**3.1 Phagocytes**

*Extracellular Vesicles and Their Importance in Human Health*

Neuron Cortical mouse neuron

Epithelial Cervical cancer (HeLa)

Epithelial (CNE1, HONE1,

Endothelial Cerebral vascular

D3)

Endothelial Brain microvascular endothelial

Dendritic cell (DC), T cell

Epithelial, endothelial

Epithelial, macrophage, endothelial

Epithelial Breast cancer

(PC12)

Pheochromocytoma

NU-GC-3, A549)

endothelial (hCMEC

Monocyte derived primary DC, T cell (Jurkat)

Melanoma (A375), (RAW264.7), dermal microvascular endothelial (HMVEC)

lymphoma (Jeko1)

Glioblastoma (U87), umbilical vein endothelial (HUVEC)

Epithelial Ovarian cancer (SKOV3)

Epithelial Breast carcinoma (BT549)

Endothelial Brain microvascular endothelial

B cell Mantle cell

*Endocytosis pathways involved in exosome internalization in various cell types.*

(MCF7)

**Recipient cell line Exosome cell of origin References**

Oligodendrocyte (Oli-neu)

(PC12)

(A431)

Normal breast epithelial cell (MCF-10A)—exosome mimetics

Macrophage (RAW264.7)

(Hek293T)

Dendritic cell Mouse primary Mouse dendritic cell [15]

Embryonic kidney cell

Ovarian cancer cell (SKOV3)

Breast carcinoma (BT549)

Embryonic kidney cell

Mantle cell lymphoma

(Hek293T)

(Jeko1)

Pheochromocytoma

Epidermoid carcinoma

EBV-infected B cells [95]

T cell (Jurkat) [75]

Glioblastoma (U87) [43]

Melanoma (A375) [46]

[115]

[114]

[90]

[96]

[89]

[87]

[97]

[44]

[87]

[61]

**Recipient cell type**

Neuron precursor cell

**Endocytosis pathway**

Caveolindependent endocytosis

Lipid raftdependent endocytosis

considered to be interchangeable with CME, but it is now understood that not all RME is dependent on clathrin [25]. Receptor-ligand interactions play a role in phagocytosis [25, 27], macropinocytosis [19], and lipid raft-dependent endocytosis [40]. Exosome internalization has been linked to multiple receptor-ligand interactions in each of these pathways [19, 20]. Each subtype of endocytosis has been

**46**

**Table 1.**

As introduced previously, some cells are uniquely designed to internalize extracellular material through phagocytosis. Those cells generally considered "professional" phagocytes are monocytes, macrophages, and neutrophils [25] with dendritic cells, osteoclasts, and eosinophils occasionally included [27]. Phagocytosis is dependent on receptor/ligand interactions, relying on a vast array of different receptors and ligands. Some of the established receptors include Fc receptors, integrins, pattern-recognition receptors, phosphatidylserine (PS) receptors, and scavenger receptors [45]. Macrophage uptake of exosomes has been shown to involve many of these receptors including scavenger receptors [46–48], PS/PS receptors [20, 48–51], lectins [17, 52, 53] and Fc receptors [54].

However, internalization of extracellular material by phagocytes does not always fit perfectly with the hallmarks of phagocytosis. Some phagocytic receptors, such as integrins (αvβ3), scavenger receptors (CD68 and CD36), and CD14, facilitate the tethering of apoptotic cells to the phagocyte surface, but then are unable to initiate internalization without other means, such as PS and PS receptor binding [55]. The PS/ PS receptor interaction also stimulates membrane ruffling and vacuole appearance classic hallmarks of macropinocytosis [55]. Phagocytes are primarily involved in phagocytosis, but this evidence supports the idea that multiple modes of endocytosis are operational in the same cell. This is not unique to apoptotic cell uptake, but has been seen with exosome internalization by microglia (phagocytic cells in the brain) exhibiting a dependence on PS in a macropinocytic manner [49, 56]. Cooperation between multiple receptors appears to be an important characteristic of endocytosis in phagocytic cells. Plebenak et al., showed that the scavenger receptor SR-B1 on macrophages, when blocked, reduces exosome uptake, but with further testing on melanoma cells this blocking was dependent both on the receptor as well as on cholesterol flux in the lipid rafts [46], broadening the endocytosis landscape of phagocytes to include lipid raft-dependent endocytosis.

The dependence of phagocytosis on extracellular- facing PS, which on healthy cells is expressed only on the cytosolic side of the membrane, is evidence that the material to be ingested influences the endocytic pathway of phagocytes. Further support of this interaction is found in the hypothesis that exosomes "target" specific recipient cells [48, 57]. Macrophage uptake (**Figure 2A**) of TEX is dependent on the presence of cellular scavenger receptors or exosomal PS [20, 46, 48, 51, 56], while non-tumor cellderived exosomes require the presence of a heterogeneity of receptors. When internalized by macrophages and monocytes, hepatic stellate cell-derived exosomes require Fc receptors [54]; B cell, dendritic cell and reticulocyte-derived exosomes use lectins [52, 53]; trophoblast-derived exosomes bind to integrins [58]; and T cell-derived exosomes need scavenger receptors [50] (**Table 2**). Costa-Silva et al., showed that when comparing TEX to normal cell-derived exosomes, Kupffer cells, liver-specific macrophages, preferentially internalized TEX [57]. The significance of the exosome surface topography is therefore influential in directing a specific endocytosis pathway. Phagocytes are responsible for internalization of extracellular material and are so

#### *Extracellular Vesicles and Their Importance in Human Health*

#### **Figure 2.**

*Cell-specific internalization of exosomes by antigen presenting cells: (A) macrophage, (B) B cell and (C) Dendritic cells each employ multiple endocytic pathways in the uptake of exosomes. Macrophages utilize multiple endocytic pathways in the uptake of exosomes. B Cells and dendritic cells (DC) both employ multiple endocytic pathways in the uptake of exsomes. Lipid rafts, integrins and adhesion molecules are used by B cells while tetraspanins and adhesion molecules are the more common receptors found in DC-exosome interactions. Intercellular adhesion molecule 1 (ICAM-1), Dendritic Cell-Specific Intercellular adhesion molecule-3- Grabbing Non-integrin (DC-SIGN).*

named based on the primary use of phagocytosis, but as seen above, other endocytic pathways are utilized, especially in the context of exosomal internalization.

#### **3.2 Antigen presenting cells**

The antigen presenting cells (APCs) include primary phagocytes such as macrophages, but also B cells and dendritic cells [59]. The immune response is heavily dependent on the recognition of foreign structures, such as peptides, for activation. These APCs sample the extracellular environment, digest and display peptides on their surface, and then present these peptides to immune cells that can execute the response. The intercellular trafficking of immune regulating proteins, such as the major histocompatibility complexes (MHC) [28], by exosomes has the potential to either stimulate or block the immune response, dependent on the exosomal contents [17]. Uptake of exosomes plays an important role in B cell and DC cell proliferation, protein presentation, and interactions with other immune cells [17].

B cells perform multiple functions as an immune cell, including presenting antigens to T cells in order to stimulate additional immune responses. B cells traditionally operate though clathrin-mediated endocytosis, relying heavily on the B-cell receptor [60]. However, when it comes to exosome internalization, B cells have shown a greater dependence on lipid rafts and various receptors, such as adhesion molecules and tetraspanins [17] than on clathrin, indicating a preference for clathrin-independent and receptor-mediated endocytosis (**Figure 2B**). In analyzing B cell uptake of exosomes, using the mantle cell lymphoma (mutated immature B cell) cell line, Jeko-1, Hazan-Halevy et al., found dynamin, epidermal growth factor receptor (EGFR), and cholesterol to be involved in exosome internalization instead of clathrin [61]. EGFR is a well-established target in cancer therapy, particularly with lung cancer [62] and its role in exosome internalization may lend clarity and power to multiple existing and future chemotherapeutics. Additional exosomal surface proteins, with receptor functions, have been identified as participants in

**49**

*Cellular-Defined Microenvironmental Internalization of Exosomes*

Lectins Lymph node cells, splenic cells,

Tetraspanins B cell, pancreatic

cell

EGFR A431 (epidermoid carcinoma cells)

ICAM-1/LFA-1 Dendritic cell, hCMEC/D3

HSPG U87 (glioblastoma cells), CAG

Cad-11 PC3-mm2 (prostate cancer cells)

CD62L Lymph node cells, splenic cells,

platelet, HeLa

Fc receptors Macrophage CT26 (colon

Integrins Macrophage, B cell Trophoblast,

CD11c Lymph node cells/splenic cells Pancreatic

CD11b Lymph node cells/splenic cells Pancreatic

CD44 Lymph node cells/splenic cells Pancreatic

CD49d/CD106 Lymph node cells/splenic cells Pancreatic

Tspan8 Endothelial cell Pancreatic

(brain endothelial cells), aortic endothelium, HUVEC

(myeloma), HUVEC, SW780 (bladder cancer cells)

SNAP 25 Neuron Mesenchymal stromal

pancreatic adenocarcinoma,

lung fibroblasts

DC-SIGN Dendritic cell Breast milk [70]

Syncytin Choriocarcinoma cells Trophoblasts [105]

adenocarcinoma, endothelial

**Protein Cell type Exosome origin References**

PS receptor Macrophage Activated T cells [50]

kidney cells)

cell lines)

Pancreatic adenocarcinoma, reticulocyte, B cell, macrophage, mesenchymal stem

carcinoma cells)

pancreatic adenocarcinoma cells

Pancreatic adenocarcinoma cells

adenocarcinoma cells

adenocarcinoma cells

adenocarcinoma cells

adenocarcinoma cells

adenocarcinoma cells

adenocarcinoma cells, T cells, macrophage

U-87 cells, myeloma cells, SW780 cells

cells

Pancreatic adenocarcinoma

Osteoblasts [104]

Dendritic cells, pancreatic

HeLa cells [19]

cell

oligodendrocytes

[47]

[49–51, 56]

[20]

[17, 48, 52, 53, 65, 89, 72, 103]

[54]

[17, 58]

[17, 48, 106]

[17]

[17]

[17]

[17]

[48, 106]

[16, 17, 37, 65, 69, 89]

[63, 99, 100, 101]

[116]

[17, 48]

Scavenger receptor Macrophage Hek293 (embryonic

Phosphatidylserine (PS) Macrophage, microglia Neuron, melanoma,

TIM4 Macrophage K562, MT4 (leukemia

pancreatic adenocarcinoma, lung fibroblast, macrophage, dendritic cell, hCMEC/ D3(brain endothelial cells),

*DOI: http://dx.doi.org/10.5772/intechopen.86020*

*Extracellular Vesicles and Their Importance in Human Health*

named based on the primary use of phagocytosis, but as seen above, other endocytic

The antigen presenting cells (APCs) include primary phagocytes such as macrophages, but also B cells and dendritic cells [59]. The immune response is heavily dependent on the recognition of foreign structures, such as peptides, for activation. These APCs sample the extracellular environment, digest and display peptides on their surface, and then present these peptides to immune cells that can execute the response. The intercellular trafficking of immune regulating proteins, such as the major histocompatibility complexes (MHC) [28], by exosomes has the potential to either stimulate or block the immune response, dependent on the exosomal contents [17]. Uptake of exosomes plays an important role in B cell and DC cell proliferation, protein presentation, and interactions with other immune cells [17]. B cells perform multiple functions as an immune cell, including presenting antigens to T cells in order to stimulate additional immune responses. B cells traditionally operate though clathrin-mediated endocytosis, relying heavily on the B-cell receptor [60]. However, when it comes to exosome internalization, B cells have shown a greater dependence on lipid rafts and various receptors, such as adhesion molecules and tetraspanins [17] than on clathrin, indicating a preference for clathrin-independent and receptor-mediated endocytosis (**Figure 2B**). In analyzing B cell uptake of exosomes, using the mantle cell lymphoma (mutated immature B cell) cell line, Jeko-1, Hazan-Halevy et al., found dynamin, epidermal growth factor receptor (EGFR), and cholesterol to be involved in exosome internalization instead of clathrin [61]. EGFR is a well-established target in cancer therapy, particularly with lung cancer [62] and its role in exosome internalization may lend clarity and power to multiple existing and future chemotherapeutics. Additional exosomal surface proteins, with receptor functions, have been identified as participants in

pathways are utilized, especially in the context of exosomal internalization.

*Cell-specific internalization of exosomes by antigen presenting cells: (A) macrophage, (B) B cell and (C) Dendritic cells each employ multiple endocytic pathways in the uptake of exosomes. Macrophages utilize multiple endocytic pathways in the uptake of exosomes. B Cells and dendritic cells (DC) both employ multiple endocytic pathways in the uptake of exsomes. Lipid rafts, integrins and adhesion molecules are used by B cells while tetraspanins and adhesion molecules are the more common receptors found in DC-exosome interactions. Intercellular adhesion molecule 1 (ICAM-1), Dendritic Cell-Specific Intercellular adhesion molecule-3-*

**3.2 Antigen presenting cells**

*Grabbing Non-integrin (DC-SIGN).*

**Figure 2.**

**48**



#### **Table 2.**

*Proteins involved in exosomal uptake.*

B cell internalization of TEX, including integrins (CD49) and cell adhesion molecules (intercellular adhesion molecule 1—ICAM-1/CD54 and CD62L) [17].

These protein interactions between the cell and the exosomal membranes are essential steps in the influence the exosome has on the recipient cell. Exosomes derived from myeloma cells, cancerous plasma (mature B) cells, are dependent on the interaction between exosomal fibronectin and cellular heparan sulfate in order to form a bond between cell and exosome, resulting in modification of intracellular signaling [63]. As seen with these cells, the effects caused by the exosomes are not entirely dependent on uptake, even though the standard operation of APCs requires internalization. Some exosome-cell binding (as opposed to internalization) may be sufficient, or specifically designed, to alter intracellular processes, including signaling, as is also seen with dendritic cell-derived exosomes and T cell function [16]. While the influence of heparan sulfate on internalization in B cells is still unclear, there is evidence linking heparan sulfate proteoglycans to exosomal internalization which indicates that while it wasn't assessed in these cells, the uptake may still be present [21–23]. Whether these differing mechanisms and protein participants of uptake in the B cell population are dependent on normal versus oncologic physiology of recipient cells, or on the origin of the exosome population (tumor-derived versus non-tumor derived) is yet to be determined.

These heterogeneous protein profiles are specific to each cell type and contribute to the comparative ability of each cell to internalize exosomes. In line with the role of B cells, it was found that they readily take in exosomes, in contrast to other immune cells such as T cells and natural killer cells [61, 64]. This suggests that certain immune cells are more effective at endocytosing exosomes than others, consistent with the primary functions of these specific cell types. Additional groups have shown that while B cells internalize exosomes, the uptake is significantly less than that of macrophages and dendritic cells, but similar to T cells [17]. This was shown in non-mutated mouse cells and may also illustrate important differences between cancer cell and normal cell internalization mechanisms.

Dendritic cells (DC) can be classified as both APCs and as phagocytes since internalization of extracellular material is a crucial part of their role in the immune system. Endocytosis pathways involved in exosome uptake in these cells have been tested with various endocytic blockers, including cytochalasin D (inhibits actin polymerization), EDTA (chelates calcium), and decreased temperature (reducing active cellular processes) [15, 37, 65, 66]. As dendritic cells mature, their mode of endocytosis changes; starting first with macropinocytosis, and then in the mature cell, receptor-mediated endocytosis and phagocytosis prevails [67] (**Figure 2C**). Despite the evidence of phagocytosis in mature DCs, it was demonstrated that immature DCs are more adept at exosomal uptake [37, 68]. Developmental preference for exosome uptake may shed light on why cancer cells, which often have

**51**

*Cellular-Defined Microenvironmental Internalization of Exosomes*

similar profiles to developing cells and are subject to continuous proliferation, are so responsive to modification by exosomes. Also, immature DCs play a role in immunologic tolerance and so are less likely to activate T cells, while mature DCs activate T cell immunity [15]. This down-regulation of the adaptive immune response by immature DCs would be advantageous for tumors and so TEX may specifically target immature DCs, explaining the increase in uptake. While the mechanism is still unknown, dendritic cells are also more likely to take up TEX or DC-derived exosomes than B and T cells, as seen with fluorescent staining *in vitro* and *in vivo* in a rat model of pancreatic adenocarcinoma [17] and flow cytometry analysis of mouse bone marrow derived cells [15]. The CD11c membrane protein present on the DC and not on the other cells, was found to be involved in the internalization of TEX, as uptake decreased in the presence of an antibody to CD11c. The expression of this protein unique to DCs may contribute to the disparity in uptake among the immune cell types [17]. Recipient cell specificity in exosome uptake and DC interconnection with immune effector cells is another potential area of immune-

Many of the studies of exosome internalization by DCs have revealed dependence on various adhesion molecules. The ubiquity of these proteins on exosomes, leukocytes, and endothelial cells promotes the non-specific internalization characteristic of DCs. The involvement of ICAM-1 and/or its ligand, lymphocyte function-associated antigen (LFA-1), in DC-exosome interaction has been shown both *in vitro* and *in vivo* [16, 17, 37, 65, 69]. These interactions are not unique to exosome uptake as DCs regularly depend on a wide range of adhesion molecules, including a dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) [70]. This particular adhesion molecule has been shown to be more effective at exosome uptake by DCs, when looking at breast milk-derived exosomes, than the ICAM-1/LFA-1 binding [71]. In addition to adhesion molecules, C-type lectin and its receptor have also been identified in DC-exosome binding [65]. These glycan binding proteins have also been identified as exosome uptake mediators in other cell types, including macrophages [52]

In addition to binding to membrane receptors, dendritic cell endocytosis is dependent on lipid rafts and the lipid components of the cell membrane, particularly with viral or bacterial uptake [73, 74]. As viruses and exosomes are similar in size, endocytosis mechanisms are often common between these two structures [22]. Lipid-dependent endocytosis is evident in exosome uptake by DCs as illustrated with DC- and T-cell derived exosomes [15, 75]. While proteins have been the most common structure analyzed in connection with exosomal uptake, the membrane cholesterol concentration of recipient cells [15] as well as the lipid profile of the exosomal membrane [75] both play a role in uptake of exosomes by dendritic cells

In addition to the previously mentioned cells, two other circulating cells/structures have also been found to endocytose exosomes, platelets and T cells. Platelets are cell fragments involved in blood coagulation that are unique in their formation as they are devoid of a nucleus and some organelles. Despite a reduced intracellular load, they are involved in binding extracellular vesicles. They do so through the interaction of cellular P-selectin and vesicular P-selectin glycoprotein ligand-1 (PSGL-1) as well as PS [72]. Data suggests that binding facilitates fusion of the exosomes to the platelets, transferring of material and enhancing platelet coagulation activity [72]. This speaks to the impact of these exosomes on intracellular

*DOI: http://dx.doi.org/10.5772/intechopen.86020*

therapeutic manipulation.

and platelets [72].

and need further clarification.

**3.3 Circulating cells**

#### *Cellular-Defined Microenvironmental Internalization of Exosomes DOI: http://dx.doi.org/10.5772/intechopen.86020*

*Extracellular Vesicles and Their Importance in Human Health*

CD169/ α2,3-linked sialic

*Proteins involved in exosomal uptake.*

C-type lectin/C-type lectin receptor

acid

**Table 2.**

B cell internalization of TEX, including integrins (CD49) and cell adhesion molecules (intercellular adhesion molecule 1—ICAM-1/CD54 and CD62L) [17].

P-selectin/PSGL-1 Platelet Macrophage [72]

Dendritic cell, brain endothelial cell (hCMEC/D3)

These protein interactions between the cell and the exosomal membranes are essential steps in the influence the exosome has on the recipient cell. Exosomes derived from myeloma cells, cancerous plasma (mature B) cells, are dependent on the interaction between exosomal fibronectin and cellular heparan sulfate in order to form a bond between cell and exosome, resulting in modification of intracellular signaling [63]. As seen with these cells, the effects caused by the exosomes are not entirely dependent on uptake, even though the standard operation of APCs requires internalization. Some exosome-cell binding (as opposed to internalization) may be sufficient, or specifically designed, to alter intracellular processes, including signaling, as is also seen with dendritic cell-derived exosomes and T cell function [16]. While the influence of heparan sulfate on internalization in B cells is still unclear, there is evidence linking heparan sulfate proteoglycans to exosomal internalization which indicates that while it wasn't assessed in these cells, the uptake may still be present [21–23]. Whether these differing mechanisms and protein participants of uptake in the B cell population are dependent on normal versus oncologic physiology of recipient cells, or on the origin of the exosome population (tumor-derived versus non-tumor derived) is yet to be

**Protein Cell type Exosome origin References** Galectin 5 Macrophage Reticulocyte [52]

Lymph node cells, splenic cells B cell [53]

Macrophage [65, 89]

These heterogeneous protein profiles are specific to each cell type and contribute

to the comparative ability of each cell to internalize exosomes. In line with the role of B cells, it was found that they readily take in exosomes, in contrast to other immune cells such as T cells and natural killer cells [61, 64]. This suggests that certain immune cells are more effective at endocytosing exosomes than others, consistent with the primary functions of these specific cell types. Additional groups have shown that while B cells internalize exosomes, the uptake is significantly less than that of macrophages and dendritic cells, but similar to T cells [17]. This was shown in non-mutated mouse cells and may also illustrate important differences

Dendritic cells (DC) can be classified as both APCs and as phagocytes since internalization of extracellular material is a crucial part of their role in the immune system. Endocytosis pathways involved in exosome uptake in these cells have been tested with various endocytic blockers, including cytochalasin D (inhibits actin polymerization), EDTA (chelates calcium), and decreased temperature (reducing active cellular processes) [15, 37, 65, 66]. As dendritic cells mature, their mode of endocytosis changes; starting first with macropinocytosis, and then in the mature cell, receptor-mediated endocytosis and phagocytosis prevails [67] (**Figure 2C**). Despite the evidence of phagocytosis in mature DCs, it was demonstrated that immature DCs are more adept at exosomal uptake [37, 68]. Developmental preference for exosome uptake may shed light on why cancer cells, which often have

between cancer cell and normal cell internalization mechanisms.

**50**

determined.

similar profiles to developing cells and are subject to continuous proliferation, are so responsive to modification by exosomes. Also, immature DCs play a role in immunologic tolerance and so are less likely to activate T cells, while mature DCs activate T cell immunity [15]. This down-regulation of the adaptive immune response by immature DCs would be advantageous for tumors and so TEX may specifically target immature DCs, explaining the increase in uptake. While the mechanism is still unknown, dendritic cells are also more likely to take up TEX or DC-derived exosomes than B and T cells, as seen with fluorescent staining *in vitro* and *in vivo* in a rat model of pancreatic adenocarcinoma [17] and flow cytometry analysis of mouse bone marrow derived cells [15]. The CD11c membrane protein present on the DC and not on the other cells, was found to be involved in the internalization of TEX, as uptake decreased in the presence of an antibody to CD11c. The expression of this protein unique to DCs may contribute to the disparity in uptake among the immune cell types [17]. Recipient cell specificity in exosome uptake and DC interconnection with immune effector cells is another potential area of immunetherapeutic manipulation.

Many of the studies of exosome internalization by DCs have revealed dependence on various adhesion molecules. The ubiquity of these proteins on exosomes, leukocytes, and endothelial cells promotes the non-specific internalization characteristic of DCs. The involvement of ICAM-1 and/or its ligand, lymphocyte function-associated antigen (LFA-1), in DC-exosome interaction has been shown both *in vitro* and *in vivo* [16, 17, 37, 65, 69]. These interactions are not unique to exosome uptake as DCs regularly depend on a wide range of adhesion molecules, including a dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) [70]. This particular adhesion molecule has been shown to be more effective at exosome uptake by DCs, when looking at breast milk-derived exosomes, than the ICAM-1/LFA-1 binding [71]. In addition to adhesion molecules, C-type lectin and its receptor have also been identified in DC-exosome binding [65]. These glycan binding proteins have also been identified as exosome uptake mediators in other cell types, including macrophages [52] and platelets [72].

In addition to binding to membrane receptors, dendritic cell endocytosis is dependent on lipid rafts and the lipid components of the cell membrane, particularly with viral or bacterial uptake [73, 74]. As viruses and exosomes are similar in size, endocytosis mechanisms are often common between these two structures [22]. Lipid-dependent endocytosis is evident in exosome uptake by DCs as illustrated with DC- and T-cell derived exosomes [15, 75]. While proteins have been the most common structure analyzed in connection with exosomal uptake, the membrane cholesterol concentration of recipient cells [15] as well as the lipid profile of the exosomal membrane [75] both play a role in uptake of exosomes by dendritic cells and need further clarification.

## **3.3 Circulating cells**

In addition to the previously mentioned cells, two other circulating cells/structures have also been found to endocytose exosomes, platelets and T cells. Platelets are cell fragments involved in blood coagulation that are unique in their formation as they are devoid of a nucleus and some organelles. Despite a reduced intracellular load, they are involved in binding extracellular vesicles. They do so through the interaction of cellular P-selectin and vesicular P-selectin glycoprotein ligand-1 (PSGL-1) as well as PS [72]. Data suggests that binding facilitates fusion of the exosomes to the platelets, transferring of material and enhancing platelet coagulation activity [72]. This speaks to the impact of these exosomes on intracellular

**Figure 3.**

*Cell-specific internalization of exosomes: (A) Platelet-exosome interactions have been linked to fusion as well as the binding to PSGL-1 and phosphatidylserine, (B) T cell are influenced through their surface interactions with exosomes.*

communication, both in the variability and specificity of recipient cells, since binding and fusion occurred preferentially in the activated platelets [72] (**Figure 3A**). The exosomes in this study came from monocytes, suggesting this interaction could be a key player in coagulation at a site of injury.

T cells are the effector cells of the immune system and intercellular communication is essential for activation. Endocytosis, while not a primary function of T cells, is important to T cell receptor signaling [76] as well as other functions. Dynamin-dependent endocytosis [76], phagocytosis [77], and RME [78] are some of the mechanisms involved in T cell interaction with its surrounding environment. In relation to exosomes, T cells operate through RME [17, 79, 80] and lipid raft-dependent endocytosis [75]. However, T cells do not always readily uptake exosomes as was found in a comparison with other blood cell types. In a peripheral blood mononuclear cell culture, when uptake by monocytes was blocked, internalization by T-cells increased [47], suggesting that T cell uptake may be an adaptive response to increased exosome concentration. When exosome uptake was compared to multiple splenic leukocytes [15] or peripheral blood leukocytes [64], T cells showed minimal internalization. T cell activity is often regulated by surface interactions with other cells, such as with the T cell receptor and the MHC II/antigen interaction with APCs. Exosomal influence on T cells may therefore operate similarly with surface interaction instead of exosome internalization (**Figure 3B**). When cultured with DC or DC-derived exosomes, T cells acquired functional surface molecules including MHC II from exosomes through direct exosome interaction with the T cell membrane, while still showing little evidence of internalization [81]. Mouse T cells do not express MHC II and after incubation with these exosomes, this protein was identified on the surface of the T cell, suggesting the binding of exosomes to cellular membranes is sufficient to transfer material, without internalization [81]. Further research into the transfer of material between exosomes and immune cells may elucidate the role exosomes play in immune regulation in the tumor microenvironment. Depending on the cell

**53**

*Cellular-Defined Microenvironmental Internalization of Exosomes*

type involved, exosome-mediated communication and manipulation may not be

*Cell-specific internalization of exosomes: (A) epithelial and (B) endothelial cells. Epithelial cells and endothelial cells show the most diversity in exosome uptake of all the cell types. Multiple receptor involved in the internalization process are expressed on both cell types, including tetraspanins, adhesion molecules, and* 

*heparan sulfate peptidoglycans (HSPG). Intercellular adhesion molecule 1 (ICAM-1).*

Epithelial and endothelial cells are responsible for lining most of the organs, spaces, and blood vessels in the body. They are in a prime position to be exposed to and actively endocytose a wide variety of extracellular material. Due to this broad selection, the specific mechanisms utilized are dependent on the cell subtype as well as the character of the endocytosed material [82–84]. With such variability, it is no surprise that exosome uptake by epithelial and endothelial cells is just as diverse (**Figure 4**). Cellular location of these cells is crucial in cancer biology as most of the TEX will be in close proximity to epithelial and endothelial cells either in the circulatory system or during paracrine spread in solid tumors. While there have been many studies on cell-exosome interaction in these cells, there is still much work needed to clearly understand all of the factors that dictate the endocytic mechanism

A unique finding in exosome studies with epithelial and endothelial cells is the dependence of uptake on intracellular signaling. Svensson et al., discovered that exosome internalization is dependent on the proper functioning of the signaling pathway, ERK1/2-HSP27 [43]. The promotion of endocytosis through intracellular signaling has been shown previously with EGFR-cSrc-ERK1/2 pathways in epithelial cells [85] and the Ras-PI3K pathway with virus uptake by fibroblasts [86]. However, little is known about how these pathways facilitate exosome internalization. The ability of exosomes to cross the blood–brain barrier and be endocytosed by the microvascular endothelial cells in the brain is also dependent on signaling. Tumor necrosis factor (TNFα) signaling, as is seen in stroke models, enhances exosome uptake [87]. Intracellular signaling may provide a regulatory mechanism

*DOI: http://dx.doi.org/10.5772/intechopen.86020*

entirely dependent on endocytosis.

**Figure 4.**

**3.4 Epithelial and endothelial cells**

of epithelial and endothelial cells from different tissues.

*Cellular-Defined Microenvironmental Internalization of Exosomes DOI: http://dx.doi.org/10.5772/intechopen.86020*

**Figure 4.**

*Extracellular Vesicles and Their Importance in Human Health*

be a key player in coagulation at a site of injury.

communication, both in the variability and specificity of recipient cells, since binding and fusion occurred preferentially in the activated platelets [72] (**Figure 3A**). The exosomes in this study came from monocytes, suggesting this interaction could

*Cell-specific internalization of exosomes: (A) Platelet-exosome interactions have been linked to fusion as well as the binding to PSGL-1 and phosphatidylserine, (B) T cell are influenced through their surface interactions* 

T cells are the effector cells of the immune system and intercellular communication is essential for activation. Endocytosis, while not a primary function of T cells, is important to T cell receptor signaling [76] as well as other functions. Dynamin-dependent endocytosis [76], phagocytosis [77], and RME [78] are some of the mechanisms involved in T cell interaction with its surrounding environment. In relation to exosomes, T cells operate through RME [17, 79, 80] and lipid raft-dependent endocytosis [75]. However, T cells do not always readily uptake exosomes as was found in a comparison with other blood cell types. In a peripheral blood mononuclear cell culture, when uptake by monocytes was blocked, internalization by T-cells increased [47], suggesting that T cell uptake may be an adaptive response to increased exosome concentration. When exosome uptake was compared to multiple splenic leukocytes [15] or peripheral blood leukocytes [64], T cells showed minimal internalization. T cell activity is often regulated by surface interactions with other cells, such as with the T cell receptor and the MHC II/antigen interaction with APCs. Exosomal influence on T cells may therefore operate similarly with surface interaction instead of exosome internalization (**Figure 3B**). When cultured with DC or DC-derived exosomes, T cells acquired functional surface molecules including MHC II from exosomes through direct exosome interaction with the T cell membrane, while still showing little evidence of internalization [81]. Mouse T cells do not express MHC II and after incubation with these exosomes, this protein was identified on the surface of the T cell, suggesting the binding of exosomes to cellular membranes is sufficient to transfer material, without internalization [81]. Further research into the transfer of material between exosomes and immune cells may elucidate the role exosomes play in immune regulation in the tumor microenvironment. Depending on the cell

**52**

**Figure 3.**

*with exosomes.*

*Cell-specific internalization of exosomes: (A) epithelial and (B) endothelial cells. Epithelial cells and endothelial cells show the most diversity in exosome uptake of all the cell types. Multiple receptor involved in the internalization process are expressed on both cell types, including tetraspanins, adhesion molecules, and heparan sulfate peptidoglycans (HSPG). Intercellular adhesion molecule 1 (ICAM-1).*

type involved, exosome-mediated communication and manipulation may not be entirely dependent on endocytosis.

### **3.4 Epithelial and endothelial cells**

Epithelial and endothelial cells are responsible for lining most of the organs, spaces, and blood vessels in the body. They are in a prime position to be exposed to and actively endocytose a wide variety of extracellular material. Due to this broad selection, the specific mechanisms utilized are dependent on the cell subtype as well as the character of the endocytosed material [82–84]. With such variability, it is no surprise that exosome uptake by epithelial and endothelial cells is just as diverse (**Figure 4**). Cellular location of these cells is crucial in cancer biology as most of the TEX will be in close proximity to epithelial and endothelial cells either in the circulatory system or during paracrine spread in solid tumors. While there have been many studies on cell-exosome interaction in these cells, there is still much work needed to clearly understand all of the factors that dictate the endocytic mechanism of epithelial and endothelial cells from different tissues.

A unique finding in exosome studies with epithelial and endothelial cells is the dependence of uptake on intracellular signaling. Svensson et al., discovered that exosome internalization is dependent on the proper functioning of the signaling pathway, ERK1/2-HSP27 [43]. The promotion of endocytosis through intracellular signaling has been shown previously with EGFR-cSrc-ERK1/2 pathways in epithelial cells [85] and the Ras-PI3K pathway with virus uptake by fibroblasts [86]. However, little is known about how these pathways facilitate exosome internalization. The ability of exosomes to cross the blood–brain barrier and be endocytosed by the microvascular endothelial cells in the brain is also dependent on signaling. Tumor necrosis factor (TNFα) signaling, as is seen in stroke models, enhances exosome uptake [87]. Intracellular signaling may provide a regulatory mechanism

to control exosome internalization. Some studies described previously have shown that fusion of exosomes to the cell membrane, without endocytosis, can influence intracellular signaling [63], but these are the first to show how intracellular signaling specifically impacts the endocytosis mechanism of exosomes. These results illustrate the complexity of exosome-cell interactions and where additional research is needed. The interdependence of exosome-cell interactions and intracellular signaling are unexplored areas with vast therapeutic potential and are necessary to better understand how extracellular vesicles influence their environment.

Other characteristics are influential in directing endocytosis in epithelial cells including vesicle size, lipid profile, and protein profile (**Figure 4A**). In epithelial cells, particle size dictates entry mechanism with macropinocytosis as one of the pathways operative at a size range that corresponds with exosomes [88]. This pattern is supported by multiple studies where exosome internalization was decreased when key aspects of macropinocytosis were targeted. Macropinocytosis was blocked with an inhibitor of Na<sup>+</sup> /H<sup>+</sup> exchange (which affects Rac1 activation and actin reorganization) in human cerebral microvascular endothelial cells (hCMEC/D3) [89] and HeLa cells, as well as with an inhibitor of phosphoinositide 3-kinase (PI3K) (influences membrane ruffling and macropinosome formation) [19, 90] with concomitant decreases in exosome internalization. Assessing the same pathway but from an activating instead of inhibiting direction, exosome internalization was stimulated by activation of epidermal growth factor receptor (which activates Rac family members) in HeLa cells [19]. Membrane extensions, or filopodia, that facilitate the formation of the macropinosome and are regulated by Rac1 activation have also been shown to influence exosome internalization in hepatocyte (Huh7) and kidney (Hek293) cells [91], furthering the support that exosomes utilize macropinocytosis in multiple epithelial cell lines.

The lipid profile of the exosomes and membrane integrity of the cell are also important contributors to vesicle uptake in several different types of epithelial and endothelial cells. While macrophages readily recognize external-facing PS, these cells can also utilize exosomal PS in the process of internalization, as was shown when pre-incubating exosomes with Annexin V inhibited uptake by HeLa cells (cervical cancer epithelial cells), A375 and A431 cells (squamous skin cancer cells) [92] and in human umbilical vein endothelial cells (HUVEC) [93]. Disruption of cellular lipid raft integrity through cholesterol depletion or sequestration reduced exosome uptake in U87 human glioblastoma epithelial cells [43], hCME/D3 human cerebral microvascular cells [89], HeLa cells [43, 90], HUVECs [43, 46], and A375 cells [46]. Lipid rafts play a key role in many of the functions of epithelial cells, including the protein binding interactions between cell and extracellular environment. Also, some of the most central components to epithelial cell function are proteins that interact closely with the environment such as integrins and adhesion molecules, and are anchored into lipid rafts.

Protein interactions are essential to epithelial and endothelial function and are closely tied to several of the most common endocytosis pathways used by these cells. Clathrin-dependent endocytosis has been shown in gastric [94], nasopharyngeal [95], breast [96], ovarian cancer epithelial cells [97] and HUVECs [98]. Caveolin-dependence was seen in breast [96] and nasopharyngeal cancer [95], however, caveolin-1 showed negative regulation in glioblastoma cell lines [43] (**Figure 4B**). General receptor-mediated uptake has been shown with several proteins including heparan sulfate peptidoglycan (HSPG) in glioblastoma cells and HUVECs [99, 100] and in the transitional epithelial cells of the bladder [101];

**55**

**Figure 5.**

*utilize multiple endocytic pathways.*

*Cellular-Defined Microenvironmental Internalization of Exosomes*

intercellular adhesion molecule (ICAM1) in hCMEC/D3 cells [89], rat aortic endothelial cells [48], and HUVECs [102]; lectins in cervical cancer [103], HUVECs [102], rat aortic endothelial cells [48] and hCMEC/D3 cells [89]; cad-11 in prostate cancer [104]; syncytin proteins in choriocarcinoma [105] and tetraspanins in an *in vivo* rat model of pancreatic cancer [48, 106]. The nature of cellular research has limited most of the epithelial endocytosis studies to cell lines, which consist entirely of transformed cells, and it is still unknown whether these trends are translatable to normal healthy epithelial and endothelial cells. While the mechanisms remain unknown, cultured primary normal epithelial cells take up TEX [107] highlighting a role for exosome intercellular communication in normal cell

The extracellular matrix (ECM) and stroma are important contributors to cellular homeostasis and function. This is particularly evident in tumors when evaluating the role of the tumor microenvironment (TME) on the survival and progression of the tumor cells. Fibroblasts are the major component of this extracellular environment. In normal physiology, they promote stromal stability, while in cancer, they contribute to altered ECM, increased angiogenesis, and metastasis [108]. These cells are in a pivotal position to interact with circulating exosomes and their internalization can have a compounding effect on the surrounding environment. Fibroblasts have been shown to participate primarily in clathrin-mediated endocytosis [109, 110] and occasionally receptor-mediated endocytosis [111]. Interestingly, RME [48, 106] and macropinocytosis [91] are the mechanisms by which fibroblasts have been shown to internalize exosomes (**Figure 5**). Tetraspanins are important proteins in fibroblast function and migration [112]. This protein family is well represented on the exosomal surface and is key to the uptake in many different cell types [48]. Additionally, evidence shows that the smaller the size of the vesicle, the more likely the fibroblast is to use receptors to internalize particles [111]. These three qualities lend support to the evidence of RME as a key pathway for fibroblasts to endocytose exosomes.

*Cell-specific internalization of exosomes: fibroblasts. Fibroblasts take up exosomes with tetraspanins and* 

*DOI: http://dx.doi.org/10.5772/intechopen.86020*

physiology.

**3.5 Fibroblasts**

### *Cellular-Defined Microenvironmental Internalization of Exosomes DOI: http://dx.doi.org/10.5772/intechopen.86020*

intercellular adhesion molecule (ICAM1) in hCMEC/D3 cells [89], rat aortic endothelial cells [48], and HUVECs [102]; lectins in cervical cancer [103], HUVECs [102], rat aortic endothelial cells [48] and hCMEC/D3 cells [89]; cad-11 in prostate cancer [104]; syncytin proteins in choriocarcinoma [105] and tetraspanins in an *in vivo* rat model of pancreatic cancer [48, 106]. The nature of cellular research has limited most of the epithelial endocytosis studies to cell lines, which consist entirely of transformed cells, and it is still unknown whether these trends are translatable to normal healthy epithelial and endothelial cells. While the mechanisms remain unknown, cultured primary normal epithelial cells take up TEX [107] highlighting a role for exosome intercellular communication in normal cell physiology.
