNAs are

abounded in exosomes, and some, even if present in the donor cells, are barely distinguished in the secreted vesicles is standing as a proof for a specific sorting mechanisms

56-59

. HnRN-

PA2B1 is a wide encounter protein and has been associated with trafficking functions for mRNA in distal sites of neurons 60. This protein contains a binding motif in the 3’UTR for an RNA transport signal (RTS or A2RE)

61

, motif that is also overlapping with EXOmotifs (se-

quences that mediate the loading of miRNAs into exosomes 59). Therefore, it is supposed that HnRNPA2B1 is playing actually a crucial role regarding the sorting process of miRNAs in exosomes. Moreover, the same protein is frequently encountered as sumoylated in the secreted vesicles, where this modification is mandatory for the uptake of miRNAs 59. Next genera8 ACS Paragon Plus Environment

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tion sequencing experiments showed that actually the most abundant RNA structures from exosomes are represented by the class of structural RNAs and also small ribosomal RNA 56. Intriguingly, Wild and colleagues showed that the binding motif that mediates the forming of SRP-RNA - SRP protein complex is actually the EXOmotif: GGAG 62.

4. Exosome isolation methods Exosomes have been isolated from different cell types with ultracentrifugation being the most common method for separation of these vesicles. Most isolation methods are based on exosome size, which – in general results in very good homogeneity of the obtained particles. Nevertheless, all methods still are not very well reproducible and it is still difficult to obtain reliable results in validation studies or when comparing data derived from different groups. An important task for the field will be to standardize the technologies and make them more comparable. The most commonly used procedures for exosomes isolation are presented in Table 1.

Table 1. Commonly used procedures for exosome isolation. General characteristics.

Ultracentrifugation Centrifugation followed by ultracentrifugation Ultrafiltration

- Separation based on selective filters

Time

Cost

General workflow

Purity

Principle

Yield

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


++

++

++

+

+

+

+

++

+++

+++

+++

++

+

+

+

++

- Pressure or centrifugation Density gradient centrifugation

- Centrifugation - Supernatant + sucrose solution - Ultracentrifugation

Precipitation polymerization

- Incubation with solution for exosome precipitation - Centrifugation - Resuspension



Magnetic-activated - Incubation with magnetic beads cell sorting

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+

++

++

++

+

+++

++

++

+

+++

++

+++

+

++

+

+++

+

++

+

+

+

+++

+

+

- Centrifugation - Elution

Immunoaffinity

- Low-speed centrifugation

capture-based

- Microplate-based immunoaffinity approach

techniques

- Binding of specific membrane biomarkers

Mass spectrometric - Monolithic silica micropipette tips with specific immunoassay

antibodies

Acoustic nanofilter - Separation bsed on size and density with ultrasound standing waves Ciliated nanowire- - Sequenstration of exosomes based on the size: on-micropillar

40 and 100 nm - Elimination of bigger molecules and debris

ExoChip

- Separtion based on immunoaffinity - Staining with membrane specific dye - Analyzation of exosomes content through Western Blot and RT-qPCR

*(“+” - low, “++” - medium, “+++” - increased; these marks does not necessarily mean that “+” represents an disadvantage and “+++” an advantage, this being context dependent – e.g. low cost – positive aspect, low purity – negative aspect)

5. Mechanisms of exosome implication in both immune activation and inhibition Exosomes are important immune-modulators, being able to trigger the activation of a diverse set of immune cells (Figure 2). In the late 90s, Rapaso et al. discovered that B cells are able to secrete and deliver exosomes containing MHC (major histocompatibility complex) class II that induces a response by T cells 63. Now, it is generally accepted that dendritic cells (DCs) release exosomes bearing MHC I and MHC II class molecules, an action that can trigger an immune response against cancer cells when the exosomes are derived from mature DCs loaded with tumor antigens. These types of exosomes are able to directly activate CD8+ T cells


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and naive CD4+ T cells through an antigen-speciﬁc manner 64. Immature DCs are also able to discharge exosomes in the microenvironment, but in this case the direct activation of T cells is hampered and can only be induced following additional processing steps by the antigen presenting cells

65

. Based on these findings, it has been suggested to use DCs derived exo-

somes (DC-Exo) as a therapeutic option in cancer. For example, the administration of such vesicles and the subsequent incorporation of the immunogenic cargo into cancer cells increased the number of IFN-γ-secreting cells in the context of human breast adenocarcinoma cells co-cultured with sensitized T-cells 65. Inversely, tumor derived exosomes hold immunosuppressive characteristics generated by their content (Figure 2). This is the case for Fas Ligand (FasL) a type II protein that have its place in the TNF family, that is often found in tumor-derived exosomes. Indeed, exosomederived FasL has been shown to promote apoptosis of T lymphocytes 66. Another central inhibitory mechanism associated with tumor-derived exosome biology is NKG2D (natural killer group 2, member D) , a receptor that normally activates a number of immune cells (NK, gammadelta (+) T cells, CD8 (+) and NKT), however, is frequently deregulated in cancer being one of the triggers for immune evasion. Cancers compromise immune activation by secreting growth factors (e.g. TGF-beta) and soluble NKG2D ligands thereby reducing the expression of NKG2D. Exosomes secreted by cancer cells are taking advantage of this mechanism and impair the activity of NK cells and CD8 (+) T cells by hijacking the expression of NKG2D present on the surface of the respective immune cells. Clayton et al. showed that this ability of cancer-derived exosomes is mediated by the presence of TGF-beta1 and NKG2D ligands and takes place in the moment of direct interactions between the vesicles and NK cells or CD8 (+) T cells 67. On the other hand, exosomes secreted by cancer cells can also act against the tumor mass, being a source of antigens for DCs inducing pathways by which an immune response can be triggered against malignant cells 68-70.



Figure 2. Exosome mediated immune response in cancer versus pathogen-host interaction. Exosomes are crucial players in the activation of an immune response but at the same time can support tumor growth by suppressing immune cell activation. In case of pathogenhost interactions exosomes secreted by dendritic cells can activate effector CD4+ T and effector CD8+ T cells through direct antigen presentation or by cross presentation that requires the involvement of another set of mature DCs. Moreover, the same exosomes released by DCs can activate endothelial cells, promote the cytotoxic activity of NKs and also trigger inflammation. In the case of malignant tumors, exosomes secreted by cancer cells can act in favor of the donor cells thereby suppressing differentiation and activation of DCs. Also, the vesicles can induce apoptosis of CD8+ T cells, inhibit the activity of NKs through downregulation of NKG2D receptors and also induce the differentiation of CD4+ T cells into T regulatory cells. In this way, cancer derived exosomes can maintain a “light” immune environment that favors the proliferation and invasiveness of cancer cells within the body. NK - Natural killer; DC – Dendritic Cell; Treg cell - Regulatory T cell; TNF - Tumor Necrosis Factor; IL-15R - Interleukin 15 Receptor; BAT-3 - HLA-B-Associated Transcript 3; IL1β - Interleukin 1 beta


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Besides these immunomodulatory roles, exosomes are also involved in other processes like retroviral spreading within cell populations, where particles like HIV-1 take advantage of the biogenesis pathway of exosomes, escape from the lysosomal degradation and infect de novo additional cells 71, 72. Exosomes are also present at the interphase between neurons and serve as ‘bridge’ in terms of exchange of molecular information. This bridging function is not only apparent under physiological conditions, but also in neurodegenerative diseases. For example, molecules like PrPsc (scrapie form of the prion protein), alpha-synuclein or fragments of amyloid precursor protein (APP) can be transferred between cells via exosomes

38

. These

structures are key elements in neurodegenerative diseases, where PrPsc is responsible for or associated with Creutzfeldt-Jacob disease, alpha-synuclein for Parkinson respectively APP for Alzheimer, all of them being found inside of secreted exosomes

73-75

. Another important

cellular system where exosomes seem to play an important role for information exchange between cells is the stem cell compartment. Stem cells also secret exosomes and in light of this property, stem cell-derived exosomes are increasingly considered for therapeutic option in the area of regenerative medicine 38. The association of exosomes and immune cells together with their content is briefly described in Table 2.

Table 2. Immuno-derived exosomes and their content Immune cell Dendritic cells

Function

Antigen presentation, Activation MHC T

cells,

Immune

Immune memory B cells

Exosome content complexes,

tolerance, B71/CD80,

B7-2/CD86,

ICAM-1

Antibody production, Antigen CD19, CD37, MHC class presenting cells for T helper II cells, memory B cells

T cells

CD8+:

Cytotoxicity,

antigen binding;

MHC-I Treg: CTLA-4;

CD73, CD4+:

CD25, CD4,

CD4+: MHC-II antigen binding, HLA Class I, TfR, TCR, helper for cytotoxic T cells, Integrin β2, Fas Ligand; helper for macrophage activation CD8+: Fas Ligand CD8, TCR Natural killer cells

Cytotoxicity

Perforin,

CD56,

Granzyme B, Fas Ligand


Ref 76-79


Macrophages

Inflammation,

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Phagocytosis, Bacterial

Phenotypic plasticity

antigens,

Cofilin-1, GNB1, Actin, Cyclophilin A

Mast cells

Immunomodulation, Antigen

Allergy, MHC

class

II,

c-Kit,

presentation, esRNA, LFA1, ICAM

Inflammation

6. Immunomodulatory roles of exosomal miRNAs One of the main cargos that can be found in exosomes consists in RNA sequences, transcripts that hold or not the ability to further codify functional proteins. The later ones, non-coding RNAs are especially composed of miRNAs and lncRNAs that are actively expelled from the donor cell in order to modulate the target cell or even more, to free the donor cell of supplementary non-coding RNAs in order to conserve the homeostatic balance between mRNAs and miRNAs (or other non-coding sequences). In the latest years of research it has been shown that the non-coding exosomal profile is actual dependent on the cellular state of the donor cell and also on the general state of the organisms as a whole. In this sense, chronic inflammation, that represents an important risk factor for several types of cancers (e.g. colon cancer), was shown to be an active modulator of the transcriptomics landscape of exosomes, including modifications in miRNAs content

80

. The RNA content analysis between RAW

264.7 cells stimulated or not with LPS detected 433 miRNAs with exosomal provenance. Within these non-coding sequences, miR-146a, miR-146b, and miR-21-3p were found as dysregulated between the two experimental scenarios, miRNAs that have been previously shown to be involved in inflammatory pathways 81, 82. The same approach was also conducted on a human derived monocytic cell line (THP-1), where miR-484 was found as significantly downregulated in LPS stimulated cells and miR-26a as upregulated with targets related to the inflammatory pathways. MiRNAs secreted by cancer cells through exosomes can modulate immune cells by binding to Toll-like receptors and mediate a pro-metastatic microenvironment: Fabbri et al. showed that miR-21 and miR-29a can target immune cells via exosomes and further bind and activate TLR8 that leads to stimulation of NFκB pathway and increased secretion of cytokines with inflammatory/prometastatic abilities 83. The role of exosomal miRNAs in activating/suppressing the immune system in malignant pathologies is still incompletely elucidated, hampering the smooth translation of exosomes


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vaccines towards the clinical area. After showing that miR-203 can prompt the immune tolerance by decreasing the expression of TLR4 in dendritic cells in prostate cancer through exosomal trafficking

84

, Que et al. implemented the possibility of depleting exosomes from

miRNA and administrate them as fortified therapeutic agents. Stimulation of dendritic cell/cytokine-induced killer cells (DC/CIKs) with modified exosomes lysate (without miRNAs) showed increased inhibitory activity on pancreatic cancer cells that in the case of LPS or naive exosomes stimulation, showing that the miRNA content can significantly influence the ability of the immune system to counteract the malignant development

85

. The miRNAs

from exosomes can also travel between immune cells; miR-146a and miR-155 are released and subsequently target dendritic cells (regulation between dendritic cells) in order to manage the cell’s response to endotoxin, and implicit regulate inflammatory related pathways. Moreover, these two miRNAs are acting in an antagonist manner, where miR-146a is reducing the expression of inflammation genes and miR-155 promotes it 86. Challenging the cells with exosomes loaded with the specific non-coding sequence revealed the same pattern of inhibition/enforcement, underling important therapeutic related options in terms of exosomes administration in cancer and also inflammatory states. Even so the data related to miRNAs enclosed in exosomes and their role in modulating the immune surveillance against or even in favor of the cancer cell is still unsecured from a clinical therapeutic point of view. The heterogeneity of the exosomal non-coding content is destabilizing the exact effect of naive exosomes administration. However, it has been previously shown that these vehicles can be experimental loaded with “custom made” cargos in order to actually take advantage of their “self” attributes and not necessarily of their content for the in

vivo delivery of immune modulatory agents (miRNAs, but also other non-coding RNAs).

6. Exosomes vaccines - DEXs Dendritic cell-derived exosomes (DEXs), as the name suggests, secreted by dendritic cells increase the fitness of primary and also secondary immune response. Their role was established in 1998 by Zitvogel et al. where they showed that DEXs present MHC class I and class II and also additional stimulatory molecules that can trigger a tumor inhibitory effect dependent of T cells

87

. This distinct cargo endow exosomes with immunostimulatory properties,

functions that are also potentiated through the presence of surface molecules that facilitate the fusion with the target cells in order to further produce an immune response. Among these proteins, α and β-chains (αMβ2), milk fat globule EGF factor 8 and the Ig superfamily member ICAM-1 are within the ones that stimulate the fusion with the receiving cell. More specif15 ACS Paragon Plus Environment


Page 16 of 35

ically, milk fat globule EGF factor 8 is actually mediating the formation of a bridge between exosomes and αvβ3 or αvβ5 integrins from the target cell

88

. Other facilitators of the exo-

somes uptaking within the population of the targeted cells could be also the proteins from the tetraspanin family like CD81, CD9 and CD63 that are present on the outer membrane of the vehicles 88, 89. Thery et al. particularly studied CD9 where they proposed that this member is the most abundant one within exosomes derived from dendritic cells and also a mediator for APCs or T cells interaction 88. Even so, the most investigated aspects regarding the proteomic profile of exosomes consists in the presence of peptide–MHC class II complex that could function as stimulators of a T cell specific response via de Ag presentation model 88, 90. DEXs previously stimulated with tumor peptides showed inhibitory effects on malignant development after exogenous administration in mice (injection). This action was rapidly translated into potential clinical use, where DEXs were proposed as potential cell-free tumor vaccine. Additionally to MHC class II complex, exosomes also presents MHC class I proteins that are associated with activation of CD8+ T cells once are delivered into the recipient dendritic cell within the microenvironment

91

. Hsc73 (heat shock protein) represents a common member

between the endocytic space of dendritic cells and exosomes, being associated in the same time with potent inhibitory effect upon tumor cells in vivo 88. The accumulation of hsc73 in DEXs could represent another immune layer of regulation involving exosomes, where these proteins were characterized as modulators of tumor rejection and cytotoxic T lymphocyte activation in mice injected with heat shock proteins (hsp) derived from the tumor microenvironment 92. It was also suggested that dendritic cells and macrophages express receptors for hsp as it was shown that these shock proteins could be actually transferred to APCs for Ag presentation

93, 94

. The presence of Mac-1 on the surface of exosomes could represent also an

immune mediator mechanism for the interaction with cells that express the ligands for Mac-1. These ligands, ICAM-1– or ICAM-2, are usually present in endothelial cells, but also in dendritic cells and lymphocytes, fact that underlines a possible mechanism of communication between secreted DEXs and target cells 88. DEXs were also studied in the context of NK cells, as they were shown to present NKG2D ligands upon secretion by immature DCs that further can activate in a direct manner these cells (NK). The ex vivo observations are sustain also by in vivo studies where DEXs were able to promote the activation and proliferation of NK cells in a NKG2D dependent manner; mechanism encountered also in a previous clinical study where 50% of the patient responded to DEX vaccination through activation of NKs

95

. Another NK dependent tumor inhibition

that renders the activity of exosomes could be through BAT3 (NKp30 ligand) that is actively 16 ACS Paragon Plus Environment

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expressed on the surface of the nanovesicles 96. Additional surface molecules like, FasL and TRAIL, mediated the stimulation of NK through DEXs, but also can potentiate the induction of apoptosis in cancer cells through activation of caspases 97. The potential of exosomes derived from dendritic cells was further explored in clinical scenario in order to asses the feasibility of this immunotherapy. Two clinical trials marked an important point for DEXs, where MAGE3+ patients with small cell lung cancers and advanced melanoma received weekly doses of DEXs enclosing MAGE malignant antigens. Even if the response from T cells was not as higher as expected, part of the patients were correlated with stable disease and also some of them presented increased NK functions

98, 99

.

These initial data paved the way towards more advanced investigation regarding the potential therapeutic value of DEXs: mDEXs derived from mature DCs can be used with a superior effect in terms of T cells activation due to the fact that express a higher amount of stimulatory molecules than those obtained from immature DCs (e.g. MHC class II, ICAM1) 100-102.

7. Therapeutic potential of exosomes in cancer - beyond immunotherapy The discovery of heterogeneous exosome involvement in malignant progression has led to the idea of targeting or using these vesicles in the context of cancer therapy (Figure 3) 14. One approach is focusing on the removal of exosomes from the circulation in order to inhibit the pro-carcinogenic actions of these agents. The microvesicular extinction could be achieved through biogenesis inhibition by prevention of microtubule formation and stability, modulation of endosomal distribution pathways, or through the administration of proton pumps inhibitors

37, 103-105

. Another form of action related to exosome-mediated therapy consist in the

usage of these molecules as nanovehicles for the targeted delivery of drugs to malignant sites, avoiding secondary cytotoxicity and immune eradication of the vehicle. Moreover, exosomes can be loaded with different nucleic acid sequences such as miRNA inhibitors or mimics, or siRNAs (small interfering RNAs) for the modulation of gene expression, but also with specific antigens in order to trigger an immune response

14, 103, 106-108

. In a recent study,

Mathiyalagan and Sahoo revealed a protocol for the isolation of exosomes and potential use as carriers of miRNA for gene therapy in cardiovascular disease. Even if the targeted pathology was not cancer, this protocol where they generated exosomes from human CD34+ stem cells containing exogenous miRNA sequences for targeted delivery can be easily translated into other health sectors 109. In another research study, Ohno et al. delivered engineered exosomes containing let-7 to EGFR expressing breast cancer cells 110. Let-7 was associated with tumor suppressor capacities in cancer and was also observed as downregulated in multiple 17 ACS Paragon Plus Environment


malignant pathologies like: colon, ovary, breast and lung cancer

Page 18 of 35

111

. The targeting mecha-

nisms was constructed based on the idea that numerous epithelial tumors express EGFR, therefore functioning as an intermediary factor for specific delivery. The downside consists in the fact that EGF, the ligand for this receptor, is extremely mitogenic, decreasing the reliability of this duo as mediators of targeted delivery strategies. However, GE11 peptide is efficiently recognized by EGFR and also is less mitogenic that EGF, being counted as the ideal peptide for this type of therapeutic based interaction

112

. Therefore, Ohno et al. modified the

exosomes in order to express the GE11 peptide and successfully delivered let-7 to EGFRexpressing cancer cells both in vitro and in vivo (RAG2–/– mice with breast cancer cells xenografts) 110. The significance of miRNA replacement or inhibition therapies has been already established with numerous studies conducted on this strategy for cancer impairment; however the constant issue remains the delivery method for this small sequences, where exosomes are gaining increased attention due to their advantageous characteristics (non-imunogenic, decreased toxicity, ability to survive in circulation and target cancer cells) 106, 108, 113-115.

Figure 3. Diagnosis and therapeutic value of exosomes. Exosomes can be used as early and non-invasive diagnostic and prognostic tools (A) due to their ubiquitous distribution in body fluids and also due to their specific cargo that can reveal the state of the donor cell. The same vesicles can also be loaded with anti-cancer drugs (B), compromising a more efficient


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delivery vehicle due to the “self” characteristics and persistence within the circulation. Another therapeutic alternative related to exosomes consist in the inhibition of the biogenesis process or even more in the depletion of the nanovesicles with different compounds (C). In this way, the pro-tumoral activity of exosomes can be significantly reduced concomitant with a decrease in cancer development or progression.

Recently, Lugini et al. discovered that exosomes derived from colorectal cancer cells can promote the malignant development by influencing the phenotype of cancer mesenchymal stromal cells (cMSCs)

116

. The modifications on the level of cMSCs are associated with tu-

mor-like changes, including increased proliferation and vesicle secretion, actions that favor the boosting of the tumor microenvironment. Moreover, two tumor markers were found as up-regulated in cMSCs following exosome uptake: vacuolar H+-ATPase (V-ATPase) and carcinoembryonic antigen (CEA). These two molecules are potent candidates for colon cancer therapy, where V-ATPase is associated with an advanced malignant state (metastatic cancer) and CEA is an established biomarker used for screening of the evolution, progression, or regression during follow up of colorectal malignancies

117, 118

. Considering the unfavorable

consequences of exosomes released by colon cancer cells on the surrounding microenvironment, it would be an attractive strategy to apply an anti-cancer therapy compromising the depletion of exosomes to reduce the pathological transformation of surrounding cells. This type of strategy was successfully applied in mouse models of colon carcinoma where administration of DMA (dimethyl amiloride) promoted the effect of CTX (cyclophosphamide) by cancer derived exosomes depletion. Moreover, a synonym compound, amiloride, capable of inducing reduction of exosome levels is currently being used for the treatment of high blood pressure. Therefore, by analyzing the blood profile of metastatic colon cancer patients that are treated with amiloride to reduce blood pressure, an inhibition of exosome formation together with inhibition of the suppressive function of MDSC-like (Myeloid-derived suppressor-like) cells was observed

119

. MDSC-like cells can inhibit T cell activation in cancer con-

tributing in this way to the development of the disease

120

. In this sense, exosomes secreted

by colon cancer cells were associated with an increased activity of MDSC like cells, an effect that was attenuated in patients treated with amiloride. The KRAS gene that is mutated in approximately 30% to 40% of colorectal cancers represents another important therapeutic pathway related to exosomes in this pathology. Cancer cells that are holding a mutant gene secret exosomes rich in proteins with tumor promoting roles like EGFR, KRAS, integrins and SRC family kinases. The surrounding wild-type cells 19 ACS Paragon Plus Environment


Page 20 of 35

that are a target of these exosomes are implicated in a pathway by which cancer cells exploit the cells in the tumor microenvironment, which rapidly acquire malignant-like phenotypes and grow at a faster rate 121. In this sense, mutant KRAS or exosomes bearing mutant KRAS proteins are a target for cancer therapy, where the silencing of the oncogene or depletion of the tumor favoring vehicles could stand as a potent anti-cancer strategy. The tumor-promoting role of exosomes secreted by cancer cells is now well established despite the fact that the exact mechanisms are not yet fully elucidated. A wide quantitative proteomics analysis from the plasma of patients with colorectal cancer revealed a differentially expressed exosomal pattern with a panel of 36 upregulated proteins and 22 downregulated ones compared to healthy controls. Moreover, a large part of the upregulated molecules demonstrated an oncogenic role regarding cancer invasiveness but also regarding the arrangement of the pre-metastatic niche. Intriguingly, the proteins with known roles regarding cell proliferation, growth and implicit tumor development were found to be downregulated, a fact that suggested a possible role of exosomes as metastatic promoters but not tumor growth supporters 122. Nevertheless, the strategy of exosomes by blocking through different strategies is still a viable one, considering the disastrous effects of metastatic cancers. Another possible course of action in terms of exosome inhibition in colorectal cancer could be represented by therapeutic targeting of ALIX (ALG 2-interacting protein X), a molecule involved in the formation of MVBs within exosome biogenesis. This hypothesis is based on the fact that ALX was found as overexpressed in colorectal adenoma and carcinoma patients compared to healthy controls, contributing to the increased release of pro-carcinogenic vesicles 123. Diminishing the levels of ALX could also alter the concentration of released exosomes from cancer cells thereby also reducing tumor progression in patients. At the same time, there are also attempts in the context of colorectal cancer. Within a Phase I clinical trial the administration of ascites-derived exosomes (Aex) is followed by granulocyte-macrophage colony stimulating factor (GM-CSF) in order to immunologically manipulate the development of colorectal cancer. The synergistic administration of these two compounds, but not Aex alone, induced a cytotoxic T cells response with anti-tumor effects. Importantly both types of therapeutic strategies (Aex or Aex + GM-CSF) were well tolerated by the patients 124. In accordance with the latest trend on phytotherapies in cancer, where the beneficial effects of natural compounds are now validated by current technologies

125-128

, there is an incipient

clinical trial that involves the combination of natural compounds with plant exosomes. More specifically, the feasibility of curcumin delivery by plant exosomes to colon cancer tissue is 20 ACS Paragon Plus Environment

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tested in order to increase the stability and bioavailability as well as to improve the delivery efficiency of the plant-derived compound (https://clinicaltrials.gov – search performed using the terms “exosomes” and “colorectal cancer”).

Although exosome based strategies are not yet standard of care in cancer therapy, the existing basic and applied research as well as the previous and ongoing clinical trials assessing their therapeutic value envision a feasible treatment option for cancer patients (Table 3). Indeed, these nano-vehicles seem to avoid the major downsides of cell-based therapies consisting majorly in increased toxicology and also activation of unwanted immune reactions. Considering the fact that exosomes naturally shift molecules like nucleic acids and proteins between cells is increasingly countering the idea that the secreted vehicles could function as the ideal delivery vehicle for drugs (small molecules, nucleic acids, chemotherapy, natural agents, etc). While offering a stable environment for the custom cargo and also being able target and fuse with the cell membranes, exosomes are now in the center of cancer therapy that compromise the specific delivery of an anti-cancer agent. At the present time there are two major conduits employing the use of exosomes in cancer and not only: engineering of secreting cells through viral or non-viral methods for the releasement of modified exosomes and direct modification of exosomes content upon secretion. Also, recent evidences suggest that a possible future strategy could represent the modification of exosomes surface in order to specifically target cancer cells while avoiding healthy ones. These aspects were comprehensively reviewed in a recent article by Gilligan and Dwyer alongside with the most significant clinical or preclinical studies made in this area 24.

Table 3. Exosomes based therapeutic strategies Type of therapy involving exosomes

Delivery vehicle

Therapeutic strategy

Principle

Example

Direct modification of secreted exosomes

Electroporation for inclusion of drug/molecule upon the appliance of an electric field Incubation of exosomes with the specific drug/molecule small enough (molecular size) to

Delivery of siRNA via plasma exosomes to monocytes and lymphocytes Delivery of curcumin loaded exosomes to myeloid cells for antiinflammatory purposes


Ref 129

130, 131


passively enter into the nanovehicle – based on concentration gradient Sonication of exosomes together with the drug or small molecules – alteration of exosomes membrane based on the mechanical force and implicit diffusion of drug into exosomes.

Extrusion of exosomes and drug mixture with a syringe-based lipid extruder equipped with a 100– 400 nm porous membranes

Page 22 of 35

Delivery of paclitaxel to MDR cancer cells – one hour after the sonication, the exosomes reshaped to the previous state and the membrane composition was not affected. However there is also the possibility that the drug will attached to the outer part of the exosomal membrane Inclusion of porphyrin into MDA-MB231 secreted exosomes – possible cytotoxicity due to alteration of the zetapotential of exosomes. No cytotoxicity in the case of exosomes secreted by RAW264.7 cells and loaded with catalase through smaller extrusion cycles (10 cycles; previous study – 30 cycles) Pour yield and efficiency compared with other methods. Efficient for generation of fused exosome-liposomes particles due to aggregation that is usually a consequence of this method

Repeated freeze and thaw cycles of exosomes and drug mix for mechanical disruption of the exosome membrane and encapsulation of the drug (cycle: room temperature followed by liquid nitrogen or -80 °C freezer; protocol: minimum 3 cycles) Chemically based meth- Copper-catalyzed azide ods for attachment of alkyne cycloaddition for drugs and molecules to the attachment of mole-


132

133, 134

135

136, 137

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purified exosomes

Modification of Incubation of donor cells the specific exosomes donor with drug/molecule in order to cell further isolate the secreted exosomes that will contain the administrated therapeutic

Transfection of donor cells with the gene of interest that will be further packed into exosomes secreted by the transfected cells

Modification of Modification of donor exosomes target- cells in order to produce exosomes containing speing specificity cific sequences for targeted delivery


cules, both small and macromolecules, to the membrane of isolated exosomes via conjugation Paclitaxel treated SR4987 cells have secreted after 24h of incubation with paclitaxel and 48 additional hours for culturing in fresh media, exosomes loaded with the administrated drug. These exosomes were further used for treatment of CFPAC-1 human pancreatic cells, showing significant antiproliferative effects. Exosomes isolated from marrow stromal cell transfected with plasmids containing miR146b were used injected into a rat model of glioma (xenograft). The custom made exosomes significantly reduced the growth of the primary brain tumor Modification of donor cells (HEK293) with a vector encoding GE11 for further expression in the membrane of the secreted exosomes. The isolated exosomes were then specifically bounding to breast cancer cells expressing EGFR due to the compatibility between GE11 and

138

139

110


Cell activation

Modification of exosomes cargo through activation of donor cell

Stimulation of donor cell with exogenous substances in order to increase the expression of specific molecules into exosomes

Control of exosomes level

Elimination of Dialysis-like strategy for exosomes from the depletion of specific vesicles (e.g. exosomes) circulation from the blood of cancer patients

Inhibition of exo- Modification of exosomes some re- release rate for a better response to treatment lease/biogenesis

Page 24 of 35

EGFR Stimulation of human blood cells with LPS increased the amount of miR-150, miR-146a and miR-181a in MVs, while others remained constant (miR-122 and miR-124a). Aethlon ADAPT™ (adaptive dialysis-like affinity platform technology) system for the hemofiltration of exosomes based on the specific size (

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