**2.2 Macropinocytosis**

*Extracellular Vesicles and Their Importance in Human Health*

exosomal internalization.

**2. Endocytosis pathways**

mentioned (**Figure 1**).

**2.1 Phagocytosis**

very influential in intercellular communication. Numerous studies have used these luminal proteins and genes to better understand tumor growth and metastasis, as well as for improving diagnostic, prognostic, and therapeutic methods [13, 14]. While there has been an exponential growth in research focused on exosome biology, clarification on the mechanisms of transport between the cell of origin and the recipient cell is essential to maximizing on exosome potential in treating and diagnosing disease. The methods by which exosomes influence the cells with which they interact are still under review. Some exosomes have been shown to fuse to the recipient cell [15, 16], while others are internalized by specific receptor-ligand interactions [17, 18] or by stimulating an indirect uptake by macropinocytosis [19]. Exosome binding to cells has been seen both as a mechanism of transferring luminal contents [15, 16] and as an initial step in the endocytosis process [17, 20]. The significance of the effects of cell-exosome binding in comparison to internalization is still unknown. Most types of endocytosis have been described in the process of exosome uptake [21], but which factors determine the specific mechanism used, are still unclear. Previous reviews have clearly identified a number of ligands and receptors involved in exosome trafficking [21–23], but little is known about the dependence of uptake mechanism on cell-type. This review presents the current understanding of the endocytosis process utilized by specific cells involved in

Endocytosis is a basic cellular function that is performed by all cell types in the process of maintaining homeostasis. Many of the molecules essential for cellular function are small enough to cross the cell membrane either passively or actively, however, other structures, such as exosomes, are too large and require a more complicated process. This general process of internalization is called endocytosis and is separated into various types based on the shape [24] and the size of particles internalized [25]. There are many well-written reviews covering the specifics of the endocytic pathways [25, 26], but here we will address them only superficially. Classification under the umbrella of endocytosis varies, but the major methods include phagocytosis, macropinocytosis, clathrin-mediated endocytosis, caveolinmediated endocytosis, and clathrin/caveolin-independent or lipid raft-mediated endocytosis [25, 26]. Receptor-mediated endocytosis (RME) is an additional type that is often considered to be a subcategory under several of those previously

Phagocytosis is the mechanism by which specialized cells (such as macrophages and monocytes) engulf large particles (>0.5 μm) by way of receptor/ligand interactions [25, 27] (**Figure 1A**). Promiscuous receptors allow for a broad range of ligand recognition and binding, facilitating a key role phagocytes play in clearing apoptotic cells [27]. Exosomes, derived from a diverse population of cells, present a vast array of available ligands that make phagocytes ideal recipient cells. This process of phagocytosis is designed to not only internalize extracellular material by enveloping it, but also to regulate the immune response by presenting degraded proteins as antigens on the phagocyte surface [25]. Tumor-derived exosomes influence immune involvement in the tumor [28, 29] which may be facilitated by this mechanism of endocytosis. Other non-phagocytic cells, such as epithelial cells, Sertoli, liver endothelial, astrocytes, and cancer cells have also been shown to perform

**42**

While phagocytosis or "cell eating" involves ingestion of large molecules, macropinocytosis ("cell drinking") internalizes slightly smaller particles (>1 μm) [25] (**Figure 1B**). This method is a way for cells to sample the external environment without specific receptors or ligands. It is a constitutive process in specialized antigen presenting cells, but is stimulated by growth factors in most others [30]. Macropinocytosis has a unique membrane ruffling process caused by projections from the cell surface encircling extracellular fluid and fusing to the membrane [25], resulting in an increased membrane surface area and volume of engulfed material. Nakase et al., showed that stimulation of the epidermal growth factor (EGF)

#### **Figure 1.**

*Endocytosis pathways involved in exosome uptake: (A) Phagocytosis, (B) Macropinocytosis, (C) Clathrinmediated endocytosis, (D) Caveolin-mediated endocytosis, (E) Lipid Raft-dependent or clathrin−/caveolinindependent endocytosis, (F) Receptor-mediated endocytosis.*

receptor, either by soluble EGF or exosome-bound, increased exosome internalization 27-fold through the activation of macropinocytosis [19].

### **2.3 Clathrin-dependent endocytosis**

The next three mechanisms, clathrin-dependent, caveolae-dependent, and clathrin/caveolae-independent, are facilitated by specific membrane proteins/ structures: clathrin, caveolae, and lipid rafts. Clathrin is an intracellular protein that forms a coat around an invaginating vesicle facilitating formation and internalization [31] (**Figure 1C**). These vesicles internalize material around 120 nm [25], which is within the exosome size range. Stimulation can occur through receptor/ ligand mediation or can be constitutive, depending on cell-type and receptor presence, but clathrin-mediated endocytosis (CME) occurs in all cell types [31]. Data continues to show that the extracellular cargo of these clathrin-coated vesicles can drive the specific mechanisms and protein interactions of internalization [32], giving way for exosome surface proteins to influence uptake. Two proteins used extensively to describe the details of CME are transferrin (Tf) and low density lipoprotein (LDL) and their respective receptors [25], which are all (except LDL) found on the surface of exosomes [33, 34]. Overexpression of transferrin receptors on cancer cells [35] may also contribute to increased exosomal uptake and clathrinmediated endocytosis in tumors, as there have been shown to be 50–80 percent more receptors on the cancer cell compared to the non-cancer cell [36].

#### **2.4 Caveolin-dependent endocytosis**

Caveolin is similar to clathrin, as it forms a coat around membrane invaginations called caveolae and facilitates the entry of extracellular material (**Figure 1D**). These are particularly prevalent on endothelial cells but have been found on a wide distribution of cell types [25]. Caveolae are about half the size of clathrin-coated vesicles, limiting their cargo to smaller structures [25] but still covering some of the exosome size range. This type of endocytosis as well as lipid raft-dependent uptake, plays a key role in lipid transport and homeostasis [25]. One of the defining factors of the exosome membrane is its slightly altered lipid profile, which has been shown to influence internalization [37]. Two proteins commonly active in caveolae-dependent endocytosis, which have also been identified on the surface of exosomes, are the insulin receptor and albumin [34, 38, 39]. The cellular insulin receptor itself has also recently been found to influence exosome uptake [18].

#### **2.5 Lipid raft dependent or clathrin-/caveolin-independent endocytosis**

Lipid dependence is not only characteristic of caveolae-dependent endocytosis, but also clathrin/caveolae-independent processes. Lipid raft-dependent (or clathrin/ caveolae-independent) endocytosis is similar to caveolae-dependent, except for the absence of the protein cav-1. Lipid rafts are 40-50 nm sections of the membrane with a high percentage of glycosphingolipids and cholesterol, and are anchoring points for many membrane proteins [40]. Lipid rafts are involved in exosome biogenesis and trafficking [41–43] and exosome uptake has been reduced by blocking lipid raft endocytosis [44] (**Figure 1E**).

#### **2.6 Receptor mediated endocytosis**

As mentioned previously, RME is an endocytosis pathway that can fit under several of the other categories (**Figure 1F**). The term and pathway were originally

**45**

*Cellular-Defined Microenvironmental Internalization of Exosomes*

Macrophage Mouse bone

Epithelial Ovarian cancer (SKOV3)

Epithelial Epidermoid

Epithelial Ovarian cancer (SKOV3)

Epithelial Breast cancer

Endothelial Cerebral vascular

(MCF7)

(hCMEC D3)

(PC12)

Epithelial Gastric cancer (AGS, MKN1)

(MCF7)

endothelial (hCMEC

Epithelial Ovarian cancer (SKOV3)

Epithelial Breast cancer

Endothelial Cerebral vascular

D3)

Endothelial Brain microvascular endothelial

Pheochromocytoma

Microglia Primary mouse Mouse oligodendrocyte

(HeLa)

carcinoma (A431), Pancreatic carcinoma (MIA PaCa-2)

Macropinocytosis Epithelial Cervical cancer

Neuron precursor cell

Clathrin-mediated endocytosis

Phagocytosis Macrophage RAW264.7 Leukemia cell (K562

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

[20]

[51]

[117]

[97]

[90]

[19]

[97]

[96]

[89]

[56]

[114]

[97]

[94]

[96]

[89]

[87]

or MT4)

(B16BL6)

(PC12)

Ovarian cancer cell (SKOV3)

Epidermoid carcinoma

Cervical cancer cell

Ovarian cancer cell (SKOV3)

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

Macrophage (RAW264.7)

(Oli-neu)

(PC12)

Epithelial Alveolar cells (A549) Dendritic cell [66]

Pheochromocytoma

Ovarian cancer cell (SKOV3)

Gastric cancer cell (AGS, MKN1)

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

Macrophage (RAW264.7)

(Hek293T)

Embryonic kidney cell

Microglia BV-2 Neuron (N2a) [49] Dendritic cell Mouse primary Mouse dendritic cell [15]

Epithelial Alveolar cells (A549) Dendritic cell [66]

(A431)

(HeLa)

Mouse CRC (CT-26) [54]

Macrophage J774 Rat reticulocyte [52] Macrophage Primary Trophoblast (Sw71) [58] Monocytes Primary Activated T cell [50]

Macrophage Peritoneal Mouse melanoma cell

Microglia MG6 Pheochromocytoma

marrow-derived

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

**Recipient cell type**

**Endocytosis pathway**


*Extracellular Vesicles and Their Importance in Human Health*

**2.3 Clathrin-dependent endocytosis**

**2.4 Caveolin-dependent endocytosis**

lipid raft endocytosis [44] (**Figure 1E**).

**2.6 Receptor mediated endocytosis**

tion 27-fold through the activation of macropinocytosis [19].

receptor, either by soluble EGF or exosome-bound, increased exosome internaliza-

The next three mechanisms, clathrin-dependent, caveolae-dependent, and clathrin/caveolae-independent, are facilitated by specific membrane proteins/ structures: clathrin, caveolae, and lipid rafts. Clathrin is an intracellular protein that forms a coat around an invaginating vesicle facilitating formation and internalization [31] (**Figure 1C**). These vesicles internalize material around 120 nm [25], which is within the exosome size range. Stimulation can occur through receptor/ ligand mediation or can be constitutive, depending on cell-type and receptor presence, but clathrin-mediated endocytosis (CME) occurs in all cell types [31]. Data continues to show that the extracellular cargo of these clathrin-coated vesicles can drive the specific mechanisms and protein interactions of internalization [32], giving way for exosome surface proteins to influence uptake. Two proteins used extensively to describe the details of CME are transferrin (Tf) and low density lipoprotein (LDL) and their respective receptors [25], which are all (except LDL) found on the surface of exosomes [33, 34]. Overexpression of transferrin receptors on cancer cells [35] may also contribute to increased exosomal uptake and clathrinmediated endocytosis in tumors, as there have been shown to be 50–80 percent

more receptors on the cancer cell compared to the non-cancer cell [36].

called caveolae and facilitates the entry of extracellular material

Caveolin is similar to clathrin, as it forms a coat around membrane invaginations

(**Figure 1D**). These are particularly prevalent on endothelial cells but have been found on a wide distribution of cell types [25]. Caveolae are about half the size of clathrin-coated vesicles, limiting their cargo to smaller structures [25] but still covering some of the exosome size range. This type of endocytosis as well as lipid raft-dependent uptake, plays a key role in lipid transport and homeostasis [25]. One of the defining factors of the exosome membrane is its slightly altered lipid profile, which has been shown to influence internalization [37]. Two proteins commonly active in caveolae-dependent endocytosis, which have also been identified on the surface of exosomes, are the insulin receptor and albumin [34, 38, 39]. The cellular insulin receptor itself has also recently been found to influence exosome uptake [18].

**2.5 Lipid raft dependent or clathrin-/caveolin-independent endocytosis**

Lipid dependence is not only characteristic of caveolae-dependent endocytosis, but also clathrin/caveolae-independent processes. Lipid raft-dependent (or clathrin/ caveolae-independent) endocytosis is similar to caveolae-dependent, except for the absence of the protein cav-1. Lipid rafts are 40-50 nm sections of the membrane with a high percentage of glycosphingolipids and cholesterol, and are anchoring points for many membrane proteins [40]. Lipid rafts are involved in exosome biogenesis and trafficking [41–43] and exosome uptake has been reduced by blocking

As mentioned previously, RME is an endocytosis pathway that can fit under several of the other categories (**Figure 1F**). The term and pathway were originally

**44**


#### **Table 1.**

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

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

**47**

*Cellular-Defined Microenvironmental Internalization of Exosomes*

**3. Cell type-specific internalization of exosomes**

[20, 48–51], lectins [17, 52, 53] and Fc receptors [54].

lipid raft-dependent endocytosis.

identified in the exosome internalization process (**Table 1**) but additional research is needed to determine the driving factors behind the specific mechanisms. One hypothesized factor is that the recipient cell type may determine the specific type of

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

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

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

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

internalization.

**3.1 Phagocytes**

identified in the exosome internalization process (**Table 1**) but additional research is needed to determine the driving factors behind the specific mechanisms. One hypothesized factor is that the recipient cell type may determine the specific type of internalization.
