**2. Exosome biogenesis, secretion and physiologic considerations**

Since their description in the process of reticulocyte maturation almost a quarter century ago (Johnstone *et al.*, 1987), exosomes; the intralumenal vesicles (ILV's) of multivesicular bodies (MVB's), have gained significant notoriety as evidenced by the nearly 10-15 fold rise in the

The Application of Membrane Vesicles for Cancer Therapy 23

Fig. 1. Tumor derived exosomes (TEX). A. Electron microscopy revealed 50-100 nm sized microvesicles (Khan et al., 2011). B. Diagram depicting a typical TEX (modified from JP

The presence of specific proteins within and on the vesicle suggest the existence of a protein sorting mechanism during its formation. Some of these proteins are shared by exosomes derived from different sources and used to identify these vesicles during proteomic analysis. In our laboratory, we have consistently used the lysosomal associated membrane protein LAMP1 (common to exosomes from a wide variety of cells) as a positive control for exosome

Fig. 2. Protein loading and release by exosomes. Endocytosed surface proteins as well as a subset of cytosolic proteins are taken by the endosomal system. Those destined for release as

exosomes or for lysosomal degradation are sorted into lumenal vesicles called

Mitchel, exosomes in cancer immunology).

multivesicular bodies (MVB).

presence in western blot analysis (Khan *et al.*, 2011).

amount of publications devoted to the subject just within the past decade (Raimondo *et al.*, 2011).

Many types of vesicles have been described in the literature having quite heterogeneous size, protein content, RNA content and origin. As a result there have been many names given to these different vesicle types (**Table 1**) (Ronquist & Brody, 1985; Ronquist & Frithz, 1986; Rooney *et al.*, 1993; Arienti *et al.*, 1997; Thery *et al.*, 2006; Lehmann *et al.*, 2008; Schiller *et al.*, 2008; Simpson *et al.*, 2008; Skog *et al.*, 2008; Dashevsky *et al.*, 2009; Di Vizio *et al.*, 2009; Haubold *et al.*, 2009; Jansen *et al.*, 2009; Nilsson *et al.*, 2009; Duijvesz *et al.*, 2010). Unfortunately, the different names given to these vesicles lead to much confusion and it is still unclear if all of the different vesicles are unique in biological function or if they represent a sliding scale of one entity (Duijvesz *et al.*, 2010). Exosomes are nanometer sized lipid bound vesicles derived from late endosomes contained in MVB's. They are characterized by their size 40-100nm, density ranging from 1.13g/ml to 1.19g/ml on a sucrose gradient (Record *et al.*, 2011) and their specific protein content (heat shock proteins, tetraspanins, Rab proteins, etc) **Figure 1**. Exosomes are secreted by both hematopoietic (eg dendritic cells, lymphocytes, mast cells) and non-hematopoietic cells such as fibroblasts, intestinal epithelium, neurons and various tumor cells. Exosomes have been isolated from serum and other biological fluids such as urine, ascitic fluid, amniotic fluid and even cerebrospinal fluid (Vella *et al.*, 2008). Exosome biogenesis involves an inward budding of the limiting membrane of late endosomes, also known as multivesicular bodies (MVBs) **(Figure 2)**, concurrent protein sorting into these budding exosomes, and subsequent splitting (scission) of these microvesicles (Keller et al., 2006). As a consequence, the nascent exosome contains the inner leaflet of the limiting membrane, membrane associated proteins, both native and recruited, and some amount of cytosol and cytosolic proteins.


Table 1. Characteristics of different types of microvessicles.

amount of publications devoted to the subject just within the past decade (Raimondo *et al.*,

Many types of vesicles have been described in the literature having quite heterogeneous size, protein content, RNA content and origin. As a result there have been many names given to these different vesicle types (**Table 1**) (Ronquist & Brody, 1985; Ronquist & Frithz, 1986; Rooney *et al.*, 1993; Arienti *et al.*, 1997; Thery *et al.*, 2006; Lehmann *et al.*, 2008; Schiller *et al.*, 2008; Simpson *et al.*, 2008; Skog *et al.*, 2008; Dashevsky *et al.*, 2009; Di Vizio *et al.*, 2009; Haubold *et al.*, 2009; Jansen *et al.*, 2009; Nilsson *et al.*, 2009; Duijvesz *et al.*, 2010). Unfortunately, the different names given to these vesicles lead to much confusion and it is still unclear if all of the different vesicles are unique in biological function or if they represent a sliding scale of one entity (Duijvesz *et al.*, 2010). Exosomes are nanometer sized lipid bound vesicles derived from late endosomes contained in MVB's. They are characterized by their size 40-100nm, density ranging from 1.13g/ml to 1.19g/ml on a sucrose gradient (Record *et al.*, 2011) and their specific protein content (heat shock proteins, tetraspanins, Rab proteins, etc) **Figure 1**. Exosomes are secreted by both hematopoietic (eg dendritic cells, lymphocytes, mast cells) and non-hematopoietic cells such as fibroblasts, intestinal epithelium, neurons and various tumor cells. Exosomes have been isolated from serum and other biological fluids such as urine, ascitic fluid, amniotic fluid and even cerebrospinal fluid (Vella *et al.*, 2008). Exosome biogenesis involves an inward budding of the limiting membrane of late endosomes, also known as multivesicular bodies (MVBs) **(Figure 2)**, concurrent protein sorting into these budding exosomes, and subsequent splitting (scission) of these microvesicles (Keller et al., 2006). As a consequence, the nascent exosome contains the inner leaflet of the limiting membrane, membrane associated proteins,

both native and recruited, and some amount of cytosol and cytosolic proteins.

Table 1. Characteristics of different types of microvessicles.

2011).

Fig. 1. Tumor derived exosomes (TEX). A. Electron microscopy revealed 50-100 nm sized microvesicles (Khan et al., 2011). B. Diagram depicting a typical TEX (modified from JP Mitchel, exosomes in cancer immunology).

The presence of specific proteins within and on the vesicle suggest the existence of a protein sorting mechanism during its formation. Some of these proteins are shared by exosomes derived from different sources and used to identify these vesicles during proteomic analysis. In our laboratory, we have consistently used the lysosomal associated membrane protein LAMP1 (common to exosomes from a wide variety of cells) as a positive control for exosome presence in western blot analysis (Khan *et al.*, 2011).

Fig. 2. Protein loading and release by exosomes. Endocytosed surface proteins as well as a subset of cytosolic proteins are taken by the endosomal system. Those destined for release as exosomes or for lysosomal degradation are sorted into lumenal vesicles called multivesicular bodies (MVB).

The Application of Membrane Vesicles for Cancer Therapy 25

complex subsequently dissociates from the cargo when the ATPase Vps 4 binds, thus

Other exosomal proteins, for which no ESCRT mechanisms have been described, may be incorporated into the exosome via varying levels of co-sorting. For example, tetraspanins may sort into exosomes due to their affinity for exosomal membrane components like sphingolipids and ceramides and may follow these phospholipids to further aggregate into lipid raft domains (de Gassart *et al.*, 2003). Protein-protein associations also contribute to co-sorting. MHC Class II molecules can associate with tetraspanins and be co-sorted into exosomal membranes (Blanc & Vidal, 2010). Some evidence also suggests lipid co-sorting into exosomes. The exosomal lipid LBPA (Lysobisphosphatidic Acid) can sort into exosomal membranes by interaction with the exosomal protein Alix. When present in media, LBPA was also shown to

Hematopoietic cells including dendritic cells, lymphocytes and mast cells, and nonhematopoietic cells such as fibroblasts, intestinal epithelial cells, neurons and various tumor cells secrete exosomes. Exosome release from cells may follow a constitutive versus an inducible mode of secretion. The constitutive pathway utilizes the trans-golgi network, after which the vesicles travel through the cytoplasm via an intricate tubular network and eventually are released into the extracellular space (Ponnambalam & Baldwin, 2003). The inducible mode involves the release of preformed vesicles contained in MVBs. Such release may be dependent on known cellular triggers of vesicle release, such as increased intracellular calcium levels (Savina *et al.*, 2003). Other triggers, such as cellular depolarization, have also been described. These events culminate in the fusion of MVB membrane with the cell

Exosomes interact with target cells via specific receptors present on these target cells (Losche *et al.*, 2004). The exosome thus exerts its effect either via receptor-receptor interaction or internalization of the exosome with subsequent interaction of the exosome

As will be described in other sections of this chapter, exosomes influence immune cells both in normal and abnormal states such as cancer. Furthermore RNA contained in exosomes could be translated within the recipient cell and as a result exert an epigenetic influence (Baj-Krzyworzeka *et al.*, 2006). In this regard, exosomes mimic viral particles by directing host cellular processes to its advantage. The role of exosomes in inflammatory conditions has recently been questioned after platelets and macrophage derived microparticles/exosomes were found in the lipid core of artherosclerotic plaques (Leroyer *et al.*, 2008). It is of interest to better understand the precise pro-inflammatory and thrombotic roles of exosomes and potentially use them as therapeutic targets in these conditions. Finally, the discovery of exosomes in urine has opened up the possibility of exosomal proteins being used as biomarkers for disease. It was recently shown that decreased levels of exosomal aquaporin-1 correlates with renal ischemia-reperfusion injury (Sonoda *et al.*, 2009). What makes this prospect exciting is the relative abundance of urine that can be obtained without an invasive

Exosomes are small membrane bound vesicles of endocytic origin that are released by most cells. The purpose for release seemed at first to be to discard membrane proteins and those

releasing the cargo protein into the budding exosome.

assist with the formation of vesicles in the MVBs (Matsuo *et al.*, 2004).

membrane and subsequent exosome release into the extracellular space.

content with the recipient cellular machinery (Record *et al.*, 2011).

procedure.

**2.3 Summary** 

**2.2 Other modes of protein sorting** 

#### **2.1 ESCRT-dependent protein sorting**

The specific protein content of exosomes can be exploited to identify these exosomes and the cell-types from which they are derived via proteomic analysis. The fact that the exosome's protein content can be used as an identifier suggests the existence of a protein sorting mechanism during its formation. Endosomal Sorting Complex Required for Transport (ESCRT), comprised of a series of three protein complexes: ESCRT I, ESCRT II, and ESCRT III **(Figure 3)**, is suspected to play a critical role in sorting proteins into exosomes at the endosomal limiting membrane.

Fig. 3. Ubiquitin marks cargo proteins for endosomal sorting.

Monoubiquitination appears to mark cargo proteins for protein sorting within the MVBs. In yeast, recruitment of ubiquitinated cargo proteins depends on a class of proteins known as class E Vps (Vacuolar Protein Sorting) proteins that have been highly conserved from yeast to mammals—at least one mammalian homolog has been identified for each yeast class member (Babst, 2005). In the yeast model, Vps 27, is thought to complex on the endosomal membrane's outer leaflet with clathrin and ubiquitin-binding proteins, such as Golgiassociated, γ-adaptin homologs, Arf-binding (GGA) proteins, to form an endosomal clathrin coat that recruits monoubiquitinated cargo to the inner leaflet of the endosomal membrane. This complex recruits the ESCRT I complex from the cytoplasm and transfers to it the ubiquitinated cargo. ESCRT I then activates ESCRT II, which in turn occasions the oligomerization of Vps 2, Vps 20, Vps 24, and Snf 7 to form the ESCRT III complex and transfers to ESCRT III the ubiquitinated cargo. ESCRT III continues to accumulate and concentrates the cargo into still-budding exosomes of the MVB (Babst, 2005). The deubiquitinating protein Doa4 is then recruited into the complex where it functions to remove the ubiquitin from the cargo protein (Luhtala & Odorizzi, 2004). The ESCRT III complex subsequently dissociates from the cargo when the ATPase Vps 4 binds, thus releasing the cargo protein into the budding exosome.

#### **2.2 Other modes of protein sorting**

24 Advances in Cancer Therapy

The specific protein content of exosomes can be exploited to identify these exosomes and the cell-types from which they are derived via proteomic analysis. The fact that the exosome's protein content can be used as an identifier suggests the existence of a protein sorting mechanism during its formation. Endosomal Sorting Complex Required for Transport (ESCRT), comprised of a series of three protein complexes: ESCRT I, ESCRT II, and ESCRT III **(Figure 3)**, is suspected to play a critical role in sorting proteins into exosomes at the

**2.1 ESCRT-dependent protein sorting** 

Fig. 3. Ubiquitin marks cargo proteins for endosomal sorting.

Monoubiquitination appears to mark cargo proteins for protein sorting within the MVBs. In yeast, recruitment of ubiquitinated cargo proteins depends on a class of proteins known as class E Vps (Vacuolar Protein Sorting) proteins that have been highly conserved from yeast to mammals—at least one mammalian homolog has been identified for each yeast class member (Babst, 2005). In the yeast model, Vps 27, is thought to complex on the endosomal membrane's outer leaflet with clathrin and ubiquitin-binding proteins, such as Golgiassociated, γ-adaptin homologs, Arf-binding (GGA) proteins, to form an endosomal clathrin coat that recruits monoubiquitinated cargo to the inner leaflet of the endosomal membrane. This complex recruits the ESCRT I complex from the cytoplasm and transfers to it the ubiquitinated cargo. ESCRT I then activates ESCRT II, which in turn occasions the oligomerization of Vps 2, Vps 20, Vps 24, and Snf 7 to form the ESCRT III complex and transfers to ESCRT III the ubiquitinated cargo. ESCRT III continues to accumulate and concentrates the cargo into still-budding exosomes of the MVB (Babst, 2005). The deubiquitinating protein Doa4 is then recruited into the complex where it functions to remove the ubiquitin from the cargo protein (Luhtala & Odorizzi, 2004). The ESCRT III

endosomal limiting membrane.

Other exosomal proteins, for which no ESCRT mechanisms have been described, may be incorporated into the exosome via varying levels of co-sorting. For example, tetraspanins may sort into exosomes due to their affinity for exosomal membrane components like sphingolipids and ceramides and may follow these phospholipids to further aggregate into lipid raft domains (de Gassart *et al.*, 2003). Protein-protein associations also contribute to co-sorting. MHC Class II molecules can associate with tetraspanins and be co-sorted into exosomal membranes (Blanc & Vidal, 2010). Some evidence also suggests lipid co-sorting into exosomes. The exosomal lipid LBPA (Lysobisphosphatidic Acid) can sort into exosomal membranes by interaction with the exosomal protein Alix. When present in media, LBPA was also shown to assist with the formation of vesicles in the MVBs (Matsuo *et al.*, 2004).

Hematopoietic cells including dendritic cells, lymphocytes and mast cells, and nonhematopoietic cells such as fibroblasts, intestinal epithelial cells, neurons and various tumor cells secrete exosomes. Exosome release from cells may follow a constitutive versus an inducible mode of secretion. The constitutive pathway utilizes the trans-golgi network, after which the vesicles travel through the cytoplasm via an intricate tubular network and eventually are released into the extracellular space (Ponnambalam & Baldwin, 2003). The inducible mode involves the release of preformed vesicles contained in MVBs. Such release may be dependent on known cellular triggers of vesicle release, such as increased intracellular calcium levels (Savina *et al.*, 2003). Other triggers, such as cellular depolarization, have also been described. These events culminate in the fusion of MVB membrane with the cell membrane and subsequent exosome release into the extracellular space.

Exosomes interact with target cells via specific receptors present on these target cells (Losche *et al.*, 2004). The exosome thus exerts its effect either via receptor-receptor interaction or internalization of the exosome with subsequent interaction of the exosome content with the recipient cellular machinery (Record *et al.*, 2011).

As will be described in other sections of this chapter, exosomes influence immune cells both in normal and abnormal states such as cancer. Furthermore RNA contained in exosomes could be translated within the recipient cell and as a result exert an epigenetic influence (Baj-Krzyworzeka *et al.*, 2006). In this regard, exosomes mimic viral particles by directing host cellular processes to its advantage. The role of exosomes in inflammatory conditions has recently been questioned after platelets and macrophage derived microparticles/exosomes were found in the lipid core of artherosclerotic plaques (Leroyer *et al.*, 2008). It is of interest to better understand the precise pro-inflammatory and thrombotic roles of exosomes and potentially use them as therapeutic targets in these conditions. Finally, the discovery of exosomes in urine has opened up the possibility of exosomal proteins being used as biomarkers for disease. It was recently shown that decreased levels of exosomal aquaporin-1 correlates with renal ischemia-reperfusion injury (Sonoda *et al.*, 2009). What makes this prospect exciting is the relative abundance of urine that can be obtained without an invasive procedure.

#### **2.3 Summary**

Exosomes are small membrane bound vesicles of endocytic origin that are released by most cells. The purpose for release seemed at first to be to discard membrane proteins and those

The Application of Membrane Vesicles for Cancer Therapy 27

ERBB2\*/HER2\* (Andre *et al.*, 2002b;

Koga *et al.*, 2005)

(Andreola *et al.*, 2002; Abusamra *et al.*, 2005b; Huber *et al.*, 2005; Taylor & Gercel-Taylor, 2005; Choi *et al.*, 2007; Simpson *et al.*, 2009)

(Andre *et al.*, 2002b; Mears *et al.*, 2004; Abusamra *et al.*, 2005b)

Andreola *et al.*, 2002; Koga *et al.*, 2005)

(Huber *et al.*, 2005; Kim

(Taylor *et al.*, 2006; Martinez-Lostao *et al.*,

*et al.*, 2005)

2010)

**Cancer Type Protein References** 

**Cervical Cancer** (heLa) Survivin, HSP 70, HSP 90 (Khan *et al.*, 2011)

TNFSF10\*

GTPases)

TNFSF10\*

TNFSF10

**Prostate** FASLG\*

**Squamous cell cancer** FASLG

superfamily member 6 (FasL).

GPA 33\*, CEACAM5\*, EFNB1\* annexins, ARFs\*, Rabs\*, ADAM10\*, CD44\*, NG2 ephrin-B1, MIF\*, bcatenin, junction plakoglobin galectin-4, RACK1\*, and tetraspanin-8, FASL\* AND TRAIL\* FASLG\*

**Glioma** Cancer EGFR\* (Al-Nedawi *et al.*, 2009)

A33\*, CEA, EGFR\*, ADAM10\*, dipeptidase 1 ephrin-B1, hsc70, tetraspanins, ESCRT proteins (integrins, annexins, Rabs, and

**Mesothelioma** PLVAP\* (Hegmans *et al.*, 2004) **Ovarian** ERBB2\* (Andre *et al.*, 2002b;

Table 2. Protein content on and within exosomes reflects their origin and establishes their functional role. Cell surface A33 antigen precursor (GPA 33); Carcinoembryonic antigenrelated cell Adhesion molecule 5 (CEACAM5); Ephrin-B1 (EFNB1); ADP-ribolysing factor (ARF), Ras-Gprotein superfamily (Rabs), A disintegrin and metaloprotease (ADAM10), Macrophage Migration inhibitory factor (MIF), Receptor for activated C-kinase (RACK1), 5,6-Dihydroxyyindole-2 carboxylic acid oxidase (TRP); Epidermal-growth factor receptor (EGFR); Receptor tyrosine-protein kinase erbB-2 (ERBB2); Plasmalemma vesicle associated protein (PLVAP); Melanocyte Protein Pmel 17 (GP100); Melanoma Antigen Recognized by T cells 1 (MART-1), Mel-CAM, TNF-ligand superfamily member 10 (TRAIL) TNF-ligand

TRP\*GP100\*MART-1\* Mel-CAM\*FASLG\*TNFSF10\*

**Breast adenocarcinoma**

MDA-MB-231, TS/A, H-2,

**Colorectal Cancer** (LIM1215, HT29, SW403, 1869COL, AND CRC28462,

(BT-474

P815)

LIM1215)

**Melanoma** (Fon and Mel-888)

believed to be resistant to lysosomal degradation. Now, it is believed that they mediate intercellular communication without the need for direct cell-to-cell interaction.

#### **3. Existence of secreted membrane vesicles in cancers**

Tumors are known to shed membrane vesicles (Taylor & Gercel-Taylor, 2005). In particular, human and mouse tumor cells have been shown to secrete tumor cell-derived exosomes (TEX), constitutively into the extracellular space (Wolfers *et al.*, 2001). The morphology, density and certain membrane markers expressed, such as LAMP1, MHC class I, HSP70 and HSP80, on the released TEX are similar to the dendritic cell-derived exosomes (DEX) (Andre *et al.*, 2002b) which will be discussed later. Despite similarities to DEX, there are differences in the molecular profiles and biological roles of TEXs, both of which give an indication of the cell of origin (Wieckowski & Whiteside, 2006). The specific protein content found on and within exosomes not only reflects their origin, but in addition, establishes their functional role (Zitvogel *et al.*, 1998) **(Table 2)**. TEX secreted from neoplastic cells express diverse tumor antigens, which signifies the type of tumor cells from where TEXs were released (Iero *et al.*, 2008). *In-vitro,* it has been shown that TEX released from breast carcinoma cells contain HER2, while carcinoembryonic antigen (CEA) was found in the exosomes secreted from colon carcinoma cells, and proteins MelanA/Mart-1 and gp100 that are expressed in melanoma cells are found on the released TEX (Andre *et al.*, 2002b; Andreola *et al.*, 2002). This phenomenon is also evident *in-vivo*, where plasma from cancer patients contain membrane vesicles that are characterized by the expression of tumor antigens which reflect the tumor of origin (Hegmans *et al.*, 2004; Mears *et al.*, 2004).

When immunocompetent and nude mice were pre-treated with murine mammary TEX, an accelerated growth of the tumor was observed (Liu *et al.*, 2006). This observation led to various studies to try to elucidate the role of secreted membrane vesicles in cancer. TEX can be described as "multi-purpose carriers" which have important roles in the communication, protection, as well as the exchange of genetic information with neighboring cells (Nieuwland & Sturk, 2010). The production and secretion of TEX is important for the tumor. They serve a protective function, have a supportive role in the survival and growth of the tumor cells, are involved in the promotion of host tissue invasion and subsequent metastasis, and facilitate evasion from the immune response (Valenti *et al.*, 2007; Anderson *et al.*, 2010). Acting in a paracrine fashion, the diverse function of TEX is speculated to be due to the various bioactive molecules found within and on the vesicles having a strong influence on the surrounding environment (Hegmans *et al.*, 2004; Mears *et al.*, 2004; van Niel *et al.*, 2006; Iero *et al.*, 2008).

The promotion of angiogenesis is due in part to the upregulation of vascular endothelial growth factor (VEGF) (Skog *et al.*, 2008) and release of matrix metalloproteinases (MMPs) in neighboring, even distant endothelial cells, which are brought by TEX containing tetraspanin family members (Gesierich *et al.*, 2006), epithelial growth factor receptor (EGFR) (Al-Nedawi *et al.*, 2009), platelet-derived tissue factor (TF) (Osterud, 2003) or developmental endothelial locus-1 protein (Hegmans *et al.*, 2004). TEX has also been implicated in the further growth of tumor by the exchange of genetic material. mRNA was detected within exosomes released from glioblastoma cells. Neighboring microvascular endothelial cells that take up the exosomes and translate the mRNA become liable for further tumor growth leading to the stimulation of angiogenesis (Skog *et al.*, 2008). In addition, tissue invasion and stromal remodeling can be facilitated by proteases and MMP transport and release via exosomes (Ginestra *et al.*, 1998; Graves *et al.*, 2004).

believed to be resistant to lysosomal degradation. Now, it is believed that they mediate

Tumors are known to shed membrane vesicles (Taylor & Gercel-Taylor, 2005). In particular, human and mouse tumor cells have been shown to secrete tumor cell-derived exosomes (TEX), constitutively into the extracellular space (Wolfers *et al.*, 2001). The morphology, density and certain membrane markers expressed, such as LAMP1, MHC class I, HSP70 and HSP80, on the released TEX are similar to the dendritic cell-derived exosomes (DEX) (Andre *et al.*, 2002b) which will be discussed later. Despite similarities to DEX, there are differences in the molecular profiles and biological roles of TEXs, both of which give an indication of the cell of origin (Wieckowski & Whiteside, 2006). The specific protein content found on and within exosomes not only reflects their origin, but in addition, establishes their functional role (Zitvogel *et al.*, 1998) **(Table 2)**. TEX secreted from neoplastic cells express diverse tumor antigens, which signifies the type of tumor cells from where TEXs were released (Iero *et al.*, 2008). *In-vitro,* it has been shown that TEX released from breast carcinoma cells contain HER2, while carcinoembryonic antigen (CEA) was found in the exosomes secreted from colon carcinoma cells, and proteins MelanA/Mart-1 and gp100 that are expressed in melanoma cells are found on the released TEX (Andre *et al.*, 2002b; Andreola *et al.*, 2002). This phenomenon is also evident *in-vivo*, where plasma from cancer patients contain membrane vesicles that are characterized by the expression of tumor antigens which reflect

When immunocompetent and nude mice were pre-treated with murine mammary TEX, an accelerated growth of the tumor was observed (Liu *et al.*, 2006). This observation led to various studies to try to elucidate the role of secreted membrane vesicles in cancer. TEX can be described as "multi-purpose carriers" which have important roles in the communication, protection, as well as the exchange of genetic information with neighboring cells (Nieuwland & Sturk, 2010). The production and secretion of TEX is important for the tumor. They serve a protective function, have a supportive role in the survival and growth of the tumor cells, are involved in the promotion of host tissue invasion and subsequent metastasis, and facilitate evasion from the immune response (Valenti *et al.*, 2007; Anderson *et al.*, 2010). Acting in a paracrine fashion, the diverse function of TEX is speculated to be due to the various bioactive molecules found within and on the vesicles having a strong influence on the surrounding environment (Hegmans *et al.*, 2004; Mears *et al.*, 2004; van Niel *et al.*, 2006; Iero *et al.*, 2008). The promotion of angiogenesis is due in part to the upregulation of vascular endothelial growth factor (VEGF) (Skog *et al.*, 2008) and release of matrix metalloproteinases (MMPs) in neighboring, even distant endothelial cells, which are brought by TEX containing tetraspanin family members (Gesierich *et al.*, 2006), epithelial growth factor receptor (EGFR) (Al-Nedawi *et al.*, 2009), platelet-derived tissue factor (TF) (Osterud, 2003) or developmental endothelial locus-1 protein (Hegmans *et al.*, 2004). TEX has also been implicated in the further growth of tumor by the exchange of genetic material. mRNA was detected within exosomes released from glioblastoma cells. Neighboring microvascular endothelial cells that take up the exosomes and translate the mRNA become liable for further tumor growth leading to the stimulation of angiogenesis (Skog *et al.*, 2008). In addition, tissue invasion and stromal remodeling can be facilitated by proteases and MMP transport and release via

intercellular communication without the need for direct cell-to-cell interaction.

**3. Existence of secreted membrane vesicles in cancers** 

the tumor of origin (Hegmans *et al.*, 2004; Mears *et al.*, 2004).

exosomes (Ginestra *et al.*, 1998; Graves *et al.*, 2004).


Table 2. Protein content on and within exosomes reflects their origin and establishes their functional role. Cell surface A33 antigen precursor (GPA 33); Carcinoembryonic antigenrelated cell Adhesion molecule 5 (CEACAM5); Ephrin-B1 (EFNB1); ADP-ribolysing factor (ARF), Ras-Gprotein superfamily (Rabs), A disintegrin and metaloprotease (ADAM10), Macrophage Migration inhibitory factor (MIF), Receptor for activated C-kinase (RACK1), 5,6-Dihydroxyyindole-2 carboxylic acid oxidase (TRP); Epidermal-growth factor receptor (EGFR); Receptor tyrosine-protein kinase erbB-2 (ERBB2); Plasmalemma vesicle associated protein (PLVAP); Melanocyte Protein Pmel 17 (GP100); Melanoma Antigen Recognized by T cells 1 (MART-1), Mel-CAM, TNF-ligand superfamily member 10 (TRAIL) TNF-ligand superfamily member 6 (FasL).

The Application of Membrane Vesicles for Cancer Therapy 29

express specific tumor antigens which reflect the protein content of the tumor, giving an indication of the tumor type. The content of these vesicles can also be useful as markers for

Fig. 4. Activation of p53 by DNA damage induces the expression of TSAP6 which enhances

Mass spectrometry-based proteomic tools coupled with advanced purification methods for exosomes, has allowed more in-depth proteome analyses, contributing immensely to our understanding of the molecular composition of exosomes. Proteomic analysis of exosomes from diverse cell types, including cancers has revealed a common set of membrane and cytosolic proteins, suggesting the evolutionary importance of these membrane particles. In addition, exosomes express an array of proteins that reflect the originating host cell. The excessive release of exosomes in tumor cells, as evidenced by their increased levels in body fluids during the late stage of a disease and their overexpression of certain tumor cell biomarkers, suggests an important role of exosomes in diagnosis and biomarker studies

By proteomic analysis we can enrich low abundance membrane proteins from underrepresented conventional cell lysates and unfractionated biological fluids. Identification of a conserved set of common proteins that are essential for vesicle biogenesis, structure and trafficking mechanisms can be explored. We can also detect cell-specific biomarkers. These concepts suggest that analyzing the composition and abundance of such

proteins in exosomes may be useful to reveal different cell behaviors.

the aggressiveness of the disease.

exosome-mediated secretion.

(Simpson *et al.*, 2009).

**4. Proteome of cancer membrane vesicles** 

Recent studies have shown that TEX provide a protective role to the cancer cells, which can be manifested in different ways. Survivin, a member of the inhibitor of apoptosis (IAP) protein family, was found to be released from tumor cells via exosomes (Khan *et al.*, 2011). The protective role of TEX can be attained by the accumulation and packaging of chemotherapeutic drugs or its metabolites into the vesicles, thus decreasing cellular levels of the drug, a factor leading to drug resistance (Shedden *et al.*, 2003; Safaei *et al.*, 2005). This phenomenon has been observed in various cancer cells. Cisplatin enhanced the shedding of the vesicle from melanoma cells (Chen *et al.*, 2006a), while doxorubicin was found in the exosomes released from ovarian carcinoma cells (Shedden *et al.*, 2003).

Despite the beneficial roles of TEX for the tumor cells and the tumor microenvironment, TEX can be a useful tool for detecting the malignant condition. Serum levels of exosomes taken from cancer patients are significantly increased. These vesicles taken from serum (Ginestra *et al.*, 1999), as well as from malignant tumor fluids, such as ascites fluids (Adams *et al.*, 2005), pleural effusions (Andre *et al.*, 2002b) and urine (Nilsson *et al.*, 2009), positively correlate with the tumor progression.

#### **3.1 Constitutive and inducible vesicle secretion in cancer and cancer therapy**

In the tumor microenvironment, various changes are taking place, which could have an effect on the release of vesicles, such as exosomes. Environmental changes, such as stress induced by chemo- and radio-therapy, can modulate TEX release and the biome they contain. This phenomenon may induce the tissues to adapt to changes taking place in the microenvironment (Thery *et al.*, 2009). Tumor cells that have undergone radiation or chemotherapy treatment have been shown to increase the release of TEX (Yu *et al.*, 2006; Lehmann *et al.*, 2008). Interestingly, when treated with chemotherapeutic agents, there is a significantly enhanced membrane vesicle secretion in chemoresistant cells compared to chemosensitive cells. This activity may be a factor leading to drug resistance (Shedden *et al.*, 2003; Safaei *et al.*, 2005).

Chemotherapy and radiation (Chen *et al.*, 2006b) treatment lead to DNA-damaging conditions. In this state, the p53 pathway is activated, leading, among various other changes physiologically, to an induced expression of the transmembrane protein tumor suppressoractivated pathway 6 (TSAP6). TSAP6 is an important cellular component as it regulates the secretion of protein via the non-classical pathway or the ER/Golgi-independent protein secretion pathway needed for the enhanced release of exosomes (Nickel, 2003; Yu *et al.*, 2006; Lespagnol *et al.*, 2008). Normally, the secretion of exosomes in various cell types happens at a low rate. However, when p53 is activated, endosomal compartment activities are activated. Simultaneously, there is an increased expression of TSAP6, inducing the release of exosomes at a higher rate (Yu *et al.*, 2009) (**Figure 4**). It is suggested that following p53 activation, exosomal release may act as a 'detoxifier' to expel unwanted chemotherapeutic agents (Shedden *et al.*, 2003; Safaei *et al.*, 2005; Chen *et al.*, 2006b; Lespagnol *et al.*, 2008). Communication to the microenvironment is the other proposed role of TSAP6 and exosomal release after p53 activation, which may act as a warning signal to the neighboring cells, the immune system, and the extracellular matrix, that there are abnormal intracellular events happening (Lespagnol *et al.*, 2008; Yu *et al.*, 2009).

#### **3.2 Summary**

TEX can be used as an important biomarker for the disease, which will give information not only on the disease progression, but on the tumor type. As previously mentioned, TEX

Recent studies have shown that TEX provide a protective role to the cancer cells, which can be manifested in different ways. Survivin, a member of the inhibitor of apoptosis (IAP) protein family, was found to be released from tumor cells via exosomes (Khan *et al.*, 2011). The protective role of TEX can be attained by the accumulation and packaging of chemotherapeutic drugs or its metabolites into the vesicles, thus decreasing cellular levels of the drug, a factor leading to drug resistance (Shedden *et al.*, 2003; Safaei *et al.*, 2005). This phenomenon has been observed in various cancer cells. Cisplatin enhanced the shedding of the vesicle from melanoma cells (Chen *et al.*, 2006a), while doxorubicin was found in the

Despite the beneficial roles of TEX for the tumor cells and the tumor microenvironment, TEX can be a useful tool for detecting the malignant condition. Serum levels of exosomes taken from cancer patients are significantly increased. These vesicles taken from serum (Ginestra *et al.*, 1999), as well as from malignant tumor fluids, such as ascites fluids (Adams *et al.*, 2005), pleural effusions (Andre *et al.*, 2002b) and urine (Nilsson *et al.*, 2009), positively

In the tumor microenvironment, various changes are taking place, which could have an effect on the release of vesicles, such as exosomes. Environmental changes, such as stress induced by chemo- and radio-therapy, can modulate TEX release and the biome they contain. This phenomenon may induce the tissues to adapt to changes taking place in the microenvironment (Thery *et al.*, 2009). Tumor cells that have undergone radiation or chemotherapy treatment have been shown to increase the release of TEX (Yu *et al.*, 2006; Lehmann *et al.*, 2008). Interestingly, when treated with chemotherapeutic agents, there is a significantly enhanced membrane vesicle secretion in chemoresistant cells compared to chemosensitive cells. This activity may be a factor leading to drug resistance (Shedden *et al.*, 2003; Safaei *et al.*, 2005). Chemotherapy and radiation (Chen *et al.*, 2006b) treatment lead to DNA-damaging conditions. In this state, the p53 pathway is activated, leading, among various other changes physiologically, to an induced expression of the transmembrane protein tumor suppressoractivated pathway 6 (TSAP6). TSAP6 is an important cellular component as it regulates the secretion of protein via the non-classical pathway or the ER/Golgi-independent protein secretion pathway needed for the enhanced release of exosomes (Nickel, 2003; Yu *et al.*, 2006; Lespagnol *et al.*, 2008). Normally, the secretion of exosomes in various cell types happens at a low rate. However, when p53 is activated, endosomal compartment activities are activated. Simultaneously, there is an increased expression of TSAP6, inducing the release of exosomes at a higher rate (Yu *et al.*, 2009) (**Figure 4**). It is suggested that following p53 activation, exosomal release may act as a 'detoxifier' to expel unwanted chemotherapeutic agents (Shedden *et al.*, 2003; Safaei *et al.*, 2005; Chen *et al.*, 2006b; Lespagnol *et al.*, 2008). Communication to the microenvironment is the other proposed role of TSAP6 and exosomal release after p53 activation, which may act as a warning signal to the neighboring cells, the immune system, and the extracellular matrix, that there are abnormal intracellular events

TEX can be used as an important biomarker for the disease, which will give information not only on the disease progression, but on the tumor type. As previously mentioned, TEX

**3.1 Constitutive and inducible vesicle secretion in cancer and cancer therapy** 

exosomes released from ovarian carcinoma cells (Shedden *et al.*, 2003).

correlate with the tumor progression.

happening (Lespagnol *et al.*, 2008; Yu *et al.*, 2009).

**3.2 Summary** 

express specific tumor antigens which reflect the protein content of the tumor, giving an indication of the tumor type. The content of these vesicles can also be useful as markers for the aggressiveness of the disease.

Fig. 4. Activation of p53 by DNA damage induces the expression of TSAP6 which enhances exosome-mediated secretion.
