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## **Meet the editor**

Professor Jin Wang is a PI at Shanghai Public Health Clinical Center, Fudan University, China. Dr. Wang has been working in the fields of cancer and infectious diseases. A major focus of his research is to develop novel early detection biomarkers of human cancers utilizing various state-of-the-art genomic technologies, and he has been able to identify several interesting candidate

biomarkers of human pancreatic cancers and liver cancer. Dr. Wang has authored/coauthored 3 patents (*U.S. WO Patent WO/2012/030,956*; *P.R. China Patent NO: 99113481.8*) and over 30 SCI publications with a citation rate of over 1000. He has received the prestigious *Anne Eastland Spears Fellowship Award* in Gastrointestinal Cancer Research from the University of Texas, MD Anderson Cancer Center in the USA, and three independent fellowships from reputed institutions including NIH/NIDDK (USA). His laboratory conducts basic and translational research in the following areas: (1) development of novel early detection biomarkers of human gastrointestinal (GI) cancers and (2) pharmacologic *ascorbic acid*, *all-trans retinoid acid*, and *ncRNAs* for treatment of cancer and infectious diseases.

### Contents

#### **Preface XI**

Lucia Paolini, Giuseppe Di Noto, Andrea Zendrini, Paolo Bergese and Doris Ricotta

#### Chapter 7 **Microvesicles Released from Human Red Blood Cells: Properties and Potential Applications 137** Duc Bach Nguyen, Thi Bich Thuy Ly and Ingolf Bernhardt

#### Chapter 8 **Exosome Isolation: Is There an Optimal Method with Regard to Diagnosis or Treatment? 163** Mustafa Kotmakçı and Gülşah Erel Akbaba

### Preface

Chapter 7 **Microvesicles Released from Human Red Blood Cells: Properties**

Duc Bach Nguyen, Thi Bich Thuy Ly and Ingolf Bernhardt

Chapter 8 **Exosome Isolation: Is There an Optimal Method with Regard to**

**and Potential Applications 137**

**VI** Contents

**Diagnosis or Treatment? 163**

Mustafa Kotmakçı and Gülşah Erel Akbaba

Exosome research is a young and rapidly changing field. It is exciting to write about it, but it is also hard to know when to stop, since new results appear at frequent intervals. This book provides a forum for novel implications of exosomes in diagnosis and treatment of cancer and infectious diseases. It is a timely reference to explore the early detection biomarkers of cancer and infectious diseases and will help develop successful therapeutic intervention in these diseases in the future.

For clarity, the eight chapters of this book have been clustered into three parts. Part I clus‐ ters Chapters 1–3, outlining the exosomes in cancer. The topics deal with the mechanisms of tumor formation under the exosomes' action, showing how circulating exosomes have come into the spotlight of research as a high potential biomarker of pancreatic cancer and multiple myeloma, and outlining the application of exosomes as transfer vector in tumor therapeu‐ tics. As a high potential source of "liquid biopsies," exosomes, which are secreted by fusing the intracellular multivesicular body with cell membrane, have relative stability and compo‐ sition covering the whole range of cancer-related biomarkers including the cells' proteins, lipids, DNA, and RNA. The emphasis in the first part of the book is on exosomal lncRNAs and miRNAs, which can serve as diagnostic and prognostic factors, complementing clinical and pathological parameters in the effort to predict the outcome of patients with pancreatic cancer. Chapter 2 discusses that multiple myeloma and monoclonal gammopathy of unde‐ termined significance (MGUS) exosomes are different in concentration, biological activity, and biochemical markers. These differences seem to be related to the free light chains associ‐ ated to exosomes and their pro-pathogenic properties. Knowledge in the field of tumor-de‐ rived exosomes advanced so quickly, which lead to numerous findings correlating these extracellular vesicles with tumor growth, metastatic process, and their close association with the immune system. Chapter 3 presents animal models in exosome research, where Dr. Melo presents that genetically engineered mouse models can be a promising approach to address the current technical limitations allowing tracing tumor-derived exosomes while retaining the animal immune system.

Part II comprises two chapters focusing on exosomes in viral infection. Chapter 4 provides an overview of our current understanding of exosome biogenesis and how this normal physiological process is hijacked by some pathogenic viruses. The potential roles of exo‐ somes in viral pathogenesis are also discussed in Chapter 4. Details relating to exosomes from Epstein-Barr virus (EBV)-associated cancers are included in Chapter 5. Current eviden‐ ces support the pathogenic roles of exosomes in EBV-related cancers as biomarkers in cancer diagnostics and therapy response. Part II highlights the potential challenges in the develop‐ ment of exosome-based biomarkers for clinical application in infectious diseases.

The last three chapters in Part III explain the formation and release of extracellular vesicles (EVs), microvesicles (MVs), and exosomes. Exosomes are a class of EVs of small size (40–120 nm) originating in multivesicular endosomes and can be released from a wide range of cells, which differ in size from MVs (50–1000 nm) and apoptotic bodies (800–5000 nm) and are secreted directly from the cell membrane in budding form. Details of how to isolate exo‐ somes from liquid biopsy and blood are given in Part III, where recent progress and remain‐ ing challenges in the isolation of EVs for diagnosis and treatment of cancer and infectious diseases are discussed.

This book is intended to provide deep coverage of topics that we feel are reasonably well understood. Rapid progress in the field of exosomes made it possible to reduce the coverage of less significant areas. The book is designed for different readers although it is hard to simultaneously cover all the areas of exosomes in this book. I believe that one should solve the basic exosome problem before starting to worry about implications of exosomes in diag‐ nosis and treatment of cancer and infectious diseases.

> **Jin Wang PhD** Professor Scientific Research Center Shanghai Public Health Clinical Center Fudan University

**Section 1**

**Exosome in Cancer**

The last three chapters in Part III explain the formation and release of extracellular vesicles (EVs), microvesicles (MVs), and exosomes. Exosomes are a class of EVs of small size (40–120 nm) originating in multivesicular endosomes and can be released from a wide range of cells, which differ in size from MVs (50–1000 nm) and apoptotic bodies (800–5000 nm) and are secreted directly from the cell membrane in budding form. Details of how to isolate exo‐ somes from liquid biopsy and blood are given in Part III, where recent progress and remain‐ ing challenges in the isolation of EVs for diagnosis and treatment of cancer and infectious

This book is intended to provide deep coverage of topics that we feel are reasonably well understood. Rapid progress in the field of exosomes made it possible to reduce the coverage of less significant areas. The book is designed for different readers although it is hard to simultaneously cover all the areas of exosomes in this book. I believe that one should solve the basic exosome problem before starting to worry about implications of exosomes in diag‐

> **Jin Wang PhD** Professor

Fudan University

Scientific Research Center

Shanghai Public Health Clinical Center

diseases are discussed.

VIII Preface

nosis and treatment of cancer and infectious diseases.

### **Novel Implications of Exosomes and lncRNAs in the Diagnosis and Treatment of Pancreatic Cancer**

Jin Wang, Xuan Zhang, Chunxia Ji, Lei Zhang, Yang Di, Wenhui Lou, Xiaoyan Zhang and Jianqing Xu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69510

#### **Abstract**

Pancreatic cancer remains a leading cause of cancer-related deaths. Most patients are present with advanced stages of the disease at the time of diagnosis; thus, surgery, which is the best curative option for this malignancy, is no longer an effective treatment modality for affected individuals. As a likely source of "liquid biopsies," exosomes, which are secreted by fusing intracellular multivesicular bodies with cell membranes, have relative stability and composition, allowing them to cover the entire range of cancer-related biomarkers, including cellular proteins, lipids, DNA, RNA, miRNA, and long non-coding RNAs (lncRNAs). To explore the early detection biomarkers of pancreatic cancer and to develop successful therapeutic intervention for this disease, assessing the implications of exosomes in pancreatic cancer patients is essential. In this chapter, we wish to focus on the possibility of using exosomes and lncRNAs in the clinical management of patients with pancreatic cancer. We will discuss the mechanisms of tumor formation under the exosomal action, demonstrate how circulating exosomes and lncRNAs have come into the research spotlight as likely biomarkers of pancreatic cancer, and discuss the applications of exosomes as transfer vectors in tumor therapeutics.

**Keywords:** exosomes, lncRNA, pancreatic cancer, biomarkers, diagnosis, therapeutic intervention

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

#### **1.1. Exosomes, ncRNAs, and lncRNAs**

Exosomes are a class of small (40–120 nm) extracellular vesicles (EVs) that originate in multivesicular endosomes [1–3] and can be released from a wide range of cells, including cancer cells [4]. Exosomes differ in size from microvesicles (50–1000 nm) and apoptotic bodies (800–5000 nm) and are secreted directly from the cell membrane in a budding form [5–7]. Late endosomes released from multivesicular bodies (MVBs) are integrated with the cell membrane in the extracellular matrix during the release of exosomes. Exosomes released into the extracellular environment can be utilized by tumor cells to alter the tumor microenvironment or to provide a favorable microenvironment for distant metastases by affecting distant organs [8–10]. Therefore, exosomes serve as efficient vehicles for long- and short-distance intercellular communication by signaling molecules in the form of lipids, proteins, DNA, RNAs, and non-coding RNAs (ncRNAs) [11]. Exosomes play an important role in signal transduction between cells.

In the complicated human genome, approximately 2% of the genomic sequence encodes proteins involved in biological progression [12], of which approximately 90% are ncRNAs. ncRNAs are described as the "noise" of the genome in their primary form, and they can be divided into two subgroups: small ncRNAs (sncRNAs) and long ncRNAs (lncRNAs) [13–16]. If RNA is <200 nt in length, the ncRNAs are defined as sncRNAs, which includes microRNAs (also called miRNAs or miRs). Conversely, long non-coding RNAs (lncRNAs) are >200 nt in length. Previous studies have reported that lncRNAs are involved in numerous physiological and pathological processes.

In recent years, an increasing number of lncRNAs have been investigated, and play a vital role in various major biological processes associated with promoting proliferation, invasion, and migration metabolism [17–19]. Increasing evidence points to important functional or regulatory roles of lncRNAs in cellular processes, including the cell cycle, proliferation, apoptosis [20–22], RNA processing [23], chromatin modification [24, 25], genomic reprogramming [26, 27], and gene imprinting [28]. They also play a role in cancers resulting from aberrant lncRNA expression. Recent findings indicate that lncRNAs are dysregulated in many kinds of cancer, including pancreatic cancer (PaCa), and they are closely related with tumorigenesis, metastasis, prognosis, and diagnosis.

#### **2. The physiological function of exosomes**

Exosomes carry a variety of substances from secreted cells, including proteins, lipids, DNA, RNA, and ncRNA [29, 30]. The intercellular communication regulated by exosomes is not only involved in regulating the physiological processes of normal cells but also participates in many pathological processes associated with disease development, including tumors [31–33]. Exosomes regulate biological activity through the rapid reaction of signal molecules on their surface or by the release of extracellular biologically active substances. Exosomal biological activity is mainly determined by its components (i.e., the exosome cell source) [8–10]. Exosomes, which use autocrine, paracrine, and endocrine signaling to exchange biological information, are involved in the transmission of substances and signals between cells.

In addition, exosomes have immunomodulatory function [34]. Antigen-presenting cell (APC)-derived exosomes can promote the proliferation of T lymphocytes and induce antitumor immune responses in vivo. Exosomes have the features of their original cells because they bring DNA, RNA, and proteins from the original cell and carry a variety of proteins on their surface. Since exosomes are released from endosomes, they carry certain endosomalspecific proteins, including GTPases, flotillin, Alix, Tsg101, CD81 and CD82, heat shock proteins Hsp70 and Hsp90, and epithelial cell adhesion molecules [35–38] that are involved in exosome formation.

If exosomes are secreted by tumor cells, they can kill the tumor cells by providing information to cytotoxic T lymphocytes by cross-reacting with antigen-presenting cells [39]. However, exosomes from tumor cells have a dual role in that they have antitumor activity and also promote tumor growth. For example, exosomes from colorectal cancer cells contain cell cyclerelated mRNAs that promote the proliferation of endothelial cells, which can induce tumor angiogenesis [40]. Exosomes obtained from gastric cancer cells promote tumor progression by activating the NF-kB pathway in macrophages [41]. In ovarian cancer, epithelial ovarian cancer (EOC) cell-derived exosomes promote ovarian cancer metastasis and deterioration by transferring CD44 to peritoneal cells [42].

#### **3. Exosomes as novel biomarkers of cancer**

**1. Introduction**

between cells.

and pathological processes.

sis, prognosis, and diagnosis.

**2. The physiological function of exosomes**

**1.1. Exosomes, ncRNAs, and lncRNAs**

Exosomes are a class of small (40–120 nm) extracellular vesicles (EVs) that originate in multivesicular endosomes [1–3] and can be released from a wide range of cells, including cancer cells [4]. Exosomes differ in size from microvesicles (50–1000 nm) and apoptotic bodies (800–5000 nm) and are secreted directly from the cell membrane in a budding form [5–7]. Late endosomes released from multivesicular bodies (MVBs) are integrated with the cell membrane in the extracellular matrix during the release of exosomes. Exosomes released into the extracellular environment can be utilized by tumor cells to alter the tumor microenvironment or to provide a favorable microenvironment for distant metastases by affecting distant organs [8–10]. Therefore, exosomes serve as efficient vehicles for long- and short-distance intercellular communication by signaling molecules in the form of lipids, proteins, DNA, RNAs, and non-coding RNAs (ncRNAs) [11]. Exosomes play an important role in signal transduction

4 Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

In the complicated human genome, approximately 2% of the genomic sequence encodes proteins involved in biological progression [12], of which approximately 90% are ncRNAs. ncRNAs are described as the "noise" of the genome in their primary form, and they can be divided into two subgroups: small ncRNAs (sncRNAs) and long ncRNAs (lncRNAs) [13–16]. If RNA is <200 nt in length, the ncRNAs are defined as sncRNAs, which includes microRNAs (also called miRNAs or miRs). Conversely, long non-coding RNAs (lncRNAs) are >200 nt in length. Previous studies have reported that lncRNAs are involved in numerous physiological

In recent years, an increasing number of lncRNAs have been investigated, and play a vital role in various major biological processes associated with promoting proliferation, invasion, and migration metabolism [17–19]. Increasing evidence points to important functional or regulatory roles of lncRNAs in cellular processes, including the cell cycle, proliferation, apoptosis [20–22], RNA processing [23], chromatin modification [24, 25], genomic reprogramming [26, 27], and gene imprinting [28]. They also play a role in cancers resulting from aberrant lncRNA expression. Recent findings indicate that lncRNAs are dysregulated in many kinds of cancer, including pancreatic cancer (PaCa), and they are closely related with tumorigenesis, metasta-

Exosomes carry a variety of substances from secreted cells, including proteins, lipids, DNA, RNA, and ncRNA [29, 30]. The intercellular communication regulated by exosomes is not only involved in regulating the physiological processes of normal cells but also participates in many pathological processes associated with disease development, including tumors [31–33]. Exosomes regulate biological activity through the rapid reaction of signal The identification of cancer-specific exosomes in bodily fluids, such as serum, plasma, and urine, will be useful for the detection of cancer and will allow for the identification of specific DNA, RNA, and protein content in the absence of contamination from non-cancerous exosomes [43]. The proteoglycan glypican-1 (GPC1) is highly expressed in tumor cell-derived exosomes. GPC1 has been shown to be a specific, sensitive marker in serum from pancreatic patients that are in both the early and late stages but not in benign pancreatic diseases [43]. CD24 and EpCAM are tumor-derived exosome markers isolated by immune-affinity techniques involving anti-CD24 and anti-EpCAM magnetic beads [44]. In serum, CD24 and EpCAM serve as early diagnosis biomarkers [44], while fibronectin can serve as an early diagnosis biomarker in plasma. The ELISA method has been used to detect fibronectin [45]. The levels of exosomal EDIL3 from breast cancer patients can be dramatically reduced with surgery, indicating that EDIL3 can also serve as a diagnostic and prognostic biomarker [46]. Survivin expression has been shown to be significantly increased in patients with prostate cancer, but lower survivin expression has been found in benign prostatic hyperplasia (BPH) and healthy subjects. Additionally, the levels of survivin in BPH and healthy subjects are not significantly different. Thus, survivin can be used as a new diagnostic indicator of prostate cancer [47].

Separated and purified exosomes not only contain mRNA and miRNA but also tRNA and some lncRNA [11, 48–50]. Six miRNAs (miR-19b-3p, miR-21-5p, miR-221-3p, miR-409-3p, miR-425-5p, and miR-584-5p) were found to be upregulated in lung adenocarcinoma [51]. Eight miRNAs (miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR-203, miR-205, and miR-214) have served as diagnostic biomarkers for ovarian cancer, and these miRNAs have also been identified in exosomes from ovarian cancer patients [52]. miRNAs can also be diagnostic biomarkers for esophageal squamous cell cancer (ESCC), as the serum levels of exosomal miR-21 from patients with ESCC are significantly higher than those of patients with benign diseases without systemic inflammation and are positively correlated with tumor progression and aggressiveness [53].

#### **4. Exosomes for therapeutic intervention in cancer**

The recent contribution by Zhang et al. reviewed the recent advances in cancer immunotherapy, exosome functions, exosome immunoregulation, and immune cell-derived exosomes [34]. As mentioned in Zhang's manuscript, exosomes cannot only transfer messages between cells by carrying RNA and proteins but also can modulate the immune response. After reviewing recent findings regarding exosomes and immunity in cancer, we have highlighted the novel insights into the development of efficient exosome-based cancer vaccines for cancer therapeutic intervention. Specifically, exosomes derived from immune cells, such as APCs, dendritic cells (DCs), and NK cells, play a crucial role in the immunomodulation of cancer, and they may be the best cancer vaccine candidates because they can inhibit the malignant activity of cancer cells and leave healthy cells unaffected [54–56]. Recently, researchers have noted that exosomes may lead to key advances in cancer therapy. Exosomes isolated from DCs have been evaluated in clinical trials as treatment for various kinds of cancers [57–59]. In a phase I clinical trial, exosomes derived from autologous DCs loaded with MAGE 3 peptides were applied as cancer therapy for stage III/IV melanoma patients [58]. Several phase I or phase II clinical trials involving exosome-based regimens have occurred in breast cancer, gastric cancer, malignant glioma, and non-small cell lung cancer patients, which demonstrates that exosomes are effective tools for the transportation of anticancer drugs [59]. Exosomes were employed to form a complex with curcumin and delivered to recipient pancreatic cancer cells, which was found to promote cytotoxicity [60]. Moreover, exosomes have been shown to deliver small, molecular anticancer drugs across the blood-brain barrier and significantly inhibit tumor growth in a brain cancer model [61, 62].

#### **5. Long non-coding RNAs as novel biomarkers in cancer**

lncRNAs modulate gene expression, while lncRNA dysregulation is associated with human cancer. lncRNAs could play a significant role in cancer progression by interacting with proteins. Since they are highly specific and easily detectable in tissue, serum, plasma, and urine, interest in exploring lncRNAs in cancer patients continues to increase. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1, also known as NEAT2), a novel lncRNA, is found on chromosome 11q13 and is well conserved among mammalian species.  MALAT-1 has an important role in regulating the metastasis of bladder cancer and can be a potential application in bladder cancer therapy [66]. The MALAT-1-mediated promotion of renal cell carcinoma (RCC) proliferation and metastasis may be due to the upregulation of Livin expression [67]. MALAT-1 promotes the proliferation of chondrosarcoma cells via activating the Notch-1 signaling pathway [68], indicates poor prognosis in non-small cell lung cancer, and induces migration and tumor growth [69]. Upregulation of MALAT-1 has been associated with survival rate, cell cycle, and migration in patients with esophageal squamous cell carcinoma (ESCC) [70]. However, the loss of MALAT1 is compatible with cell viability and normal development [71]. On the other hand, MALAT-1 is downregulated in preeclampsia and regulates the proliferation, apoptosis, migration, and invasion of JEG-3 trophoblast cells [72]. MALAT-1 is also expected to be a potential therapeutic target in prostate cancer [73]. As another critical oncogenic lncRNA in human cancers [74, 75], the lncRNA HOTTIP promotes tumor growth, inhibits cell apoptosis [76], contributes to the progression of prostate cancer [77] and non-small cell lung cancer [78] by regulating HOXA13, and increases the chemoresistance of osteosarcoma cells by activating the Wnt/β-catenin pathway [79]. HOTTIP is upregulated and associated with poor prognosis in patients with osteosarcoma [80]. Overexpression of HOTTIP can promote tumor invasion and predict poor prognosis in gastric cancer [81]. This accumulating evidence indicates that long non-coding RNAs have immense potential as powerful, non-invasive tumor markers. However, overexpression of HOTTIP inhibits glioma cell growth by brain and reproductive expression [82].

Separated and purified exosomes not only contain mRNA and miRNA but also tRNA and some lncRNA [11, 48–50]. Six miRNAs (miR-19b-3p, miR-21-5p, miR-221-3p, miR-409-3p, miR-425-5p, and miR-584-5p) were found to be upregulated in lung adenocarcinoma [51]. Eight miRNAs (miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR-203, miR-205, and miR-214) have served as diagnostic biomarkers for ovarian cancer, and these miRNAs have also been identified in exosomes from ovarian cancer patients [52]. miRNAs can also be diagnostic biomarkers for esophageal squamous cell cancer (ESCC), as the serum levels of exosomal miR-21 from patients with ESCC are significantly higher than those of patients with benign diseases without systemic inflammation and are positively correlated with tumor progression

The recent contribution by Zhang et al. reviewed the recent advances in cancer immunotherapy, exosome functions, exosome immunoregulation, and immune cell-derived exosomes [34]. As mentioned in Zhang's manuscript, exosomes cannot only transfer messages between cells by carrying RNA and proteins but also can modulate the immune response. After reviewing recent findings regarding exosomes and immunity in cancer, we have highlighted the novel insights into the development of efficient exosome-based cancer vaccines for cancer therapeutic intervention. Specifically, exosomes derived from immune cells, such as APCs, dendritic cells (DCs), and NK cells, play a crucial role in the immunomodulation of cancer, and they may be the best cancer vaccine candidates because they can inhibit the malignant activity of cancer cells and leave healthy cells unaffected [54–56]. Recently, researchers have noted that exosomes may lead to key advances in cancer therapy. Exosomes isolated from DCs have been evaluated in clinical trials as treatment for various kinds of cancers [57–59]. In a phase I clinical trial, exosomes derived from autologous DCs loaded with MAGE 3 peptides were applied as cancer therapy for stage III/IV melanoma patients [58]. Several phase I or phase II clinical trials involving exosome-based regimens have occurred in breast cancer, gastric cancer, malignant glioma, and non-small cell lung cancer patients, which demonstrates that exosomes are effective tools for the transportation of anticancer drugs [59]. Exosomes were employed to form a complex with curcumin and delivered to recipient pancreatic cancer cells, which was found to promote cytotoxicity [60]. Moreover, exosomes have been shown to deliver small, molecular anticancer drugs across the blood-brain barrier and significantly

and aggressiveness [53].

**4. Exosomes for therapeutic intervention in cancer**

6 Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

inhibit tumor growth in a brain cancer model [61, 62].

**5. Long non-coding RNAs as novel biomarkers in cancer**

lncRNAs modulate gene expression, while lncRNA dysregulation is associated with human cancer. lncRNAs could play a significant role in cancer progression by interacting with proteins. Since they are highly specific and easily detectable in tissue, serum, plasma, and urine, interest in exploring lncRNAs in cancer patients continues to increase.  Here, we have identified some interesting circulating lncRNAs (also known as exosomal lncRNAs), including MALAT-1, PVT1, HOTAIR, H19, UCA1, and TUG1, as novel biomarkers in various cancers. MALAT-1 in urine may serve as a potential biomarker for predicting prostate cancer risk. The application of the MALAT-1 model can prevent 30.2–46.5% of unnecessary biopsies in high-grade cancers [83]. PVT1 expression has been shown to be significantly elevated in non-small cell lung cancer (NSCLC), and high PVT1 expression has been associated with poor overall survival and disease-free survival in NSCLC patients; therefore, PVT1 could serve as a promising biomarker for the diagnosis and prognosis of NSCLC. PVT1 knockdown could remarkably inhibit NSCLC cell proliferation [84]. HOTAIR has been shown to be significantly higher in breast cancer patients, and circulating HOTAIR DNA levels were 2.15-fold higher in patients compared with those of healthy controls in one study, which demonstrates a moderate correlation between its expressions in tumor tissues. Plasma HOTAIR levels have been found to be significantly reduced after surgery [85, 86], indicating that plasma HOTAIR might serve as a potential biomarker for diagnosing breast cancer.  Biomarkers PaCa (blood) [121]

GC, BC

PaCa

5. Apoptosis Colorectal cancers [141]

(tissue)

tissues)

3.  Regulator of the metastasis

3. Diagnostic and prognostic biomarker

target

growth

3. Increases chemoresistance

4. Progression and prognosis

proliferation and invasion

2. Progression and prognosis

4. Modulates thyroid cancer cell proliferation

6. Novel biomarker for diagnosis and prognosis

prognostic biomarkers

uc.345 1. Promotes tumorigenesis PaCa [122]

HOTTIP 1. Inhibits glioma cell

PVT1 1. Promotes cell

LINC-PINT 1. Diagnostic and

4. Potential therapeutic

2. Cell growth, apoptosis, migration, and invasion

MALAT-1 1. Promotes cell

**Table 1.** Long non-coding RNAs (lncRNAs) as potential biomarkers for cancer.

indicated that H19 might serve as a potential biomarker for early detection and prediction of prognosis of breast cancer and gastric cancer. The expression of H19 was remarkably increased in breast cancer and gastric cancer tissues. H19 expression has been shown to be significantly correlated with invasion depth, advanced TNM stage and regional lymph node metastasis in gastric cancer. Additionally, elevated expression levels of H19 have been shown to contribute to the poor overall survival and disease-free survival of gastric cancer patients [87]. This makes H19 closely associated with progressive gastric cancer, and it could be a potential non-invasive diagnostic gastric cancer biomarker for management. Better performance could be achieved using both carcinoembryonic antigen (CEA) and H19 simultaneously [88]. Plasma H19 levels have been shown to be significantly decreased in postoperative breast cancer samples compared to those in preoperative samples [89]. Urothelial cancer-associated 1 (UCA1), originally identified as a lncRNA in bladder cancer, has been proven to play a pivotal role in bladder cancer progression and embryonic development. Upregulation of the lncRNA UCA1 and the lncRNA WRAP53 has been observed in hepatocellular carcinoma (HCC), and CA1 might serve as a novel serum biomarker for HCC. Moreover, the expression levels of UCA1 and WRAP53 in tissue have been shown to be strongly correlated with their levels in sera. Further, the combination of UCA1 and WRAP53 with serum alpha fetoprotein could improve sensitivity to 100% [90]. Further, meta-analysis also found that higher levels of UCA1 were correlated with shorter progression-free survival (PFS) and overall survival (OS) times in cancer [91], indicating that circulating lncRNAs, such as MALAT-1, PVT1, HOTAIR, H19, UCA1, and WRAP53, could serve as novel biomarkers for the early detection and the prediction of prognosis of cancer.

#### **6. Exosomes and lncRNAs in the diagnosis and treatment of pancreatic cancer**

Pancreatic cancer is one of the most lethal tumors, and its main tumor type is that of adenocarcinoma [92–94]. Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer-related deaths in both males and females in the USA, is usually asymptomatic [186], and PDAC is one of the most lethal malignant neoplasms worldwide [89, 95, 96]. Statistical analysis indicated that death rates rose from 2001 to 2010 [97]. In America, approximately 53,000 people were diagnosed with pancreatic cancer in 2016, and pancreatic cancer was responsible for 41,750 deaths in the USA [98] in that same year. Additionally, the incidence of pancreatic cancer has shown an increasing trend year-by-year in China, and pancreatic cancer has become one of the top 10 causes of cancer-related deaths [99].

It is well known that pancreatic cancer has a poor prognosis because it is usually diagnosed after the cancer has already spread, leading to poor patient outcomes. Pancreatic ductal adenocarcinoma patients have a 5-year survival rate of ~5% [100]. Survival can be improved if tumors are detected at an early stage, and the 5-year survival rate is 50% if tumors are <2 cm in size [101]. However, there have been no reliable biomarkers to accurately diagnose, image, or predict the tumor classification and biological behavior of pancreatic cancer until now. Thus, it is urgent to screen potential biomarkers and treatment-related biomarkers, such as exosome-derived proteins, DNA (exoDNA), miRNAs (exosomal miRNAs), and lncRNAs (exosomal lncRNAs), for the early detection of pancreatic cancer. Allenson found that KRAS mutations in the exoDNA of control, localized, locally advanced, and metastatic PDAC patients were 7.4, 66.7, 80, and 85%, respectively, which demonstrates that KRAS in exosomes could be applied to diagnose PDAC [102]. Takikawa also confirmed that pancreatic stellate cell (PSC)-derived exosomes stimulate the proliferation and migration of pancreatic cancer cells and upregulate the mRNA expression of the chemokine (C-X-C motif) ligands 1 and 2 in pancreatic cancer cells [103]. Over the last few years, non-coding RNAs, especially

indicated that H19 might serve as a potential biomarker for early detection and prediction of prognosis of breast cancer and gastric cancer. The expression of H19 was remarkably increased in breast cancer and gastric cancer tissues. H19 expression has been shown to be significantly correlated with invasion depth, advanced TNM stage and regional lymph node metastasis in gastric cancer. Additionally, elevated expression levels of H19 have been shown to contribute to the poor overall survival and disease-free survival of gastric cancer patients [87]. This makes H19 closely associated with progressive gastric cancer, and it could be a potential non-invasive diagnostic gastric cancer biomarker for management.  Promotes EMT BC [197] 4. Suppress metastasis Epithelial OC [198]

cancer

6. Enhances drug resistance BC, bladder cancer, GC,

OSCC, ESCC [195, 196]

BC [199, 200]

[118, 191–194]

[192, 201–205]

[171, 207–217]

[218–222]

[229, 230]

[219, 223–226]

tumorigenesis, enhances cell proliferation, migration

10 Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

5. Modulates cell growth and apoptosis, and epigenetic regulation

7. Promotes glutamine

8. As diagnostic and prognostic markers

proliferation, migration

2. Poor prognosis and promotes metastasis

3. Associated with chemotherapy resistance and poor prognosis

4. Acts as a tumor suppressor in human

5. Affects apoptosis and insulin secretion

6. As biomarker for poor

**Table 1.** Long non-coding RNAs (lncRNAs) as potential biomarkers for cancer.

glioma

prognosis

metabolism

2. Contributes to the progression and prognosis

UCA1 1. Promotes the

TUG1 1. Promotes cell

exosomal lncRNAs and exosomal miRNAs, have become a new diagnostic, prognostic, and predictive tool for pancreatic cancer. Exosomal miR-155, miR-196a, miR-17-5p, miR-10b, and miR-21 have good sensitivity and specificity in the serum of PaCa patients and can be useful serum biomarkers for pancreatic cancer [104, 105]. Not only can single exosomes be a diagnosis biomarker, but combined exosomal miRNAs, such as miR-1246, miR-4644, miR-3976, and miR-4306, can also increase sensitivity and specificity for the diagnosis of pancreatic cancer.

 However, few studies have explored the potential use of exosomal lncRNAs in pancreatic cancer detection and prognosis. MALAT-1, HOTTIP, PVT1, and HOTAIR, which are secreted from PDAC cells to bodily fluids, such as blood, pancreatic juice, cystic fluid, and urine, are some of most widely studied lncRNAs in pancreatic cancer (**Figure 1**). As a potential oncogenic lncRNA, MALAT-1 involves in proliferation, migration, and invasion and promotes the undifferentiated phenotype of pancreatic tumor cells [106]. MALAT-1 can also promote the tumorigenicity of pancreatic cancer cells, increase the proportion of pancreatic cancer stem cells, maintain a selfrenewing capacity, and decrease chemosensitivity to anticancer drugs. Moreover, MALAT-1 has potential effects on the stem cell-like phenotypes of pancreatic cancer cells, which suggests that MALAT-1 has a novel role in tumor stemness [107]. The lncRNA HOTTIP enhances pancreatic cancer cell proliferation, survival, and migration and has been implicated in pancreatic cancer diagnosis and prognosis [108]. The overexpression of HOTAIR has been described as a poor prognostic factor in PDAC and can also be a novel non-invasive salivary biomarker for the early diagnosis of PaCa with PVT1 expression [109]. Increased expression of the lncRNA PVT1 is associated with poor prognosis in pancreatic cancer patients [110]. PVT1 expression is

**Figure 1.** Exosomal lncRNAs secreted from PDAC cells as potential biomarkers of pancreatic cancer.

significantly increased in PDAC and is correlated with tumor progression. Moreover, patients with high PVT1 expression levels have been shown to have shorter overall survival times compared to those with low PVT1 expression levels, which implies that PVT1 could be a potential molecular biomarker for predicting the prognosis of patients with PDAC [110]. H19 has been shown to be overexpressed in PDAC tissues and to be correlated with the histological grade of PDAC. Knockdown of H19 can suppress cell viability, proliferation, and tumor growth, while H19 overexpression can enhance cell viability, proliferation, and tumor growth [111]. UCA1 expression has been shown to be significantly upregulated in PaCa tumor tissues and to be significantly correlated with malignant potential factors, such as tumor size, depth of invasion, CA19-9 levels, and tumor stage. Highly expressed UCA1 has been shown to be an independent prognostic biomarker of PaCa, leading to an obviously shorter 5-year overall survival (OS). Downregulation of UCA1 could effectively inhibit cell proliferative activities, which implies that UCA1 could be a potential prognostic biomarker and therapy target of PaCa [112].

exosomal lncRNAs and exosomal miRNAs, have become a new diagnostic, prognostic, and predictive tool for pancreatic cancer. Exosomal miR-155, miR-196a, miR-17-5p, miR-10b, and miR-21 have good sensitivity and specificity in the serum of PaCa patients and can be useful serum biomarkers for pancreatic cancer [104, 105]. Not only can single exosomes be a diagnosis biomarker, but combined exosomal miRNAs, such as miR-1246, miR-4644, miR-3976, and miR-4306, can also increase sensitivity and specificity for the diagnosis of pancreatic cancer.  However, few studies have explored the potential use of exosomal lncRNAs in pancreatic cancer detection and prognosis. MALAT-1, HOTTIP, PVT1, and HOTAIR, which are secreted from PDAC cells to bodily fluids, such as blood, pancreatic juice, cystic fluid, and urine, are some of most widely studied lncRNAs in pancreatic cancer (**Figure 1**). As a potential oncogenic lncRNA, MALAT-1 involves in proliferation, migration, and invasion and promotes the undifferentiated phenotype of pancreatic tumor cells [106]. MALAT-1 can also promote the tumorigenicity of pancreatic cancer cells, increase the proportion of pancreatic cancer stem cells, maintain a selfrenewing capacity, and decrease chemosensitivity to anticancer drugs. Moreover, MALAT-1 has potential effects on the stem cell-like phenotypes of pancreatic cancer cells, which suggests that MALAT-1 has a novel role in tumor stemness [107]. The lncRNA HOTTIP enhances pancreatic cancer cell proliferation, survival, and migration and has been implicated in pancreatic cancer diagnosis and prognosis [108]. The overexpression of HOTAIR has been described as a poor prognostic factor in PDAC and can also be a novel non-invasive salivary biomarker for the early diagnosis of PaCa with PVT1 expression [109]. Increased expression of the lncRNA PVT1 is associated with poor prognosis in pancreatic cancer patients [110]. PVT1 expression is

12 Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

**Figure 1.** Exosomal lncRNAs secreted from PDAC cells as potential biomarkers of pancreatic cancer.

In addition, high expression levels of the lncRNA HOXA13 have been shown to be correlated with lymph node metastasis, poor histological differentiation, and decreased overall survival in PDAC patients. The knockdown of HOXA13 resulted in proliferation arrest and impaired cell invasion in pancreatic cancer [113]. Using microarray analysis, HOTTIP was confirmed to be one of the most significantly upregulated lncRNAs in PDAC [113]. HOTTIP has been shown to be overexpressed in pancreatic cancer, and knockdown of HOTTIP in pancreatic cancer cells decreased proliferation, induced apoptosis, and decreased migration [108]. Using an Arraystar Human lncRNA Microarray, HOTTIP-005, XLOC\_006390, and RP11-567G11.1 were found to be the most increased lncRNAs in PaCa. Elevated HOTTIP-005 and RP11-567G11.1 expression could serve as poor prognostic markers for patients with PaCa. Plasma HDRF and RDRF (HOTTIP-005- and RP11-567G11.1-derived RNA fragments in plasma/serum) have also shown to be significantly increased in patients with PaCa, which demonstrates that HDRF and RDRF levels could be promising indicators for distinguishing patients with PC [114]. As an oncogenic lncRNA, uc.345 has been shown to promote tumor progression and to serve as a poor predictor for OS in pancreatic cancer patients. uc.345 was found to be upregulated in tumor tissues, and higher uc.345 expression levels have been associated with cancer invasion and metastasis, which could be an independent risk factor for the OS of pancreatic cancer patients [115]. The lncRNA IRAIN plays an important role in many malignancies, and upregulation of IRAIN has been shown to be significantly correlated with tumor size, the TNM classification of malignant tumors (TNM) stage, and lymph node metastasis in PaCa patients. The knockdown of IRAIN significantly induced cell apoptosis and inhibited cell proliferation in PaCa cells [116]. The lncRNA TUG1 has been shown to be highly expressed in pancreatic tissue compared with its expression in other organ tissues, and downregulation of TUG1 has been shown to affect apoptosis and insulin secretion in pancreatic β cells [117]. CCDC26 might be identified as a novel oncogene in PaCa by regulating proliferating cell nuclear antigen (PCNA) and Bcl2 expression. CCDC26 is significantly upregulated in PaCa, and it is correlated with tumor size, tumor number, and reduced OS [118]. Univariate and multivariate analysis showed that CCDC26 expression can be an independent prognostic factor of OS in patients with PaCa; therefore, CCDC2 could serve as a novel biomarker and therapeutic target of PC for cancer in the future [118]. LINC-ROR has been shown to be upregulated in PaCa tissues, and overexpression of LINC-ROR promoted cell proliferation, migration, invasion, and metastasis both in vitro and in mouse models. LINC-ROR acts as an important regulator of ZEB1 and might represent a novel therapeutic target [119]. The lncRNA LINC-PINT (p53-induced transcript) could also regulate tumor cell viability and proliferation. However, the expression levels of LINC-PINT have been shown to be lower in plasma and tumor tissue samples in PaCa patients. LINC-PINT has been shown to be more sensitive than CA19-9 in detecting PaCa, which suggests that LINC-PINT could be used for distinguishing the cause of malignant obstructive jaundice [120]. The lncRNA HMlincRNA717 has also been shown to be downregulated in pancreatic cancer and associated with overall survival, suggesting that HMlincRNA717 could be a potential prognostic biomarker for pancreatic cancer progression [121]. As a potential tumor suppressor, the long intergenic non-coding RNA (lincRNA) LINC00673 has been associated with pancreatic cancer risk. A G>A mutation at rs11655237 of LINC00673 created a target site for miR-1231 binding, which diminished the effect of LINC00673 in an allele-specific manner and conferred susceptibility to PaCa [122].

All the abovementioned exosomal lncRNAs could serve as diagnostic and prognostic factors to complement clinical and pathological parameters in predicting the outcome of patients with pancreatic cancer. Although there are an increasing number of clinical assays for studying exosomes, determining clinical applications for lncRNAs and exosomes is a long ways off. No matter how exosomes have become the most effective cancer vaccines, future research to investigate exosomal lncRNAs as biomarkers for the early detection of pancreatic cancer and to assess the validity and quality of the exosomes as effective vaccines for pancreatic cancer will be valuable. To achieve this long-term goal, further understanding of exosome biology, especially of the molecular mechanisms of tumor- and immune cell-derived exosomes as cancer vaccines, is required.

#### **Acknowledgement**

This research was supported by a grant from the National Natural Science Foundation of China (81672383) and a grant (16PJ1408800) from the Shanghai Pujiang Program in Shanghai, China.

#### **Abbreviations**

Novel Implications of Exosomes and lncRNAs in the Diagnosis and Treatment of Pancreatic Cancer http://dx.doi.org/10.5772/intechopen.69510 15

#### **Author details**

and metastasis both in vitro and in mouse models. LINC-ROR acts as an important regulator of ZEB1 and might represent a novel therapeutic target [119]. The lncRNA LINC-PINT (p53-induced transcript) could also regulate tumor cell viability and proliferation. However, the expression levels of LINC-PINT have been shown to be lower in plasma and tumor tissue samples in PaCa patients. LINC-PINT has been shown to be more sensitive than CA19-9 in detecting PaCa, which suggests that LINC-PINT could be used for distinguishing the cause of malignant obstructive jaundice [120]. The lncRNA HMlincRNA717 has also been shown to be downregulated in pancreatic cancer and associated with overall survival, suggesting that HMlincRNA717 could be a potential prognostic biomarker for pancreatic cancer progression [121]. As a potential tumor suppressor, the long intergenic non-coding RNA (lincRNA) LINC00673 has been associated with pancreatic cancer risk. A G>A mutation at rs11655237 of LINC00673 created a target site for miR-1231 binding, which diminished the effect of

14 Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

LINC00673 in an allele-specific manner and conferred susceptibility to PaCa [122].

All the abovementioned exosomal lncRNAs could serve as diagnostic and prognostic factors to complement clinical and pathological parameters in predicting the outcome of patients with pancreatic cancer. Although there are an increasing number of clinical assays for studying exosomes, determining clinical applications for lncRNAs and exosomes is a long ways off. No matter how exosomes have become the most effective cancer vaccines, future research to investigate exosomal lncRNAs as biomarkers for the early detection of pancreatic cancer and to assess the validity and quality of the exosomes as effective vaccines for pancreatic cancer will be valuable. To achieve this long-term goal, further understanding of exosome biology, especially of the molecular mechanisms of tumor- and immune cell-derived exosomes as cancer vaccines, is required.

This research was supported by a grant from the National Natural Science Foundation of China (81672383) and a grant (16PJ1408800) from the Shanghai Pujiang Program in Shanghai, Jin Wang<sup>1</sup> \*, Xuan Zhang1 , Chunxia Ji1 , Lei Zhang2 , Yang Di<sup>3</sup> , Wenhui Lou<sup>2</sup> , Xiaoyan Zhang1 and Jianqing Xu1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Scientific Research Center, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China

2 General Surgery Department, Zhongshan Hospital, Fudan University, Shanghai, China

3 Department of Pancreatic Surgery, Pancreatic Disease Institute, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China

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## **Exploiting Exosomes for Differential Diagnosis of Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance**

Lucia Paolini, Giuseppe Di Noto, Andrea Zendrini, Paolo Bergese and Doris Ricotta

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69398

#### **Abstract**

Multiple myeloma (MM) is a plasma cell dyscrasia characterized by a clonal plasma cell proliferation. Usually, all MM are preceded by an asymptomatic premalignant stage termed monoclonal gammopathy of undetermined significance (MGUS). Differential diagnosis requires the evidence of end-organ damage, but recently new biomarkers are emerging to help clinicians to distinguish MM from the premalignant phase. Circulating exosomes in serum seem to be a powerful tool to be analyzed for liquid biopsy, and in this chapter, we show that MM and MGUS exosomes are different in concentration, biological activity, and biochemical markers. These differences seem to be related to the free light chains (FLCs) associated with exosomes and their propathogenic properties. The cellular processing FLC-decorated exosomes and their ability to activate proinflammatory mechanisms are different in MM and MGUS patients. These elements can be evaluated to create an innovative multiparameter panel to monitor MGUS to MM switching.

**Keywords:** multiple myeloma, exosomes, biomarkers, MGUS

#### **1. Introduction**

This chapter aims to show the possibility of differential typization of multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) serum-derived exosomes exploiting an innovative biochemical and bio-physical approach, in order to evaluate new biomarkers for the differential diagnosis among these two conditions.

MM is a plasma cell dyscrasia characterized by a clonal plasma cell proliferation. The worldwide MM incidence is 3.29/100,000 and increases with age. In the 80–84 years old population, the rate is 37.1, while the median age of diagnosis is 73 years [1]. Usually, almost all MM are preceded by an asymptomatic premalignant stage termed MGUS [2]. MGUS is present in roughly 3–4% of the population over the age of 50 years and it is associated with a risk of progression to MM of approximately 1% [3]. MGUS is clinically silent and is usually diagnosed incidentally when a monoclonal (M) protein is detected during laboratory work-up of patients who have a wide spectrum of clinical conditions. The diagnosis of MGUS requires the absence of hypercalcemia, renal failure, anemia, and bone lesions (CRAB features) that can be attributed to the underlying plasma cell disorder.

Thus, over the years, the diagnosis of MM instead required evidence of end-organ damage attributable to the neoplastic clone of plasma cells in order to prevent MGUS patients from unnecessary and toxic chemotherapy. Furthermore, the MM diagnosis requires bone marrow examination like osteo-medullary biopsy to prove the presence of clonal bone marrow plasma cells [3]. Identification of new biomarkers of malignancy is fundamental to prevent end-organ damage in selected patients who are at imminent risk of symptomatic progression.

In this scenario, liquid biopsy could provide an alternative to invasive procedure and improve screening and early detection of cancer [4]. Liquid biopsy is defined as the search of biomarkers [5] in peripheral blood, and in recent years, exosomes have arisen as a powerful tool to understand cancer biology. Exosomes are vesicles with a size of 50–150 nm, which are secreted by cells into the extracellular space and play an important role in cell communication as cargoes of several specific proteins and RNAs. Exosomes are to date considered playing a pivotal role in information transfer in hematological malignancies [6] and recent studies show that specific exosomal microRNAs are involved in pathogenesis and have a prognostic role in MM [7]. In this chapter, we will analyze the biochemical and biological characteristics of MM and MGUS FLC and their cellular processing through exosomes. We show that free light chain (FLC)-decorated exosomes from patients carry peculiar bio-physical and biological qualities that allow the distinction of the malignant and premalignant condition.

#### **2. MM and MGUS FLC cellular processing**

In this section, we describe the potential pathogenic properties of serum FLC from MM and MGUS patients, correlating them with exosomal mediated cell-to-cell communication. About 80% of MM originates from intact immunoglobulin, non-IgM, MGUS and 20% from lightchain immunoglobulin MGUS [2]. Since 2009, the FLC quantitative assay (developed from both the Binding Site and Siemens) has been recommended by the International Myeloma Working Group to predict risk of progression in MGUS, the evaluation and management of MM [8]. This immunonephelometric assay allows the quantification of the kappa and lambda light chains untied to the heavy chains in serum [2, 9]. Calculation of the related kappa/lambda FLC ratio is an important parameter because about a third of patients with MGUS and more than 90% of patients with MM have altered FLC ratios that indicate excess production of a clonal FLC by the proliferating plasma cell population [2].

Thus, MGUS and MM patients can show similar serum FLC concentration with comparable FLC kappa/lambda ratio and the presence of FLC in urine is often confirmed by urine immunofixation [10]. Kappa and lambda FLCs have been long-considered a by-product of plasma cells, but evidences show that they possess intrinsic pathogenic characteristics [10] as in the case of AL amyloidosis, light-chain deposition disease, MM, and altered FLC ratio is associated with higher risk of progression in MGUS.

MM is a plasma cell dyscrasia characterized by a clonal plasma cell proliferation. The worldwide MM incidence is 3.29/100,000 and increases with age. In the 80–84 years old population, the rate is 37.1, while the median age of diagnosis is 73 years [1]. Usually, almost all MM are preceded by an asymptomatic premalignant stage termed MGUS [2]. MGUS is present in roughly 3–4% of the population over the age of 50 years and it is associated with a risk of progression to MM of approximately 1% [3]. MGUS is clinically silent and is usually diagnosed incidentally when a monoclonal (M) protein is detected during laboratory work-up of patients who have a wide spectrum of clinical conditions. The diagnosis of MGUS requires the absence of hypercalcemia, renal failure, anemia, and bone lesions (CRAB features) that

36 Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

Thus, over the years, the diagnosis of MM instead required evidence of end-organ damage attributable to the neoplastic clone of plasma cells in order to prevent MGUS patients from unnecessary and toxic chemotherapy. Furthermore, the MM diagnosis requires bone marrow examination like osteo-medullary biopsy to prove the presence of clonal bone marrow plasma cells [3]. Identification of new biomarkers of malignancy is fundamental to prevent end-organ

In this scenario, liquid biopsy could provide an alternative to invasive procedure and improve screening and early detection of cancer [4]. Liquid biopsy is defined as the search of biomarkers [5] in peripheral blood, and in recent years, exosomes have arisen as a powerful tool to understand cancer biology. Exosomes are vesicles with a size of 50–150 nm, which are secreted by cells into the extracellular space and play an important role in cell communication as cargoes of several specific proteins and RNAs. Exosomes are to date considered playing a pivotal role in information transfer in hematological malignancies [6] and recent studies show that specific exosomal microRNAs are involved in pathogenesis and have a prognostic role in MM [7]. In this chapter, we will analyze the biochemical and biological characteristics of MM and MGUS FLC and their cellular processing through exosomes. We show that free light chain (FLC)-decorated exosomes from patients carry peculiar bio-physical and biologi-

damage in selected patients who are at imminent risk of symptomatic progression.

cal qualities that allow the distinction of the malignant and premalignant condition.

In this section, we describe the potential pathogenic properties of serum FLC from MM and MGUS patients, correlating them with exosomal mediated cell-to-cell communication. About 80% of MM originates from intact immunoglobulin, non-IgM, MGUS and 20% from lightchain immunoglobulin MGUS [2]. Since 2009, the FLC quantitative assay (developed from both the Binding Site and Siemens) has been recommended by the International Myeloma Working Group to predict risk of progression in MGUS, the evaluation and management of MM [8]. This immunonephelometric assay allows the quantification of the kappa and lambda light chains untied to the heavy chains in serum [2, 9]. Calculation of the related kappa/lambda FLC ratio is an important parameter because about a third of patients with MGUS and more than 90% of patients with MM have altered FLC ratios that indicate excess production of a

can be attributed to the underlying plasma cell disorder.

**2. MM and MGUS FLC cellular processing**

clonal FLC by the proliferating plasma cell population [2].

FLCs are able to disrupt the normal physiology of different organs, such as heart, kidney, and lungs. They are occasionally responsible for severe organ damage [11], and these multiple effects are probably related to their primary sequence or posttranslational modification [12] that can alter their biochemical properties, giving them particular affinity for some tissues with respect to others or activating different turnovers inside the cells [10]. This is confirmed for FLC present in serum of patients with MM and MGUS conditions: experiment *in vitro* showed different FLC internalization rate in endothelial, myocardial, and epithelia cell lines for the MM and MGUS FLC. Considering that FLCs circulate in the blood stream and that they interact with the vascular bed to reach the target tissue, Human Vein Endothelial Cells (HVEC), Rat heart myoblast (H9C2), and Human epithelioid cervix carcinoma (HeLa) cell lines are good models for organs usually involved in FLC tissue damage, respectively, for the endothelial, cardiac, and epithelial compartment. In Di Noto et al. [10], cells were incubated with serum of MM and MGUS patients at constant FLC concentration and results showed a faster internalization rate of MM FLC (after 1 h up to 16 h) compared with MGUS FLC in HeLa, HVEC, and H9C2, respectively (**Figure 1**). This suggests that cells are able to uptake FLC from the extracellular environment and FLCs from MM and MGUS patients have different affinity to these cell types even though all of them can be internalized after 16 h of exposure. In contrast, serum from healthy patients, without altered FLC values and kappa/ lambda ratio did not show any similar properties probably because it did not contain FLC with propathological characteristics.

Considering these data, we could hypothesize that the interaction of FLC with peripheral districts depends on high-specific receptors expressed on certain cellular lines and/or that each patient has an individual clinical pattern due to the paraprotein molecular structure: thus, the differences between MM and MGUS can be related to peculiar FLC properties that can also alter their processing inside the cells [13].

It has been shown that FLC from AL amyloidosis patients induces oxidative stress and FLC internalization in cardiac fibroblasts enhances sulfatation of secreted glycosaminoglycans (GAGs) suggesting that the intracellular trafficking pathway of FLC could be correlated with the amyloidogenic potential of paraproteins [14]. Differences in FLC from MM and MGUS patients are not limited at the interaction with different cell type, but also their processing inside the cell is diverse: after intracellular internalization, they are rerouted in the extracellular melieu in different form, soluble or via pelletable extracellular vesicles (EVs).

HVEC and H9C2 cells were incubated for 4 h with serum from MM or MGUS patients containing a final FLC concentration of 20 μg/mL. After this initial step, cells were treated with trypsin, an enzyme able to cleave peptide chains, in order to eliminate FLC remained attached at the cell surface. Cells were, then, left in fresh medium (without FLC). After 16 h, the medium was collected

**Figure 1.** FLCs intracellular uptake. HeLa, HVEC, and H9C2 cell lines were incubated with serum of MM, MGUS at a final FLC concentration of 20 μg/mL and healthy donor (c). After incubation for 0, 1, 2, 6, and 16h cells were processed and analyzed by Western blot with anti-FLC antibody. Adapted from Di Noto et al. [10].

and submitted to a "three step centrifugation" protocol (800 × g 30 min, 16,000 × g 45 min, and 100,000 × g 2 h). This protocol allows the separation of different elements in biological fluids based on their size and density [15], exploiting the gravity force: the first centrifuge allowed to pellet cell debris and large vesicles like apoptotic bodies (P1), the second (P2) microvesicles budded from the plasma membrane (also called ectosomes, with diameters from 150 to 500 nm), while the ultracentrifugation step (P3) pellets smaller extracellular vesicles, like exosomes (50–150 nm) released through multivesicular bodies (MVBs) in the endosomal pathway [15]. FLCs from MM are internalized in cells after 4 h and subsequently released in the medium after 16 h (present in the P3), while MGUS FLCs are only present in cellular homogenate and in the third centrifuge superExploiting Exosomes for Differential Diagnosis of Multiple Myeloma and Monoclonal Gammop... http://dx.doi.org/10.5772/intechopen.69398 39

**Figure 2.** Extracellular rerouting of internalized FLC. HVEC cells were treated as described in the text. H: Homogenate, SN3: supernatant after 100,000 × g centrifugation, P3: pellet after 100,000 × g centrifugation. WB with antibodies anti-FLC. Adapted from Di Noto et al. [10].

natant (SN3). Cell incubated with healthy serum (c) did not give any signal as expected (**Figure 2**). Thus, the MM FLCs, after being internalized, are processed by the cells in a different way than MGUS and released outside the cell in vesicles that are pelleted at 100,000 × g, probably exosomes.

#### **2.1. EVs from cell biochemical characterization**

and submitted to a "three step centrifugation" protocol (800 × g 30 min, 16,000 × g 45 min, and 100,000 × g 2 h). This protocol allows the separation of different elements in biological fluids based on their size and density [15], exploiting the gravity force: the first centrifuge allowed to pellet cell debris and large vesicles like apoptotic bodies (P1), the second (P2) microvesicles budded from the plasma membrane (also called ectosomes, with diameters from 150 to 500 nm), while the ultracentrifugation step (P3) pellets smaller extracellular vesicles, like exosomes (50–150 nm) released through multivesicular bodies (MVBs) in the endosomal pathway [15]. FLCs from MM are internalized in cells after 4 h and subsequently released in the medium after 16 h (present in the P3), while MGUS FLCs are only present in cellular homogenate and in the third centrifuge super-

**Figure 1.** FLCs intracellular uptake. HeLa, HVEC, and H9C2 cell lines were incubated with serum of MM, MGUS at a final FLC concentration of 20 μg/mL and healthy donor (c). After incubation for 0, 1, 2, 6, and 16h cells were processed

WB anti-FLC

and analyzed by Western blot with anti-FLC antibody. Adapted from Di Noto et al. [10].

0 1h 2h 6h 16h

38 Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

HeLa

HVEC

H9C2

MGUS

c

MM

MGUS

c

MM

MGUS

c

MM

One of the criteria to characterize a population of vesicles and determine their nature is the immunoblot analysis or Western blot (WB) [16, 17]. According to the guidelines, it is important to show that at least three of the common exosomal markers are present in the sample. These protein lists are continuously updating and they include protein involved in exosomes formation (Alix, Hsp70, Annexin V, and Annexin XI), protein enriched in exosomes (tetraspanin CD63, CD81, CD9, and ADAM10) and from the endosomal compartment (TSG101, Syntenin-1, and VPS4B). WB analysis should also include negative controls: signal of protein is not enriched in exosomes (i.e., GM130 for the Cis-Golgi network, Calnexin for the endoplasmic reticulum) to verify the preparation purity from non-endosomal origin membrane. Especially for EVs deriving from cells, it is important to compare on the same gel identical amounts of protein from exosomes and from total lysates prepared from the producing cells to show an enrichment of the markers [16, 18]. In this scenario, Di Noto et al. [10] showed the presence of the exosomal markers Annexin V, Hsp70, Caveolin 1, Lamp 1, and Tubulin in P3 derived from HVEC and H9C2 culture medium, after MM FLC internalization (**Figure 3**). We observed a Caveolin 1 positive signal in P3 of MGUS treated cells loading four times the amount of MM treated cells. Thus, the P3 preparation from cells contains exosomes and according to the semiquantitative analysis, with different amount from cells exposed to MM or MGUS serum.

It is important to highlight that in this study, Di Noto et al. analyzed the presence of the c-src protein in the cell-derived exosomes. C-src is a tyrosin kinase linked with inflammatory environment and osteolytic bone disease in MM [19] and Di Noto et al. [10] could show that the kinase was present only in exosomes generated during malignant FLC processing, while non-malignant FLCs are unable to induce c-src exosomal recruitment. This is the first

HVEC

**Figure 3.** Biochemical characterization of cell-derived exosomes. HVEC and H9C2 cells were incubated with serum from MM, MGUS and healthy donor (c) patients for 4 h at 37°C. The serum was diluted to a final FLC concentration of 20 μg/ mL for all samples. Cells were then washed with PBS 1× and treated with trypsin as described in Di Noto et al. and left in fresh medium for 16 h. Medium was harvested, centrifuged at 800 × g for 30 min, 16,000 × g for 45 min and finally, ultracentrifuged at 100,000 × g for 2 h (P3). WB analysis of cell extracts (homogenate, H) and pellets (P3) with different exosome markers (C-src, Annexin V, Hsp 70, Caveolin 1, and Lamp 1). Adapted from Di Noto et al. [10].

evidence of biochemical diversity between exosomes from cells incubated with MM and MGUS serum.

### **3. EVs detection in MM and MGUS patients' serum**

#### **3.1. FLC content of serum-derived EVs**

showed the presence of the exosomal markers Annexin V, Hsp70, Caveolin 1, Lamp 1, and Tubulin in P3 derived from HVEC and H9C2 culture medium, after MM FLC internalization (**Figure 3**). We observed a Caveolin 1 positive signal in P3 of MGUS treated cells loading four times the amount of MM treated cells. Thus, the P3 preparation from cells contains exosomes and according to the semiquantitative analysis, with different amount from cells exposed to

40 Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

It is important to highlight that in this study, Di Noto et al. analyzed the presence of the c-src protein in the cell-derived exosomes. C-src is a tyrosin kinase linked with inflammatory environment and osteolytic bone disease in MM [19] and Di Noto et al. [10] could show that the kinase was present only in exosomes generated during malignant FLC processing, while non-malignant FLCs are unable to induce c-src exosomal recruitment. This is the first

HVEC

3 3 3 3 3 3

3 3 3 3 3 3

H9C2

**Figure 3.** Biochemical characterization of cell-derived exosomes. HVEC and H9C2 cells were incubated with serum from MM, MGUS and healthy donor (c) patients for 4 h at 37°C. The serum was diluted to a final FLC concentration of 20 μg/ mL for all samples. Cells were then washed with PBS 1× and treated with trypsin as described in Di Noto et al. and left in fresh medium for 16 h. Medium was harvested, centrifuged at 800 × g for 30 min, 16,000 × g for 45 min and finally, ultracentrifuged at 100,000 × g for 2 h (P3). WB analysis of cell extracts (homogenate, H) and pellets (P3) with different

exosome markers (C-src, Annexin V, Hsp 70, Caveolin 1, and Lamp 1). Adapted from Di Noto et al. [10].

MM or MGUS serum.

MM

MGUS

c

MM

MGUS

c

One of the main disadvantages of experiments using immortalized cell lines is that it can be challenging to extrapolate from the results of *in vitro* work back to the biology of the intact organism [20].

For this reason, it was important to confirm the results obtained after studying the FLC processing in human cell lines and verify the presence of the EVs also *in vivo*, analyzing directly patients' serum. After the three step centrifugation protocol on serum, EVs were found in samples from MM and MGUS patients, and MM vesicles show a higher amount of FLC in the P3 fraction than MGUS and healthy patients, estimated around 2% of all the FLC present in serum [10]. These data are consistent with a previous study on urinary exosomes from AL amyloidosis, MM, and MGUS patients [21]. The presence of small amount of FLC in P3 of MGUS serum can be explained with the high variability of EVs production *in vivo* respect of the more restricted type of EVs deriving from a cell culture. Nevertheless, MGUS EVs present different characteristics with respect to MM EVs as explained further in the chapter.

#### **3.2. Serum EVs biochemical characterization**

Similar to exosomes from cells, EV preparations from serum must be validated by WB analysis for exosomal markers: P3 from the serum of MM and MGUS patients were blotted for Hsp70, Annexin V, and Tubulin giving positive signals. It is also to note that in the serum, the MGUS exosomes do not contain c-src, confirming that this protein is a marker of malignancy in exosomes from MM patients (**Figure 4A**) [10, 22].

#### *3.2.1. Discontinuous sucrose gradient*

Exosomes can be distinct from other type of vesicles for their density, ranging from 1.077 to 1.19 g/mL [6, 16]. Exosomes from MM serum loaded on a discontinuous sucrose gradient from 15 to 60% can be detected in four fractions (from 6 to 9) with density from 1.084 to 1.18 g/mL, according to the markers Hsp70, Annexin V, CD63, TSG101, and CD81 (**Figure 4B**) [6, 10, 22, 23]. FLC and c-src are detected in the same fractions, confirming their association with MM exosomes [10].

In our lab experience and according to guidelines [16], it is best to perform this separation with samples containing high amount of exosomes, for example serum with respect to cellular medium. Usually, exosomes spread in 4–5 gradient fractions and this dilution can decrease

Density g/mL 1.05 1.08 1.11 1.14 1.18 1.22 1.31 1.39

**Figure 4.** Serum exosome biochemical characterization. (A) P3 obtained from healthy donors (c), MGUS and MM patients were analyzed by WB with different exosome markers. (B) P3 from MM patient was loaded on top of a 15–60% discontinuous sucrose gradient. Twelve fractions of equal volume were collected and analyzed by WB using different exosomal markers and anti-FLC antibodies. Exosomal markers and FLC signals are visible in the same fractions. (C and D) P3 from healthy donors (c), MGUS and MM serum were loaded in a discontinuous sucrose gradient. Fractions from 6 to 9 were collected and incubated with magnetic beads coupled with anti c-src (C) or Annexin V (D) antibodies. Only exosomes deriving from MM serum are both c-src and Annexin V positive. Adapted from Di Noto et al. [10, 22].

the exosomes protein concentration under the limit of detection with available substrate, usually femtomolar, used for the chemiluminescent reaction in WB.

#### *3.2.2. C-src and annexin V MM immunocapture*

The immunocapture assay performed with magnetic beads coupled with antibodies anti-c-src (**Figure 4C**) confirmed the ability to precipitate only exosomes (Annexin V positive) from MM serum-containing FLC. On the other hand, anti-Annexin V beads captured MGUS and MM exosomes, but only MM exosomes were positive for c-src signal.

#### *3.2.3. Exosome lipid composition*

The lipid composition of the two exosomal populations was analyzed by a thin-layer chromatography showing that both MM and MGUS exosomes are composed by phosphatidylcholine and sphingomyelin, two well-known types of lipids that compose exosome membranes [15]. It is to note that to reveal the P3 MGUS lipids content, we loaded twice the protein concentration than MM P3 (see also Section 3.4).

#### *3.2.4. Flow cytometry characterization*

MM and MGUS exosomes were incubated with FACS magnetic beads coupled to CD63 antibody and stained with a commercial membrane labeler (PKH26). Fluorescent signal was analyzed by flow cytometry revealing the presence of exosomes with the same profile of CD63 expression level [22].

#### **3.3. Serum exosomes morphological analysis**

According to the guidelines, in addition to the biochemical characterization described above, to claim the presence of EVs, in particular exosomes, in a preparation, a "single vesicles characterization" is recommended to provide indication of the heterogeneity of the sample [16, 18].

MM and MGUS exosomes preparation both from P3 and gradient fractions were analyzed by atomic force microscopy (AFM), scanning electron microscopy (SEM), and scanning helium ion microscopy (HIM). AFM allows the analysis of vesicles that tend to remain in their original 3D shape. SEM and HIM have a better resolution, but the samples are manipulated. They need to be fixed and dehydrated and these steps can create aggregates and artifacts. In our case, however, all of these techniques allowed to visualize vesicles ranging from 50 to 300 nm according to the heterogeneity of the sample: P3 contains exosomes and bigger vesicles, while gradient fractions are more monodispersed with a size range between 50 and 150 nm (**Figure 5**).

#### **3.4. MM and MGUS exosome quantification**

the exosomes protein concentration under the limit of detection with available substrate, usu-

Hsp70 C-src CD63

D

Immunocapture anti C-src c MGUS MM

Annexin V

FLC

C-src

FLC

c MGUS MM

Immunocapture anti Annexin V

Annexin V

**Figure 4.** Serum exosome biochemical characterization. (A) P3 obtained from healthy donors (c), MGUS and MM patients were analyzed by WB with different exosome markers. (B) P3 from MM patient was loaded on top of a 15–60% discontinuous sucrose gradient. Twelve fractions of equal volume were collected and analyzed by WB using different exosomal markers and anti-FLC antibodies. Exosomal markers and FLC signals are visible in the same fractions. (C and D) P3 from healthy donors (c), MGUS and MM serum were loaded in a discontinuous sucrose gradient. Fractions from 6 to 9 were collected and incubated with magnetic beads coupled with anti c-src (C) or Annexin V (D) antibodies. Only exosomes deriving from MM serum are both c-src and Annexin V positive. Adapted from Di Noto et al. [10, 22].

Tsg101

FLC

The immunocapture assay performed with magnetic beads coupled with antibodies anti-c-src (**Figure 4C**) confirmed the ability to precipitate only exosomes (Annexin V positive) from MM serum-containing FLC. On the other hand, anti-Annexin V beads captured MGUS and MM

The lipid composition of the two exosomal populations was analyzed by a thin-layer chromatography showing that both MM and MGUS exosomes are composed by phosphatidylcholine and sphingomyelin, two well-known types of lipids that compose exosome membranes [15]. It is to note that to reveal the P3 MGUS lipids content, we loaded twice the protein concentra-

ally femtomolar, used for the chemiluminescent reaction in WB.

c MGUS MM C

42 Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

Hsp70 C-src Tubulin

Annexin V

FLC

5 6 7 8 9 10 11 12

exosomes, but only MM exosomes were positive for c-src signal.

*3.2.2. C-src and annexin V MM immunocapture*

Density g/mL 1.05 1.08 1.11 1.14 1.18 1.22 1.31 1.39

*3.2.3. Exosome lipid composition*

MM

B

A

tion than MM P3 (see also Section 3.4).

Exosome quantification in a sample is one of the most discussed issues among experts in the field. How is it possible to quantify accurately something with the heterogeneous composition (lipids, protein, and nucleic acids) among a mixture of similar elements (protein aggregated from serum, circulating RNA, and vesicles with similar size, but different origin)?

Bradford assay was used to determine the amount of the total exosomes protein in the samples, and with this method, P3 from MM serum had almost double protein concentration than P3 from MGUS and healthy patients.

Further on, it has been discovered that exosome preparation protein content can be influenced by many elements, i.e. fetal bovine serum in culture medium, single, and aggregated proteins. One of the best solutions is to purify the sample as much as possible from contaminants before every type of measurements, i.e. with a sucrose gradient. Even though what is Bradford assay measuring: protein outside or inside exosomes, or both?

Thus, we decide to measure the activity of an enzyme known to be enriched within exosomes, acetylcholinesterase [24], in a P3 preparation from healthy, MGUS, and MM serum. This assay confirmed that MM P3 preparation contains more exosomes than MGUS and controls, but how much is it specific?

Our lab contributed to solve this set of problems with a nanotechnological approach. We developed a cost-effective and fast colorimetric assay for probing protein contaminants and determining the concentration of EV preparations [25].

**Figure 5.** Serum exosome morphological analysis. P3 from MM serum were analyzed by AFM, HIM, and SEM in order to visualize vesicle populations. MM and MGUS sucrose gradient fractions from 6 to 9 were examined by AFM (topography and phase mode) and HIM (only MM sample). MM scale bars are 300 nm for AFM pictures, 500 nm for HIM pictures, and 100 nm for SEM picture. MGUS scale bars are 700 nm for AFM pictures. Adapted from Paolini et al. [6] and Di Noto et al. [10].

0.00

The assay exploits colloidal gold nanoplasmonics and the fact that nanoparticle (NP) aggregation at lipid membranes is modulated by the presence of a protein corona around the NPs. When a pure exosome preparation is incubated with a gold NP solution, the NPs cluster at the exosome membrane. Clustering is associated with NP LSPR red-shift, which is proportional to the exosome molar concentration and using a calibration line made of phosphatidylcholine liposome, can therefore be exploited for titrating the solution. If the preparation is not pure from single or aggregated proteins, NPs tend to interact with these elements and do not aggregate on exosome surface. Thus, to obtain a precise exosome quantification, as described, sample must be as pure as possible.

This assay, moreover, can determine the sample purity from protein contaminants with a limit of detection of 0.005 μg/μL.

Using this assay, we probed MM and MGUS exosomes collected from the sucrose gradient fractions from 6 to 9, and we could state that exosome preparations were pure from protein contaminants. In this way, our assay could titrate the exosome concentration in each sample and results showed a four-fold increase of exosomes in MM preparation in comparison with MGUS and healthy control (**Figure 6**) [23]. These data confirmed the previous analyses of exosomes from serum and it corresponds to the production of exosomes in the cells after MM and MGUS serum exposure.

Exploiting Exosomes for Differential Diagnosis of Multiple Myeloma and Monoclonal Gammop... http://dx.doi.org/10.5772/intechopen.69398 45

**Figure 6.** MM and MGUS exosome serum quantification. Exosomes were titrated using the colorimetric nanoplasmonic assay as described in Maiolo et al. [25]. Each column represents the mean value of exosomes isolated from serum of different patients (10 healthy donors (c), 5 MGUS and 10 MM) +/− Standard deviation.  Adapted from Di Noto et al. [23].

#### **4. MM and MGUS exosome biological effects**

The assay exploits colloidal gold nanoplasmonics and the fact that nanoparticle (NP) aggregation at lipid membranes is modulated by the presence of a protein corona around the NPs. When a pure exosome preparation is incubated with a gold NP solution, the NPs cluster at the exosome membrane. Clustering is associated with NP LSPR red-shift, which is proportional to the exosome molar concentration and using a calibration line made of phosphatidylcholine liposome, can therefore be exploited for titrating the solution. If the preparation is not pure from single or aggregated proteins, NPs tend to interact with these elements and do not aggregate on exosome surface. Thus, to obtain a precise exosome quantification, as described,

**Figure 5.** Serum exosome morphological analysis. P3 from MM serum were analyzed by AFM, HIM, and SEM in order to visualize vesicle populations. MM and MGUS sucrose gradient fractions from 6 to 9 were examined by AFM (topography and phase mode) and HIM (only MM sample). MM scale bars are 300 nm for AFM pictures, 500 nm for HIM pictures, and 100 nm for SEM picture. MGUS scale bars are 700 nm for AFM pictures. Adapted from Paolini et al.

AFM HIM

44 Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

topography phase

SEM

P3

Gradient fractions

Gradient fractions

This assay, moreover, can determine the sample purity from protein contaminants with a

Using this assay, we probed MM and MGUS exosomes collected from the sucrose gradient fractions from 6 to 9, and we could state that exosome preparations were pure from protein contaminants. In this way, our assay could titrate the exosome concentration in each sample and results showed a four-fold increase of exosomes in MM preparation in comparison with MGUS and healthy control (**Figure 6**) [23]. These data confirmed the previous analyses of exosomes from serum and it corresponds to the production of exosomes in the cells after MM

sample must be as pure as possible.

21.55

21.55 nm

MGUS

MM

MM

0.00

0 nm

[6] and Di Noto et al. [10].

limit of detection of 0.005 μg/μL.

and MGUS serum exposure.

Exosome role in MM is started to be unveiled in the past few years. MM-derived EVs have been demonstrated to have a biological effect on other cell types, such as to induce phenotypical changes in osteoclasts, influence bone marrow microenvironment, and promote the production of proangiogenic factors, like IL-6 and VEFG, in endothelial cells [26].

Interestingly, MM and MGUS serum-derived exosomes showed different biological effects on endothelial and myocardial cell lines: MM exosomes have a higher internalization rate than MGUS and treatment with MM exosomes was able to induce a significantly higher proliferation rate compared with MGUS in both cell lines.

This effect is dependent on active exosomes endocytosis in cells, mediated by the presence of specific pathogenic FLC on the surface of MM exosome as demonstrated with immunofluorescence and cytofluorometry analyses (**Figure 7A and B**). In fact, MM exosomes incubated with antibodies anti-FLC, which mask the interaction with cells, decrease their internalization rate and the induced cell proliferation in both cell lines (**Figure 7C**). Furthermore, MM exosomes docking and processing by cells is influenced by the presence of heparan sulfate proteoglycans (HSPGs) on the cell surface. Incubating MM exosome with heparin, a structural analog of HSPGs, saccharide chains, decreased MM exosome uptake in HVEC cells. MM exosome/heparin interaction is being confirmed in dose-response experiments at surface plasmon resonance (SPR) spectroscopy. These data revealed a probably cooperative binding mechanism of FLC and HSPGs even if exosomes, which are characterized by different FLC decorations, have distinct binding affinities for cell-associated HSPGs and this is reflected in different cellular uptake [22, 23].

**Figure 7.** MM exosome biological effect is FLC and HSPG mediated. (A) Fluorescent microscopy and flow cytometry analyses of HVEC cells incubated with MGUS or MM PKH67-labeled exosomes. To demonstrate the FLC and HSPGs involvement in MM exosome uptake, MM vesicles were incubated with anti-FLC antibody (MM+ anti FLC) or heparin before cells incubation. Scale bars 5 μm. (B) PKH67 fluorescence intensity measurement of internalized exosomes from MGUS and MM serum. MM exosomes uptake in HVEC cells decrease after anti-FLC antibody (MM+ anti FLC) or heparin treatments. (C) Proliferation induction by MM exosomes is decreased after incubation with anti-FLC antibody both in HVEC and H9C2 cell lines. Adapted from Di Noto et al. [22].

It has been demonstrated that MM EVs are related to proinflammatory environment in cells. Immunofluorescence and biochemical separation techniques demonstrated that cells treated with exosomes from MM serum show a c-src intracellular redistribution at the plasma membrane with respect to MGUS exosome treatment. These data are consistent with cellular *ex novo* secretion of c-src positive exosomes after MM serum exposure and this phenomenon is reduced after cellular uptake inhibition, incubating MM exosomes with anti-FLC antibodies and heparin.

Similarly, the activation and nuclear translocation of the transcription factor Nf-kB, involved in the cellular responses after stress, infection, and inflammatory stimuli, is not visible in the cell treated with MGUS exosomes. MM exosome's induced effect is decreased after treatment with anti-FLC antibody and heparin (**Figure 8**).

It is to note that the Nf-kB translocation is dependent on the preparation purity: the biological activity of exosome preparations from MM patient serum is influenced by residual contaminants, which may escape the purification procedure. These contaminants probably can interfere with the exosomes-cell membrane interaction or inhibit the Nf-kB translocation. They can be Exploiting Exosomes for Differential Diagnosis of Multiple Myeloma and Monoclonal Gammop... http://dx.doi.org/10.5772/intechopen.69398 47

**Figure 8.** Nf-kB nuclear translocation is MM exosomes induced. (A) HVEC immunofluorescence imaging of Nf-kB nuclear translocation after incubation with MGUS, MM exosomes, or MM exosomes incubated with anti-FLC antibody (MM + anti FLC) or heparin (MM + heparin). Cell were fixed and incubated with DAPI for nuclear staining and anti-Nf-kB antibody as described in Di Noto et al. [22]. Scale bars 5 μm. (B) Quantification of nuclear Nf-kB fluorescence intensity in HVEC cells after MGUS, MM exosomes, MM exosomes incubated with anti-FLC antibody (MM + anti FLC) or with heparin (MM + heparin) treatment. (C) WB analysis of cell extracts (H) and nuclear compartment (N) was performed with anti Nf-kB antibody and nuclear markers (Lamin A/C, Histone H3) after different incubations. Adapted from Di Noto et al. [22].

separated from exosomes after a discontinuous gradient, but they cannot be detected with conventional techniques (i.e. WB) and need a combination of proper bioanalytical methods and nanoscale characterization [6].

#### **5. Conclusion**

It has been demonstrated that MM EVs are related to proinflammatory environment in cells. Immunofluorescence and biochemical separation techniques demonstrated that cells treated with exosomes from MM serum show a c-src intracellular redistribution at the plasma membrane with respect to MGUS exosome treatment. These data are consistent with cellular *ex novo* secretion of c-src positive exosomes after MM serum exposure and this phenomenon is reduced after cellular uptake inhibition, incubating MM exosomes with anti-FLC antibodies and heparin. Similarly, the activation and nuclear translocation of the transcription factor Nf-kB, involved in the cellular responses after stress, infection, and inflammatory stimuli, is not visible in the cell treated with MGUS exosomes. MM exosome's induced effect is decreased after treatment

**Figure 7.** MM exosome biological effect is FLC and HSPG mediated. (A) Fluorescent microscopy and flow cytometry analyses of HVEC cells incubated with MGUS or MM PKH67-labeled exosomes. To demonstrate the FLC and HSPGs involvement in MM exosome uptake, MM vesicles were incubated with anti-FLC antibody (MM+ anti FLC) or heparin before cells incubation. Scale bars 5 μm. (B) PKH67 fluorescence intensity measurement of internalized exosomes from MGUS and MM serum. MM exosomes uptake in HVEC cells decrease after anti-FLC antibody (MM+ anti FLC) or heparin treatments. (C) Proliferation induction by MM exosomes is decreased after incubation with anti-FLC antibody

C

Grow factor HVEC H9C2

MM MM MM MM + anti FLC

+ anti FLC

 MGUS MM MM MM + anti FLC

+heparin

PKH67 fluorescence

It is to note that the Nf-kB translocation is dependent on the preparation purity: the biological activity of exosome preparations from MM patient serum is influenced by residual contaminants, which may escape the purification procedure. These contaminants probably can interfere with the exosomes-cell membrane interaction or inhibit the Nf-kB translocation. They can be

with anti-FLC antibody and heparin (**Figure 8**).

both in HVEC and H9C2 cell lines. Adapted from Di Noto et al. [22].

A B

46 Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

PKH67

MGUS

MM

MM + anti FLC

MM + heparin

> In this chapter, we have shown that MM and MGUS patients can be distinguished at the nanoscale level. Although their FLC serum content may be similar, FLC biochemical characteristics are very different: they are internalized and processed in cells in distinct ways and only pathogenic FLCs are rerouted in the extracellular compartment in c-src positive exosomes. It is important to note that exosome presenting MM FLC activates a proinflammatory process: the presence of circulating c-src positive exosomes has been demonstrated only in serum of MM patients, electing this kinase as a new marker of malignancy for the differential diagnosis between MGUS and MM. Similarly only MM exosomes have the ability to induce Nf-kB translocation in cells. These parameters could be implemented with the exosome concentration in serum, binding affinity with heparin and ability to induce proliferation in HVEC and H9C2 cell lines to create an innovative multiparameter panels to monitor MGUS to MM switching. We are aware that these data need to be confirmed in a larger cohort of patients and

we still do not know how precocious these phenomena are with respect to the MGUS to MM switch using of the actual diagnostic criteria. Recently, new biomarkers are emerging to help clinicians to distinguish MM from the premalignant phase. According to recent guidelines, extreme bone marrow clonal plasmacytosis (>60%), marked elevation of serum FLC ratio of 100 or higher (provided involved free light-chain level ≥100 mg/L) and/or presence of more than one focal lesion on whole-body magnetic resonance imaging (MRI) can defined as MM even in the absence of CRAB features [2, 3].

In the future, monitoring MGUS and MM exosome parameters could support, as a liquid biopsy, the classical classification methods and help in early diagnosis to prevent the development of end-organ damage for patients who are at the highest risk [2, 3].

#### **Acknowledgements**

We thank all the colleagues that contribute to this work: Annalisa Radeghieri, Sara Busatto, Daniele Maiolo, Debora Berti, Elisa Polini, Elisabetta Lottini, Marco Chiarini, Elena Laura Mazzoldi, Viviana Giustini, Alessandra Dossi, Antonella Bugatti, Alesssandro Montanelli, Marco Rusnati, Andrea Caneschi, and Luigi Caimi. This work was supported by the University of Brescia Research through the found "ex 60%" and by MIUR (Ministero Italiano dell'Università e della Ricerca). Fondazione Cariplo and Regione Lombardia supported the publication through the found "New Opportunities and ways towards ERC", grant 2014– 2256. The authors declare no conflict of interests.

The articles [6, 10] are licensed under the Creative Common Public License. For public license and disclaimer of warranties refer to this link https://creativecommons.org/licenses/by/4.0/ legalcode.

#### **Author details**

Lucia Paolini\*, Giuseppe Di Noto, Andrea Zendrini, Paolo Bergese and Doris Ricotta

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy

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Terpos E, Kyle RA, Anderson KC, Durie BG, Miguel JF. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncology. 2014;**15**(12):e538–e548. DOI: 10.1016/S1470-2045(14)70442-5

we still do not know how precocious these phenomena are with respect to the MGUS to MM switch using of the actual diagnostic criteria. Recently, new biomarkers are emerging to help clinicians to distinguish MM from the premalignant phase. According to recent guidelines, extreme bone marrow clonal plasmacytosis (>60%), marked elevation of serum FLC ratio of 100 or higher (provided involved free light-chain level ≥100 mg/L) and/or presence of more than one focal lesion on whole-body magnetic resonance imaging (MRI) can defined as MM

In the future, monitoring MGUS and MM exosome parameters could support, as a liquid biopsy, the classical classification methods and help in early diagnosis to prevent the develop-

We thank all the colleagues that contribute to this work: Annalisa Radeghieri, Sara Busatto, Daniele Maiolo, Debora Berti, Elisa Polini, Elisabetta Lottini, Marco Chiarini, Elena Laura Mazzoldi, Viviana Giustini, Alessandra Dossi, Antonella Bugatti, Alesssandro Montanelli, Marco Rusnati, Andrea Caneschi, and Luigi Caimi. This work was supported by the University of Brescia Research through the found "ex 60%" and by MIUR (Ministero Italiano dell'Università e della Ricerca). Fondazione Cariplo and Regione Lombardia supported the publication through the found "New Opportunities and ways towards ERC", grant 2014–

The articles [6, 10] are licensed under the Creative Common Public License. For public license and disclaimer of warranties refer to this link https://creativecommons.org/licenses/by/4.0/

Lucia Paolini\*, Giuseppe Di Noto, Andrea Zendrini, Paolo Bergese and Doris Ricotta

Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy

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