**6. Extracellular vesicles and cancer metastasis**

Extracellular vesicles (EVs) are nanoparticles released by different types of cells that contain a lipid bilayer structure [38, 39]. There are three major types of EVs: namely exosomes, apoptotic bodies and microvesicles [39, 40]. However, other types such as oncosomes, cytoplasts and exomeres have also been identified. These subtypes are characterised based on their sizes, biogenesis, origin (tumour-derived, stromal cellderived etc), functions (immune-suppressing/stimulating-EVs, pro-apoptotic EVs etc)

### *Stromal Cells and Extracellular Vesicles DOI: http://dx.doi.org/10.5772/intechopen.106721*

and surface markers (CD63+ , CD9+ , CD81+ , or EpCAM+ EVs) [38, 41]. Despite this heterogeneous population of EVs, each EV is unique thus the dynamic function of EVs is due to their highly heterogeneous characteristics, which makes it difficult to accurately differentiate these EV subtypes [42].

Apoptotic bodies are the largest EV in size and are produced by dying cells [38]. These EVs contain many intercellular materials such as intracellular fragments, cellular organelles, and cytosolic contents [38, 40]. Microvesicles are the second largest EV in size and originate from the outward budding or fusion of the cytoplasm membrane and are later released into the extracellular space [43]. They majorly contain lipids such as sphingolipids, cholesterol, and phosphatidylserine [40, 44]. Both apoptotic bodies and microvesicles are sometimes collectively called ectosomes and often originate via direct outward budding or blebbing of the plasma membrane [40]. Lastly, exosomes are bilayered membrane small extracellular vesicles of 40–200 nm size that are derived from the fusion of multivesicular bodies (MVB) into the plasma membrane and resulting release of intraluminal vesicles (ILVs) into the extracellular space through exocytosis [45, 46]. Therefore, any factor that may affect the plasma membrane may positively or negatively influence formation of these EVs.

These membrane-bound organelles function as important mediators of intercellular communication mechanism and often harbour bioactive molecules such as metabolites, proteins, RNA, DNA, and lipids that often reflect the parent cell [39, 47]. The lipid membrane of these EVs serves a protective shield for enclosed nucleic acids thereby protecting them from degradation by extravesicular nucleases [48]. Much of the RNA composition are from miRNAs, a class of non-coding RNAs that mediate post-transcriptional gene silencing in many biological processes [47, 49]. Once released, these vesicles are taken up by recipient cells and could influence the pathological and physiological functions in the recipient cells by activating different signalling pathways [49–52]. These EVs deliver genetic information to recipient cells, which affect signalling transduction pathways thereby regulating target gene expression and determining the function and fate of recipient cells such as apoptosis, growth, cell cycle, migration, and differentiation [49, 53, 54]. Internalisation of these vesicles into the recipient cells occur by endocytic process via phagocytosis, fusion with the cell membrane and interaction with receptors on the cell membrane [48, 55].

During tumorigenesis, the bidirectional cell-to-cell communication between tumour and healthy cells within the TME is one of the mechanisms that enable cancer progression and metastasis, and EVs mediate this intercellular communication [56]. EVs released by cancer cells are increasingly found circulating in body fluids such as blood, urine, saliva, ascitic fluid and milk whereby they enhance the proliferation and invasion of tumour cells in autocrine and paracrine manner [44, 55]. The hypoxic or metastatic status of the tumours plays an important role in sorting the loading of composition of EVs, which affects the functions of tumour-derived EVs in the TME [24, 56]. EVs shuttle regulatory molecules, including lipids, nucleic acids and proteins that induce the reprogramming and remodelling of the stroma by facilitating the development of a tumour-supportive environment [39, 47, 57, 58]. These tumourderived EVs within the hypoxic microenvironment also drive Warburg effect thereby driving conversion of glucose mainly into lactate to meet energy requirements to ensure tumour survival [57, 58]. They also regulate the metabolism of lipids and amino acids by cancer cells to build biomass and provide more energy.

This leads to immunogenic stress thereby initiating immune changes within the TME and influencing cancer progression. Tumour-derived EVs inhibit immune response, promote the transformation of CAFs, and reprogram endothelial cells

function thus creating an anti-tumoral environment. Tumour-derived EVs interact with the host immune system and cause functional and phenotypic changes in immune cells such as natural killer (NK) cells, macrophages, T-cells, and B-cells thereby affecting the immune system homeostasis [57, 59]. EVs released by tumour cells also induce immunosuppressive or tumour-associated macrophages by NF-κB mediated metabolism and secretion of VEGF, IL-6, TNF-α and G-CSF thereby leading to cancer metastasis [60, 61]. Tumour-derived EVs also increase neutrophil mobilisation and activate regulatory T-cells that protects the tumour from CD8<sup>+</sup> T-cell mediated killing [59, 62, 63]. In addition, tumour-derived EVs activate or suppress NK cells depending on the type of tumour and express FasL and TRAIL on their membrane thereby directly influencing the apoptosis of CD8<sup>+</sup> cells [59, 62, 63]. However, tumour-derived EVs can also activate dendritic cells via delivery of tumour-derived antigens and stimulate a CD8<sup>+</sup> -mediated antitumour response.

Tumour-derived EVs also regulate the pro-tumoral function of endothelial cells by sustaining the constant delivery of nutrients and oxygen from the vascular endothelium [58, 60, 64]. Under hypoxic conditions, tumour-derived EVs also promote the regulation of endothelial cell proliferation, migration, sprouting, branching, as well as tubular-like structure formation via delivery of miRNAs, mRNAs, and proteins hence tumour-derived EVs promote angiogenesis in different types of cancer, including hepatocellular carcinoma, colorectal cancer, cervical cancer, nasopharyngeal carcinoma, glioma, and lung cancer. Neoangiogenesis, secretion of growth factors and EVs, and inflammatory cells recruitment induce the formation of pre-metastatic niches, where new tumour cells extravasate, get arrested or colonise [43, 57, 58, 61]. This further ensures tumour metastasis. In addition to pre-metastatic niches, EVs are also involved in other processes of tumour metastasis such as EMT and organ-specific metastasis.

Under hypoxic conditions, tumour-derived EVs stimulate the transition of stromal cells into CAFs via TGF-β, which in turn increase shedding of EVs and induce ECM remodelling, angiogenesis, migration, and invasion of cancer cells via different signalling pathways [58, 60, 64]. Tumour-derived EVs enhance the ability of CAFs in response to metabolic environment by activating MYC signalling pathway in stromal cells resulting in rapid tumour growth. These EVs-bound factors modify the phenotype of cancer cells or tumour stromal cells to support the aggressive phenotype and tumour progression. CAFs regulate tumour microenvironment and transfer proteins, metabolites such as tricarboxylic acid (TCA) intermediates and lipids utilised by cancer cells via EVs to facilitate and promote tumour growth under nutrient deprivation conditions [15, 31, 59, 65]. CAFs-derived EVs also enhance EMT via release of factors such as fibronectin and vimentin that trigger the loss of tumour cell adhesion, as well as differentiation of osteoblasts and proliferation of osteoclasts, which regulate the microenvironment of bone metastasis.
