The Role of Cancer-Associated Fibroblasts and Extracellular Vesicles in Tumorigenesis

Extracellular vesicles (EVs) play a key role in the communication between cancer cells and stromal components of the tumor microenvironment (TME). In this context, cancer cell-derived EVs can regulate the activation of a CAF phenotype in TME cells, which can be mediated by several EV cargos (e.g., miRNA, proteins, mRNA and lncRNAs). On the other hand, CAF-derived EVs can mediate several processes during tumorigenesis, including tumor growth, invasion, metastasis, and therapy resistance. This review aimed to discuss the molecular aspects of EV-based cross-talk between CAFs and cancer cells during tumorigenesis, in addition to assessing the roles of EV cargo in therapy resistance and pre-metastatic niche formation.


Introduction
Tumorigenesis is a multi-step process dependent on several modifications at cellular and tissue levels. These modifications lead to sustained proliferative signaling, evasion from growth suppressors and cell death, replicative immortality, and induction of angiogenesis, invasion, and metastasis [1,2]. Malignant tumors are characterized by high cellular heterogeneity, including cancerous and non-cancerous cells, and non-cellular components, composing the tumor microenvironment (TME) [3]. Malignant cells can recruit stromal cells to remodel tissue structure and to secrete growth-promoting stimuli and intermediate metabolites. As a result, stromal components (e.g., fibroblasts, endothelial cells, and pericytes) contribute to multiple processes of tumorigenesis, which include tumor growth, invasion, metastasis, and therapy resistance [3,4].
Cancer-associated fibroblasts (CAFs) are recognized as one of the most abundant stromal cells in the TME. Under the influence of parenchymal cells, stromal cells (including resident fibroblasts, mesenchymal stem cells, and adipocytes) can undergo activation into a CAF-phenotype [5]. CAFs are responsible for remodeling the extracellular matrix (ECM) during tumor progression and metastasis, where they actively participate in proteolysis, crosslinking, and assembly of the ECM, which facilitate malignant cell migration and invasion [6]. Moreover, CAFs also interact with other cells in the TME through direct cell-to-cell contact [7] and paracrine signaling [8].
Extracellular vesicles (EVs) are membrane-enclosed vesicles secreted by most cellular types. These particles play an essential role in the cross-talk between malignant cells and resident cells of the TME [9]. EVs derived from malignant cells can influence the activation of stromal cells into CAFs due to the uptake of EV contents by target cells, including proteins [10] and microRNAs (miRNAs) [11].
While outside of the main scope of the present review, these interactions between CAF and the immune system and inflammation have been previously reviewed [22,23].
Direct interactions between CAFs and cancer cells via soluble factors and EV-mediated paracrine signaling or cell-to-cell communications are vastly explored in literature. Studies show that CAFs can influence processes involved in virtually all major hallmarks of cancer [30,49,59]. The effects of soluble factors' signaling and cell-cell communications in the cross-talk between cancer cells and CAFs were previously reviewed by other researchers [5,60,61], and therefore, will not be further discussed. Interactions between cancer cells and CAFs mediated by EV cargo are detailed Sections 4 and 5 of this review.

Extracellular Vesicles (EVs)
EVs are a class of phospholipid-bilayer-enclosed membranes carrying a variety of biological molecules, including nucleic acids, proteins, and lipids. EVs are released by virtually all cell types in an evolutionarily conserved manner across eukaryotes and prokaryotes [62,63]. EVs can act on local and distant sites and can be found circulating in a wide range of biological fluids, including blood, urine, bile, nasal secretions, and saliva [64]. EVs can be broadly assigned to three main categories based on origin: exosomes, microvesicles (MVs), and apoptotic bodies.
Exosomes (30-150 nm) are formed through endosomal trafficking, beginning as late endosomes that mature into multivesicular bodies (MVBs). Intervesicular bodies (ILVs) are formed through the inward budding of the limiting membrane of MVBs [62,65]. The most well-characterized pathway for ILV biogenesis is through an endosomal sorting complex required for transport (ESCRT) complex-dependent mechanism. ESCRT (-0, -I, -II) recognizes and sequesters ubiquitinated proteins on the endosomal membrane, followed by inward budding and scission by ESCRT-III [63,66]. Cargo clustering may also occur through syntenin along with the ESCRT accessory protein ALG-2-interacting protein X (ALIX), with only ESCRT-III being required for ILV biogenesis [67,68]. ILV formation has also been shown to occur through ESRT-independent mechanisms via tetraspanins (CD63) [62,69], and a lipid mechanism involving ceramides made by sphingomyelinases (nSMases) [62,70]. MVBs may be trafficked to the lysosome for degradation, or they may be trafficked by guanosine triphosphatases (GTPases) Rab27a and Rab27b towards the plasma membrane for docking and fusion [62]. After the MVB fuses with the plasma membrane, its ILVs will be released into the extracellular space as exosomes [63] (Figure 1).
Once released into the extracellular milieu, EV-mediated communication may act in an autocrine or paracrine manner. Once at the recipient cells, EVs may remain at the plasma membrane, fuse with the plasma membrane, or be internalized by the target cell. Endocytosis of EVs may be clathrin-mediated or clathrin-independent (macropinocytosis or phagocytosis), or facilitated via caveolae or lipid rafts [62]. Intracellular communication via EVs occurs during homeostasis and under pathological conditions, including cancer. The cargo carried by EVs is selectively rather than indiscriminately packaged. This suggests a biological role for the contents that are packaged, since selection can be driven to create conditions that either promote or inhibit malignancy [73].
There are several techniques employed currently to isolate EVs. Differential ultracentrifugation (DU) is widely used as a low-cost, high-throughput method to isolate EVs from large sample sizes. DU is often combined with other purification techniques such as ultrafiltration or density gradient centrifugation to yield increased particle purity. Size-exclusion chromatography yields EV pellets of high purity but dilutes samples, which need to then be re-concentrated. Immuno-affinity and microfluidics are techniques based on EV characteristics such as surface markers (e.g., CD63) but there is currently no marker that can accurately discern between various EV subtypes. Polymer-based precipitation captures EVs in polymer nets based on size using simple centrifugation, making it useful for clinical usage. EV samples are further characterized commonly through transmission electron microscopy (TEM), nanoparticle tracking analysis, Western blots for common markers (TSG101, Alix, CD63, CD81, Floatillin), and flow-cytometry [74,75]. Microvesicles (50-1000 nm) are a heterogeneous group that includes EVs such as oncosomes, migrasomes, and arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles (ARMMs) [62,71]. MVs are formed through the outward budding of the plasma membrane ( Figure  1). Oncosomes are large EVs associated with cancer. Migrasomes are involved in the transportation of multivesicular cytoplasmic content during cell migration [62]. Additionally, apoptotic bodies (500-4000 nm) result from the disintegration of the plasma membrane of apoptotic cells [67,72].
Once released into the extracellular milieu, EV-mediated communication may act in an autocrine or paracrine manner. Once at the recipient cells, EVs may remain at the plasma membrane, fuse with the plasma membrane, or be internalized by the target cell. Endocytosis of EVs may be clathrin-mediated or clathrin-independent (macropinocytosis or phagocytosis), or facilitated via caveolae or lipid rafts [62]. Intracellular communication via EVs occurs during homeostasis and under pathological conditions, including cancer. The cargo carried by EVs is selectively rather than indiscriminately packaged. This suggests a biological role for the contents that are packaged, since selection can be driven to create conditions that either promote or inhibit malignancy [73].
There are several techniques employed currently to isolate EVs. Differential ultracentrifugation (DU) is widely used as a low-cost, high-throughput method to isolate EVs from large sample sizes. DU is often combined with other purification techniques such as ultrafiltration or density gradient  (30-150 nm) formation begins in late endosomes that maturate into multivesicular bodies (MVB), including the formation of intervesicular bodies (ILVs) through the inward budding of the MVs limiting membrane. MVB can fuse with the plasma membrane and release the ILVs into the extracellular space as exosomes. Alternatively, MVB can suffer degradation by fusing with lysosomes. EVs can contain numerous biomolecules, including protein, lipids, DNA, and RNA.
Damaged and diseased cells, including cancer, have been shown to shed higher amounts of EVs compared to their healthy counterparts. This heightened production may be linked to the extensive metabolic reprogramming that cancer cells undergo [76]. The mechanisms regulating EV production are unclear; however, EV formation and release have been inhibited by targeting biological molecules involved in EV trafficking (calpeptin, manumycin A, Y27632) or lipid metabolism (D-pantethine, imipramine, GW4869) [77]. In regard to fibroblasts, GW4869 which targets nSMases to inhibit exosome generation and release, has been extensively used in CAF-EV research [13,[78][79][80][81][82][83].

Role of EV Contents in CAF Activation
The transfer of molecular cargo from cancer to stromal cells via EVs is a key regulator of CAF differentiation. Stromal cells, particularly fibroblasts, take up large numbers of cancer cell-derived EVs when compared to epithelial cells, making them important targets of EV-mediated cross-talk [91]. Additionally, endothelial cells, pericytes, and mesenchymal stem cells can also be induced to a CAF phenotype by cancer-derived EVs [92][93][94][95][96][97]. The ability of cancer-derived EVs to promote CAF phenotype has been linked to several types of molecular cargo, including miRNAs, proteins, and to a lesser extent, messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) ( Table 1 and Figure 2). nt. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 1 of Figure 2. Summary of the extra-cellular vesicle (EVs)-mediated cross-talk between cancer cells and cancer-associated fibroblasts (CAFs). Cancer cells can influence stromal cells to activate a CAF phenotype through the release of EVs, which carry several cargos, including proteins, micro-RNAs (miRNA), and long noncoding-RNA (lncRNA). Specific cancer cells-derived EV cargos can also influence a pro-angiogenic or pro-inflammatory phenotype in CAF, and the induction of therapy resistance and pre-metastatic niche formation. At the same time, CAF-derived EVs cargos can influence cancer cells to increase epithelial-to-mesenchymal transition (EMT), growth, invasion, metastasis, motility, stemness, colony formation, apoptosis inhibition, glycolysis, and therapy resistance. Figure 2. Summary of the extra-cellular vesicle (EVs)-mediated cross-talk between cancer cells and cancer-associated fibroblasts (CAFs). Cancer cells can influence stromal cells to activate a CAF phenotype through the release of EVs, which carry several cargos, including proteins, micro-RNAs (miRNA), and long noncoding-RNA (lncRNA). Specific cancer cells-derived EV cargos can also influence a pro-angiogenic or pro-inflammatory phenotype in CAF, and the induction of therapy resistance and pre-metastatic niche formation. At the same time, CAF-derived EVs cargos can influence cancer cells to increase epithelial-to-mesenchymal transition (EMT), growth, invasion, metastasis, motility, stemness, colony formation, apoptosis inhibition, glycolysis, and therapy resistance.

miRNA
MiRNAs are small (≈18-22 nucleotides) non-coding RNAs that regulate gene expression at a post-transcriptional level. MiRNAs are among the most abundant cargo found in EVs; as such, it is unsurprising that EV-mediated miRNA transfer from cancer to stromal cells plays an important role in promoting CAF differentiation. To date, at least two dozen miRNAs have been found to be responsible for promoting EV-mediated CAF differentiation in a variety of cancer types [11,95,[98][99][100][101][102][103][104][105][106][107][108][109][110][111][112][113][114][115][116]. Uptake of miRNA-containing EVs into recipient cells results in altered expression of key signaling pathways, many of which have a known oncogenic or tumor-suppressive role, which leads to the adoption of a CAF phenotype. For instance, miR-21 has been found to be highly expressed in EVs derived from various cancer types, including multiple myeloma and oral, colon, liver, and prostate cancer [95,99,103,104,114].
Uptake of miR-21-containing EVs into recipient fibroblasts actively promotes CAF differentiation via an increase in PI3K signaling [99,104]. The same study found that CAF differentiation correlated with increased expression of additional oncogenic proteins in recipient cells, including β-catenin, signal transducer and activator of transcription 3 (STAT3), the mammalian target of rapamycin (mTOR), and TGF-β [99]. STAT3 signaling was found to be activated by miR-155 and miR-210, which are expressed at high levels in EVs of several cancer types, the uptake of which by recipient fibroblasts promotes CAF differentiation [98,105]. STAT3 signaling has long been associated with angiogenesis, and in agreement, STAT3 signaling in fibroblasts was found to drive the expression of the pro-angiogenic factors VEGF, MMP-9, and fibroblast growth factor 2 (FGF2), prompting CAFs to adopt a pro-angiogenic phenotype [98]. Although miR-155 and miR-210 both promote a STAT3-dependent pro-angiogenic phenotype in recipient fibroblasts, they do so by impacting different intracellular targets (suppressor of cytokine signaling 1 (SOCS1) and tet methylcytosine dioxygenase 2 (TET2), respectively). This observation demonstrates how tumor cells can achieve similar outcomes via different mechanisms.
In addition to the activation of oncogenic pathways, several studies have shown that EVs miRNAs can induce a CAF phenotype in recipient cells by inhibiting the tumor suppressor p53 and related proteins [101,102,115]. High expressions of miR-125b, miR-155, miR-1249-5p, miR-6737-5p, and miR-6819-5p in cancer cell-derived EVs have been shown to drive CAF differentiation in recipient fibroblasts via inhibition of the p53 pathway [101,102,115]. Interestingly, loss of p53 signaling in colon cancer cells resulted in increased expression of EV microRNAs that target p53 in recipient cells [101]. This result suggests that the onset of CAF differentiation in many tumors may correlate with p53 inhibition in cancer cells, a common and early event in tumorigenesis. Whereas inhibition of p53 in fibroblasts promotes a CAF phenotype, inhibition of another tumor suppressor, breast cancer type 1 susceptibility protein (BRCA1), allows cancer cell-derived EVs to promote an oncogenic phenotype in recipient fibroblasts that is similar to that of the inducing cancer cells [117][118][119]. This oncogenic phenotype is characterized by mesenchymal-to-epithelial transition, deregulation of gene and miRNA expression that mimics that of the parental tumor, and the ability to form solid tumors in mice [117][118][119]. Further investigation is necessary to determine why the inhibition of these tumor suppressors results in different outcomes. EVs secreted by normal ovarian cells, but not ovarian cancer cells, deliver miR-124 to fibroblasts and inhibit their differentiation into CAFs. Breast cancer cell-derived EVs contain high levels of miR-9 that promote migration, invasion, and CAF differentiation in recipient fibroblasts. In turn, fibroblasts secrete miR-9 in EVs that can inhibit E-cadherin in epithelial cells.

Ringuette Goulet
(2018) [ Prostate cancer cell-derived exosomes contain Hyal1, a hyaluronidase that is transferred to recipient stromal cells. Uptake of Hyal1-positive exosomes greatly increased stromal cell motility, enhanced adhesion to type IV collagen, and increased FAK phosphorylation and integrin engagement. Exosomes secreted by melanoma cells induced CAF activation in embryotic fibroblasts and increased cell migration.

Proteins
Whereas EV miRNAs function by post-transcriptional regulation of gene expression in recipient cells, EV proteins are thought to stimulate receptors at the recipient cell surface or to become active in the recipient cell upon EV uptake. Indeed, given the high degree of overlap between pathways targeted by EV miRNAs and proteins, these and other cargo types may work together to promote a CAF phenotype as described above for STAT3. Another example of EV cargo cooperation is provided by alterations of the TGF-β/SMAD signaling pathway. Multiple cargos, including miR-21, miR-769-3p, TGF-β protein, and TGF-β mRNA have been found to drive a CAF phenotype in recipient cells via TGF-β signaling [10,92,96,97,99,103,[120][121][122]130]. These cargos can also coexist within the same EVs [99]. Vesicle-bound TGF-β may be tethered to the EV membrane or may exist within the soluble compartment of the EV; the preferred form likely depends upon the characteristics of the parental cell [10,92,122]. Regardless of whether TGF-β is located on the EV surface or in the EV lumen, EV-bound TGF-β is a potent stimulator of SMAD signaling, which drives expression of genes required for CAF differentiation, including the known CAF marker α-SMA [10,97,122]. In turn, newly differentiated CAFs secrete high levels of TGF-β that promote a CAF phenotype in nearby cells and can promote increased metastasis of tumor cells, thus driving enhanced tumorigenesis [103].
In addition to TGF-β, several other EV proteins have been found to regulate CAF differentiation, including the integrin very late antigen-4 (VLA-4) and the virally-encoded oncoprotein latent membrane protein 1 (LMP1) [127,136]. Specifically, VLA-4 has been reported to be expressed at high levels in EVs obtained from melanoma cells and has been reported to trigger ERK1/2 signaling in recipient cells, which results in up-regulation of CAF markers and inflammatory factors [136]. Interestingly, oncogenic LMP1, encoded by the Epstein-Barr Virus (EBV), has been found to be incorporated into the EVs of virally-infected cells and transferred to recipient fibroblasts, resulting in induction of a CAF phenotype via activation of NF-κB signaling [127]. Further, LMP1 uptake has been reported to promote aerobic glycolysis in CAFs. This allows fibroblasts to release high levels of lactate and other glycolytic intermediates that promote tumorigenesis by providing tumor cells with nutrients and by acidifying the TME, a mechanism known as the reverse Warburg effect [127]. These results demonstrate how EV-mediated transfer of a single protein can have a profound effect on fibroblast differentiation and function.
Several additional proteins have been described that alter CAF function without necessarily driving CAF differentiation. For instance, the proteins desmoglein 2 and AHNAK have been found to regulate the release and packaging of EVs in head and neck cancer and breast cancer, respectively [133,134]. The knockdown of these proteins results in a decrease in EV release and the ability of EVs to promote recipient cell proliferation and migration [133,134]. Still, other EV proteins have been found to promote various CAF functions, including aerobic glycolysis (integrin subunit beta 4 (ITGB4)) and CAF recruitment to metastatic tumors (Lin-28 homolog B (LIN28B)), resistance to apoptosis (WW and C2 domain containing 2 (WWC2) and survivin), increased migration or proliferation (hyaluronidase-1 (Hyal1), sphingosine-1-phosphate receptor 2 (S1PR2)), and expression of MMPs (CD147) [123][124][125][126]128,129,131,132,135]. These results demonstrate the wide range of effects that cancer cell-derived EVs have on cells of the TME.

Other Cargos (mRNA and lncRNA)
Several studies demonstrate roles in tumorigenesis for other EV cargo types, including mRNA and lncRNA. Indeed, melanoma cell-derived EVs exhibited differential expression of over 1600 transcripts when compared to fibroblast-derived EVs, suggesting an important role for mRNA incorporation into EVs [138]. Specific mRNAs, including those encoding TNF-α, TGF-β, and IL-6, have been found to be transferred from cancer cells to fibroblasts and to regulate fibroblast differentiation and function [130]. Interestingly, various cancer cell lines have been found to package human telomerase reverse transcriptase (hTERT) mRNA into their EVs; uptake of this cargo by recipient fibroblasts results in telomerase-positive fibroblasts that exhibit increased proliferation, protection from late senescence, and protection from DNA damage [139]. Additionally, the lncRNA Gm26809 was found in melanoma-derived EVs and induced expression of CAF markers in recipient cells [137]. Although reports of mRNA and lncRNA transfer remain sparse, current data suggest an important role for these cargo types in EV-mediated CAF induction.
There is a lack of evidence supporting any anti-tumor effects by CAF-EVs specifically; however, some studies have shown possible anti-tumor effects by CAFs [157]. For instance, one study targeted the extensive fibrosis and stromal myofibroblasts associated with pancreatic ductal carcinomas. The deletion of α-SMA myofibroblasts in pancreatic ductal carcinomas mice models enhanced hypoxia, EMT, CSCs, and chemoresistance, and reduced animal survival. A decrease in immune surveillance and increased Treg infiltration were also observed, leading the authors to suggest that the fibrosis associated with pancreatic ductal carcinomas is part of a host immune response [158].

miRNA
Most studies examined the transfer of EV miRNAs from CAFs to cancer cells [12,79,81,[83][84][85][86]88,89,[142][143][144]148,150,152,[154][155][156]. A miRNA that has been reported across multiple studies is miR-21, which is enriched in CAF-derived EVs in breast, ovarian, and colorectal cancers [12,143,150]. In these studies, mir-21 acted as an oncogenic miRNA (oncomiR), by promoting tumorigenesis in cancer cells. In other instances, it was the repression of specific miRNAs in CAF-derived EVs that conferred pro-tumorigenic behaviors in recipient cells. For example, in head and neck cancer (HNC), miR-3188 acts as a tumor suppressor by inhibiting cell proliferation and promoting apoptosis in recipient cancer cells. The loss of miR-3188 in CAF-derived exosomes promotes tumor progression through the de-repression of B-cell lymphoma 2 (BCL2). Clinically, this has been reflected in the observed low levels of circulating miR-3188 in plasma samples from HNC patients [85].

Proteins
Several studies have observed that the protein contents of CAF-derived EVs are able to induce changes in recipient cancer cells, including increased migration, invasion, EMT, and metastasis [13,80,84,87,90,140,141,145,146,149,151,153]. Specific protein cargo has been shown to stimulate pro-tumor activity but can also regulate the uptake of CAF-derived EVs by recipient cancer cells. In pancreatic cancer, cell aggressiveness characterized by an increase in proliferation and metastasis is dependent on the uptake of annexin A6 (ANXA6) positive CAF-derived exosomes. Loss of ANXA6 has been reported to destabilize the ANXA6/low-density lipoprotein receptor-related protein 1 (LRP1)/thrombospondin-1 (TSP1) complex, which leads to reduced uptake of CAF-derived exosomes by cancer cells, and a reduction in their proliferative and migratory abilities. ANXA6 enrichment in CAF-derived exosomes is reflected in the serum-derived exosomes of pancreatic ductal adenocarcinoma patients, where elevated ANXA6 levels are associated with higher tumor grades and poor clinical outcomes [90].
In addition to the transfer of protein, CAF-derived EVs may carry protein markers that influence their uptake by specific cancer cell types. One study found that CAF-derived exosomes are taken up by scirrhous-type gastric cancer cells but not poorly differentiated or intestinal-type gastric cancer cells. These exosomes are positive for the surface marker CD9 when secreted from CAFs, but not when secreted by normal fibroblasts. Interestingly, these CAF-derived exosomes are negative for other common exosome surface markers, CD63 and CD81. CD9+ CAF-derived exosomes stimulate migration and invasion in scirrhous-type gastric cancer cells, and promote increased MMP-2 activity [87]. In line with this study, other studies corroborate the ability of CAFs to promote the degradation of the ECM through up-regulation of metalloproteinases. CAFs can undermine control mechanisms that regulate MMPs and A disintegrin and metalloproteases (ADAMs) under normal physiological conditions. For instance, in gastric cancer, miR-139 is down-regulated in CAF-derived exosomes, while its target MMP-11 is enriched, leading to heightened invasive and metastatic abilities of recipient cancer cells [84]. Another study reported the induction of the CAF phenotype in breast, lung, head and neck, and renal cancer cell lines through the knockdown of TIMP, a modulator of ECM integrity via post-translational regulation of MMPs and ADAMs. TIMP-less fibroblasts released exosomes rich in ADAM10, and were able to accelerate tumor growth in vivo in all the mentioned cancers except for renal cancer, which lacks a major stromal component [145].

Other Cargo (lncRNA)
A limited number of studies showcased that CAF-derived EVs may also confer pro-tumorigenic properties, including proliferation, chemoresistance, stemness, and metabolic reprogramming to malignant cells through the transfer of lncRNAs [78,82,147]. Recent findings showed lncRNA small nucleolar RNA host gene 3 (SNHG3) functioned as an endogenous sponge for miR-330, thereby modulating the expression of pyruvate kinase M1/2 (PKM), which led to a down-regulation in cancer cell mitochondrial activity and promoted cancer growth and glycolysis [78].
Overall, further investigations are needed to understand the bidirectional signaling and molecular cargo transfer network established through EVs between cancer cells and CAFs within the TME.  CAF-derived exosomes deliver miR-98-5p to ovarian cancer cells, contributing to cisplatin resistance by promoting cell proliferation and colony formation, and inhibiting cell apoptosis; CAF-derived exosome miR-98-5p targets CDKN1A, leading to its down-regulation in recipient ovarian cancer cells.
Zhang (2017) [155] HCC EVs; size NR Life Technologies exosome precipitation kit miR-320a Decreased levels of miR-320a in CAF-derived exosomes promote cell proliferation, migration, and metastasis; loss of miR-320a leads to de-repression of its target PBX3, activating MAPK pathway and up-regulation of CDK2 and MMP2; in vivo, miR-320a overexpression suppresses tumor growth and metastasis.
Li (2017)   miR-409 expression correlated with higher Gleason score in prostatic and bone tissue; miR-409 overexpression in normal fibroblasts induces CAF-like phenotype; CAF-derived EV mediated transfer of miR-409 to prostate epithelium resulted in repression of tumor suppressors (RSU1, STAG2), promotion of EMT and cell growth, and increased survival. CAF-derived exosomes taken up by scirrhous-type of gastric cancer but not in the other types; CD9-positive CAF-derived exosomes promoted migration and invasion in scirrhous-type gastric cancer cells through MMP-2 activation.

30-150 nm Ultracentrifugation TGFβ1
TGFβ1 is significantly up-regulated in exosomes from CAFs in ovarian cancers with omental metastasis; TGFβ1 promotes EMT in ovarian cancer through activation of SMAD2/3 signaling.

CAF-Derived EVs Contents in Therapy Resistance
CAF-derived EVs introduce a novel mechanism of acquired resistance to anticancer treatment (i.e., chemotherapy or hormonal therapy). Therapy resistance can occur through EVs cargos delivered from CAFs to other cells in the TME and may involve the regulation of several signaling pathways by these cargos [159].
CAFs demonstrate a high degree of resistance to chemotherapeutics and can transfer this resistance to neighboring cancer cells via EV miRNAs. For instance, exosomes derived from gemcitabine-resistant CAFs contain high levels of miR-106a that, when taken up by recipient pancreatic cancer cells, promotes chemoresistance through the inhibition of the tumor suppressor TP53INP1 [160]. In ovarian cancer, CAF-derived exosomes express high levels of miR-98-5p that is transmitted to cells of the TME, leading to the development of cisplatin resistance by cancer cells through direct inhibition of cyclin-dependent kinase inhibitor 1A (CDKN1A, p21) [148]. Further, miR-21 is secreted via EVs from cancer-associated adipocytes and CAFs, leading to inhibition of APAF1 in recipient cells of the TME [150]. MiR-21 mediated repression of APAF1 inhibits cell cycle arrest and promotes resistance to chemotherapy [150]. As an additional example of how CAF-cancer cell cross-talk can increase chemoresistance, activation of the ubiquitin-specific protease 7 (USP7)/heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) axis in gastric cancer cells exposed to paclitaxel and cisplatin increases the secretion of miR-522 in CAF-derived exosomes, leading to ferroptosis suppression in tumor cells and culminating in increased chemoresistance [161].
In addition to miRNAs, CAF-derived EV lncRNAs have also been linked to increased chemoresistance. For instance, colorectal cancer-associated lncRNA (CCAL) is an oncogenic lncRNA highly expressed in CAFs associated with colorectal cancers, and its transference to the TME via exosomes is associated with chemoresistance [14]. Similarly, lncRNA H19 is significantly up-regulated in CAF-derived exosomes in colorectal cancer and acts as a competing endogenous RNA sponge to tumor suppressive miR-141 [82]. Inhibition of miR-141 promotes the stemness of cancer cells and leads to the activation of the Wnt/β-catenin signaling pathway [82]. Transmission of exosomal H19 from CAFs to the neighboring cells is strongly associated with tumor development and resistance to oxaliplatin [82].
Cancer stem cells (CSCs) have also been shown to exhibit a high degree of resistance to common cancer treatments [162]. Interestingly, CAF-derived exosomes were found to increase the proportion of CSCs and induce their tumorigenic capability in colorectal cancer patient-derived xenografts, which led to the development of 5-fluorouracil and oxaliplatin resistance [163]. Inhibition of CAF-derived EVs decreased the proportion of CSCs and abrogated their tumorigenic potential [163]. In agreement, CAF-derived exosomes were found to transfer Wnt proteins to colorectal cancer cells, thereby reprogramming them into CSCs and increasing their resistance to chemotherapy [164]. In addition, incubation of luminal breast cancer cells exposed to hormonal therapy with miR-221-containing CAF-derived exosomes resulted in the activation of the ER/Notch feed-forward loop, the generation of CD133 CSCs with low expression levels of estrogen receptor alpha, and consequently, the development of de novo hormonal therapy resistance [165]. Reciprocally, CSC-derived EVs up-regulate the β-catenin/mTOR/STAT3 pathway and increase mRNA and protein levels of TGF-β1 [99]. These EVs can transform normal fibroblasts into CAFs with enhanced oncogenic potential; in turn, these CAFs then increase chemoresistance in oral cancer cells [99]. Although these results are intriguing, additional studies are required to fully elucidate the mechanisms underlying CSC-CAF interactions and their relationship to drug resistance.

EVs and Pre-Metastatic Niche Formation
Evidence supports the concept that primary pro-metastatic cancer cells can promote metastasis by inducing changes in the microenvironment of distant organ sites, namely, at pre-metastatic niches [166]. In this context, EV exchange between primary tumor cells and the pre-metastatic niche was shown to play an important role as a communication medium, being able to influence several processes in the pre-metastatic site (e.g., angiogenesis, ECM remodeling, immune response, and inflammation) [167]. For instance, in vivo experiments showed that EVs that originated from nasopharyngeal cancer carrying LMP1 were able to increase the expressions of fibronectin, S100A8, and VEGFR1 in lung and liver tissues [127]. Additionally, there is evidence that EVs might mediate organ-specific tropism of metastasis. The pattern of integrin expression in tumor-derived exosomes was associated with adhesion to specific ECM molecules and cell types; for example, ITGα6β4 and ITGα6β1 expression lead to lung tropism, while ITGαvβ5 expression mediates liver tropism [168].
Pre-metastatic niche formation is frequently associated with the activation of fibroblasts in a distant site by tumor cell-derived EVs. In this regard, metastatic breast cancer cells release significantly higher amounts of transglutaminase-2 and fibrillar fibronectin, which is associated with an enhanced growth-supportive phenotype in lung fibroblasts and metastatic niche formation in lung tissues [169]. Triple-negative breast cancer-derived EVs are also proposed to mediate pre-metastatic changes in ECM and soluble components of lung tissue, including enhanced expression of fibronectin and periostin in lung fibroblasts [170]. Exosomal miR-155 and miR-219 secreted by melanoma cells are able to induce metabolic reprograming in normal fibroblasts by favoring glycolysis over oxidative phosphorylation, leading to an acidified extracellular pH, which is associated with pre-metastatic niche formation [108]. Furthermore, breast cancer cell-derived EVs containing miR-122 are understood to down-regulate glucose consumption in lung fibroblasts, astrocytes, and neurons, with miR-122 inhibition restoring glucose uptake and decreasing the incidence of metastasis in vivo [171].
Cancer cell-derived EVs are also associated with pro-inflammatory profiles in pre-metastatic niche fibroblasts. Metastatic hepatocellular carcinoma cells secrete miR-1247-3p via exosomes, leading to activation of the β1-integrin-NF-κB pathway in fibroblasts and promoting a pro-inflammatory profile in these fibroblasts, which was associated with pre-metastatic niche formation and lung metastasis [111]. EVs derived from colorectal cancer cells containing integrin beta-like 1 (ITGβL1) stimulate the activation of resident fibroblasts in distant organs via TNFAIP3-mediated NF-κB pathway. As a result, the activated fibroblasts secrete pro-inflammatory cytokines and promote metastatic cancer growth [172]. Furthermore, EVs secreted by metastatic melanoma cells induce a pro-inflammatory profile in lung fibroblasts without any differences in wound healing functions, while also triggering pro-inflammatory signaling in astrocytes [138].
Fewer studies have assessed the ability of CAF-derived EVs to promote pre-metastatic niche formation. In salivary adenoid cystic carcinoma, CAF-derived EVs were able to induce pre-metastatic niche formation in lung tissue, including enhanced matrix remodeling, periostin expression, and lung fibroblast activation [173]. However, the role of CAF-derived EVs in pre-metastatic niche formation remains to be elucidated in further studies.

Conclusions
Extracellular vesicles are proven to play important roles as mediators of intercellular communication, and therefore, have been targets of increasing interest in cancer research. EVs affect different molecular pathways in the cross-talk between cancer cells, but also between these cells and the stromal components of the TME. Different cargos contained in EVs secreted by cancer cells are able to influence TME cells to exert pro-tumorigenic functions, which include induction of CAF, pro-inflammatory, and pro-angiogenic phenotypes. The activation of a CAF phenotype in stromal cells can alter the contents of their secreted EVs. CAF-derived EVs have been shown to promote tumor progression by influencing cancer cells to develop more aggressive characteristics, including increased growth, migration, invasion, metastasis, and therapy resistance. Additionally, evidence shows that EVs derived from cancer cells and CAFs are able to influence the microenvironments at distant sites and promote pre-metastatic niche formation. This line of research inquiry holds promise for clinical utility via possible identification of novel biomarkers and therapeutic targets, and the possible use of EVs as a vector for delivery of therapeutic agents [155,156,174].
It is important to emphasize that the majority of evidence regarding EVs' role in tumorigenesis has been derived from in vitro and animal model studies, with limited data regarding the utility of these findings for clinical applications. Although EV-based research seems promising to clarify the molecular mechanisms involved in the cross-talk between CAFs and cancer cells during tumorigenesis, new tools and/or research methods need to be developed to apply the findings in clinical settings.