The Function, Underlying Mechanism and Clinical Potential of Exosomes in Colorectal Cancer

Colorectal cancer (CRC) is a lethal malignancy worldwide. Exosomes are extracellular vesicles derived from the endosomal pathway of nearly all cells and can be found in body fluids. They can be considered an intercellular system in the human body that can mediate near-and long-distance intercellular communication due to their features and functions. Investigations have revealed that exosomes are participated in different processes, physiologically and pathologically, especially in cancer. However, the clinical value of exosomes and their mechanisms of action in CRC are unclear and have not been systematically assessed. The purpose of this review is to discuss how exosomes play a role in the occurrence and development of CRC, with a particular focus on the functions and underlying mechanisms of tumor-derived exosomes as well as non-tumor-derived exosomes. We also describe the evidence that exosomes can be used as diagnostic and prognostic markers for CRC. In addition, the possibilities of exosomes in CRC clinical transformation are also discussed.


Introduction
Worldwide, colorectal cancer (CRC) is a common malignant tumor [1].According to the Global Cancer Statistics 2020, CRC is the third most commonly diagnosed cancer, the second leading cause of cancer death in women, and the third leading cause of incidence and mortality in men [2].It is a malignancy originating from the mucosal epithelium and glands of the large intestine.Histologically, there are several subtypes of CRC, and approximately 90% of CRCs are adenocarcinomas [3].Patients with CRC often present with anemia, blood in the stools, aberrant bowel movement, and weight loss [4].It has been demonstrated that the occurrence of CRC is related to dietary patterns, metabolism, and inflammation [5].The initiation and progression of CRC may also be affected by environmental and genetic factors [6].Thus, CRC is the result of a complex multi-step process involving numerous factors and genes.For patients with CRC, the systemic treatment regimens utilized for clinical management include tumor resection, 5-Fluorouracilbased chemotherapy, radiotherapy, anti-angiogenic treatment, targeted therapy, and immunotherapy [7][8][9].Although the death rate of CRC has decreased due to earlier screenings and improved treatments, more than one-third of patients die within 5 years of initial diagnosis, with liver metastases being the most fatal cause of death [10][11][12].
Exosomes are extracellular vesicles (EVs) with diameters ranging from 40 to 160 nm.They are derived from the endosomal pathway of nearly all cells, and they can be found in cell culture fluids, breast milk, blood, urine, pleural effusion, saliva, and other body fluids [13][14][15].Exosomes carry specific cargo consisting of DNA, RNA, cytosolic and cell-surface proteins, metabolites, and lipids.They can be thought of as an intercellular system in the human body that mediate communication between cells and influence a variety of cell biological behaviors [13,16].During the generation of exosomes, the plasma membrane is double invaginated, and intracellular multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs) are formed.MVBs fuse to the plasma membrane, and ILVs are ultimately secreted as exosomes through exocytosis.The uptake of exosomes is not random but depends on the surface proteins on the membrane of exosomes and recipient cells [17].When exosomes encounter suitable recipient cells, exosomes can adhere to the surface of recipient cells through the interaction of ligands and receptors.They can directly fuse with cell membranes or be endocytosed by recipient cells and release the cargo into target cells (Fig. 1) [13,14].Numerous studies have shown that exosomes are involved in different physiological and pathological processes, such as inflammatory responses, cancer development, metastasis, and immunity [17,18].Recently, it has been demonstrated that exosomes play a role in the occurrence, development, invasion, metastasis, tumor microenvironment (TME) remodeling, chemoresistance, and other processes in CRC [19].This paper reviews the functions and underlying mechanisms of action of tumor-derived and non-tumor-derived exosomes in CRC, intending to summarize the previous studies on potential biomarkers and effective targets for the treatment of CRC.

Physiological and Pathological Functions of Exosomes
Exosomes were first reported as a "type of small vesicles" in 1983, and the name "exosome" was first used in 1989 [20,21].They were initially believed to be superfluous membrane vesicles during cell maturation, with the effect of regulating membrane function, removing cellular debris, and eliminating surface molecules [14].However, exosomes have successfully attracted attention among researchers because of their special roles in multiple facets of cell activity.
Exosomes can be secreted by the donor cells through exocytosis and accepted by the recipient cells through endocytosis or directly fusing to cell membranes.Thus, exosomes act as a medium in intercellular communications and transmit information to a large number of cells and locations, physiologically or pathologically (Fig. 2).
Exosomes are involved in the signal transduction of the nervous system due to reciprocal signal transfer among sensory and motor neurons, interneurons, and glial cells [22].Moreover, they play an important role in the aging process [23].Exosomes also play an important role in reproductive development.They are involved in gamete maturation, fertilization, embryo implantation, immunological communication between the mother and the fetus, and fetal protection by regulating local and/or systemic immune responses, organ development, and melanin synthesis [24][25][26][27][28]. Additionally, exosomes are related to cell proliferation, homeostasis, and maturation, such as in hepatocytes and reticulocytes [20,29].Exosomes are involved in immune response and infection [30,31].They participate in antigen presentation and induce the activation of T and/or B cells [32].The contents of exosomes are involved in regulating innate and adaptive immune responses.It has been demonstrated that the DNA of some intracellular bacteria sorted into exosomes is able to activate innate immune responses or lower antibacterial defenses [33].
Numerous studies have demonstrated that exosomes are widely involved in the occurrence and development of cancers.They are a double-edged sword in tumor growth and progression.For example, exosomes derived from M2 macrophages can inhibit the migration and invasion of glioma cells [34].Alternatively, the impact of tumor exosomal DNA on inflammatory responses can indirectly worsen cancer [35].In addition, tumor-derived exosomes can promote immune evasion by cancer cells and generate an immunosuppressive microenvironment [17,36].Exosomes derived from tumor cells may be absorbed by surrounding cells and transform the microenvironment into one that is prone to tumor development [37].They also participate in tumor growth, metastasis, apoptosis, metabolic reprogramming, extracellular matrix degradation, stromal reprogramming, immune surveillance escape, and drug resistance [23,38,39].In addition, exosomes are involved in neurodegenerative diseases, cardiovascular disease, and pathogenic infections [14,40].

The Underlying Mechanisms of Exosome Functions in CRC
Numerous studies have explored the relationship between exosomes and the pathogenesis of different cancers.The data demonstrated that exosomes have a relationship with the hallmarks of cancer, such as sustaining proliferation signaling, activating invasion and metastasis, inducing angiogenesis, metabolic reprogramming, and immune evasion [41].We reviewed the functions and mechanisms of exosomes in CRC, including CRC cell-generated exosomes and non-tumor cell-produced exosomes.

Invasion and Metastasis
Invasion and metastasis are the leading causes of cancer-associated death in patients with CRC; however, the molecular mechanisms underlying tumor invasion and metastasis are complex and elusive [94].Numerous studies  CRC-derived exosomal miR-106b-3p promotes metastasis by down-regulating DLC-1 expression.Serum exosome miR-106b-3p could be a potential molecular biomarker for prognosis and may be a target for the treatment of CRC [61].
MicroRNA-21-5p is highly enriched in CRC-derived exosomes and is essential for creating a liver proinflammatory phenotype and liver metastasis of CRC by inducing an inflammatory premetastatic niche through the miR-21-TLR7-IL-6 axis, therefore promoting liver metastasis [63].Exosomal miR-193a and let-7g facilitate cancer primary CRC progression and peritoneal metastasis by targeting MMP16 and CDKN1A [98].It is also demonstrated that hypoxia may promote the release of Wnt4-rich exosomes which are derived from tumor cells.The Wnt4-rich exosomes are delivered to normoxic cells and facilitate the migration and invasion of normoxic CRC cells [64].Exosomal IRF-2 remodels the lymphatic network in a sentinel lymph node (SLN) and may predict the development of CRC lymph node (LN) metastases [99].Exosomes rich in circPABPC1 are transported from CRC cells to CRC cells and are involved in the progression of CRC.In the nucleus, circ-PABPC1 initiates the transcription of HMGA2 by recruiting KDM4C to its promoter and reducing the H3K9me3 modification.In the cytoplasm, it inhibits the degradation of ADAM19 and BMP4, which is mediated by miR-874 and miR-1292.Thus, circPABPC1 facilitates liver metastasis in CRC via upregulating the expression of HMGA2, BMP4, and ADAM19 [100].Exosomes carrying circ-ABCC1 from CD133 + cells have the ability to mediate cell stemness and metastasis in CRC [101].LncRNA PCAT1, which is from CRC exosomes, can promote the EMT and liver metastasis of CRC.Because exosome lncRNA PCAT1 can influence miR-329-3p and the activity of the Netrin-1-CD146 complex in circulating tumor cells (CTCs) [65].Exosomal-miR-10a derived from CRC cells reduced the expression of IL-6, IL-8, and IL-1β in normal human lung fibroblasts (NHLFs), thereby reducing the proliferative and migratory activities of primary NHLFs.This will help us understand the mechanism underlying the process of CRC lung metastasis [66].Exosomal miR-1255b-5p targets human telomerase reverse transcriptase and inhibits its expression in CRC cells, ultimately suppressing tumor progression and liver metastasis of CRC [67].Exosomal lncRNA 91H enhances CRC metastasis by modifying HNRNPK expression in CRC [49].

Angiogenesis
Angiogenesis is critical for tumor growth, survival, and progression.The process is very complex, including the degradation of the vessel's basement membrane, the activation, proliferation, and migration of vascular endothelial cells, and reconstruction to form new blood vessels and networks [102].Tumor-derived exosomes are largely involved in this process [103].This has been demonstrated in the angiogenesis of CRC.For example, exosomal miR-25-3p, which is CRC cell-derived, directly targets KLF2 and KLF4 and regulates VEGFR2, ZO-1, claudin5, and occludin expression in endothelial cells, consequently promoting vascular permeability and angiogenesis [68].Exosomal miR-21-5p, which is secreted by CRC cells, induces angiogenesis and vascular permeability by targeting KRIT1 [69].MiR-1229, which is derived from the CRC cell exosomes, inhibits the expression of HIPK2 protein, thus activating the VEGF pathway and promoting angiogenesis [70].Exosomal Wnt4 derived from CRC cells increases βcatenin nuclear translocation in endothelial cells and promotes angiogenesis in CRC [71].

Immune Evasion
Exosomes are important in modulating tumor immune response and have a dual role.They may activate immune responses or exhibit strong pro-tumor immune reactions [17].Numerous studies have revealed CRC tumor-derived exosomes (CTDEs) can function as mediators of host antitumor immune responses and tumor cell immune evasion.For example, miR-208b is secreted by colon cancer cells and sufficiently transported to recipient T cells.In T cells, miR-208b, which is delivered by exosomes, directly targets PDCD4 and induces immune evasion [72].The CRC cellderived exosomal lncRNA SNHG10s can be taken up by NK cells and suppress the function of NK cells by upregulating INHBC expression.It has been suggested that exosomal lncRNA SNHG10 could lead to the inhibition of NK cells, ultimately contributing to immune escape [73].Exosomes containing Lnc-HOXB8-1:2 are secreted by neuroendocrine differentiated CRC cells, and Lnc-HOXB8-1:2 from exosomes competitively binds hsa-miR-6825-5p as ceRNA relieves the inhibitory effect of hsa-miR-6825-5p on CXCR3 expression, upregulates its expression, and then leads to TAM infiltration and M2 polarization, and promotes immune evasion of CRC [74].CRNDE-h, which is derived from tumor cells, could inhibit ubiquitination and degradation of RORγt and promote Th17 cell differentiation in CRC [75].CRC stem cell-derived exosomes (CRCSC-exos) deliver miR-17-5p to CRC cells.The high expression of miR-17-5p in CRCSC exosomes promotes tumor cell growth by promoting PD-L1 inhibition of SPOP and inhibits the anti-tumor immunity of CRC [76].Tumorderived HOTAIR could bind to PKM2 and inhibit its ubiquitination degradation, leading to the activation of STAT3 and expression of PDL1, thereby polarizing B cells toward a regulatory feature and suppressing CD8 + T cell activity [77].

TME Remodeling
The tumor microenvironment (TME) is a complex internal environmental system around tumor cells, which plays an essential role in tumorigenesis.CTDEs in the TME are essential in the formation and reprogramming of the TME.For example, miR-146a-5p and miR-155-5p contained in CRC cell exosomes can be secreted by CRC cells and taken up by the cancer-associated fibroblasts (CAFs).Then, they may activate CAFs through JAK2-STAT3/NF-κB signaling.Reciprocally, the activation of CAFs may further facilitate the invasion of CRC cells [78].CRC cell-derived exosomal HSPC111 can be engulfed by CAFs.In CAFs, it may phosphorylate ACLY and promote the expression of acetyl-CoA.Acetyl-CoA accumulation further increases H3K27 acetylation and promotes CXCL5 expression in CAFs.Interestingly, CXCL5 from CAFs can reinforce exosomal HSPC111 excretion from CRC cells and promote liver metastasis via the CXCL5-CXCR2 axis [79].Exosomal ANGPTL1 derived from CRC cells is mainly taken up by Kupffer cells (KCs).Then, ANGPTL1 can downregulate MMP9 levels by inhibiting the JAK2-STAT3 signaling pathway in KCs [80].Tumor exosomal tri-phosphate RNAs sustain neutrophil survival by inducing the expression of IL-1β through a pattern recognition-NF-κB signaling axis.CRCSC-secreted CXCL1 and CXCL2 then attract CRCSC-primed neutrophils to promote tumorigenesis of CRC cells via IL-1β [81].CRC cell-derived exosomes containing miR-10b can be transferred to fibroblast cells and activate fibroblasts to become CAFs via the PI3K/Akt pathway [82].Studies also revealed that CRC tumor-derived exosomes contribute to generating phenotypically and functionally distinct subsets of CAFs by reprogramming their proteome and may facilitate tumor progression [77].Exosomes that carry miR-203 can be secreted from CRC cells, and they can be swallowed by monocytes.In monocytes, miR-203 can promote the expression of M2 markers, suggesting that miR-203 may facilitate the differentiation of monocytes to M2-TAMs [83].

Metabolic Reprogramming
Metabolic reprogramming is an adaptive mechanism that enables tumor cells to regulate the flow of their energy to meet their needs for rapid growth.It is manifested by an increase in glucose uptake and an enhancement of glycolysis under aerobic conditions, as well as high production of lactate.These metabolic changes are also known as the "Warburg effect" [104].A growing number of studies have shown that exosomes can mediate metabolic reprogramming in cancer [105].As in CRC, hsa-miR-101-3p, which is derived from CRC cell exosomes, is associated with metabolic reprogramming in CRC by targeting HIPK3 [84].Moreover, mutant KRAS exosomes are able to cause a Warburg-like effect on recipient colonic epithelial cells [85].

Therapy Resistance
It has been demonstrated that the delivery of exosomal cargos between different cancer cells is associated with tumor drug resistance [106].Exosomes are used as carriers for cell-to-cell communication in the tumor microenvironment, however; drug-resistant tumor cells can also use this property to develop resistance to sensitive cells [107].Therapy-resistance mechanisms mediated by CT-DEs are summarized as follows: hsa_circ_0005963 is involved in chemoresistance in CRC.The exploration of underlying mechanisms showed that hsa_circ_0005963 could be a sponge for miR-122 [86].Tumor-secreted miR-208b directly targets PDCD4 and promotes Treg expansion, and it may be associated with a decrease of oxaliplatin (OX)based chemosensitivity in CRC [72].It is also demonstrated that exosomes from RKO/R cells are able to promote acquired 5-FU resistance in CRC, which is mainly related to p-STAT3 contained in the exosomes [87].Circ_0000338 is present in exosomes and improves the chemoresistance of CRC cells by targeting miR-217 and miR-485-3p [88].Exosome miR-46146 can be an important promoter of OX resistance by targeting PDCD10 and may be a potential target for OX resensitization by CRC cells [89].MiR-19b can be present in exosomes secreted by CRC cells.By delivering miR-19b, CRC-derived exosomes enhance the radiation resistance and stemness characteristics of CRC cells.Accordingly, miR-19b inhibition can enhance the efficacy of radiotherapy and reduce the stemness characteristics of CRC, suggesting that miR-19b inhibition may be a promising strategy for sensitization of CRC cells to radiotherapy [90].Exosomal circ-FBXW7 can improve the chemoresistance of CRC to OX by direct binding to miR-128-3p, which provides a promising treatment strategy for patients with OXresistant CRC [91].Exosomal circATG4B plays an important role in chemoresistance in CRC.The underlying mechanisms show that it can competitively bind to TMED10, prevent TMED10 from binding to ATG4B, and induce increased autophagy, ultimately promoting chemotherapy resistance [108].Exosomal long non-coding RNA HOTTIP derived from mitomycin-resistant CRC cells can be transferred into the parental cells and increase the resistance of CRC cells to mitomycin via impairing miR-214-mediated degradation of KPNA3 [92].

Non-Tumousr-Derived Exosomes in CRC
Although tumor cells are the main cells in the tumor microenvironment (TME), there are also many other cells, such as fibroblasts, immune cells, endothelial cells, etc. [109].Exosomes secreted by non-CRC cells in the TME may also affect the fate of CRC cells.Here, we summarize the functions and mechanisms of non-tumor-derived exosomes in CRC (Fig. 4; Table 2, Ref. ).
For example, CAFs are the main stromal cells in TME.CAFs can transfer exosomes that are rich in miR-92a-3p directly to CRC cells and increase the expression of miR-92a-3p in CRC cells significantly.In CRC cells, miR-92a-3p directly targets FBXW7 and MOAP1, ultimately promoting the stemness, EMT, metastasis, and chemotherapy resistance of CRC cells [110].CAFs can deliver H19 in exosomes to CRC cells, promoting the stemness and resistance of CRCs.Mechanistically, H19 activates the βcatenin pathway by serving as a competing endogenous RNA sponge of miR-141, which could inhibit the stemness and chemoresistance of CRC cells after delivery to CRC cells [111].CAFs-derived exosomes contain a large number of miR-93-5p, which can promote tumor growth in irradiated nude mice [112].Exosomal LINC00659 could be secreted by CAFs and taken up by CRC cells.Then, it could target the miR-342-3p/ANXA2 axis and promote cell proliferation, invasion, and migration of CRC [113].Exosomal circEIF3K derived from CAFs promotes the progression of CRC via miR-214/PD-L1 axis [114].CAFsderived exosomes contain miR-181d-5p, which is associated with 5-FU sensitivity.It has been demonstrated that miR-181d-5p inhibits 5-FU sensitivity through the METTL3/miR-181d-5p/NCALD axis in CRC cells [115].CAFs-derived exosome miR-181b-3p promotes the occur- rence and development of CRC by regulating SNX2 expression [116].CAFs-secreted exosome circN4BP2L2 promotes the CRC cell stemness and oxaliplatin resistance through EIF4A3/PI3K/AKT/mTOR pathway [117].CAFs can transmit miR-590-3p to CRC cells through exosomes.In CRC cells, miR-590-3p enhances the radioresistance of CRC by regulating the CLCA4-dependent PI3K/Akt signaling pathway [118].CAFs exosomes are directly swallowed by CRC cells, and miR-625-3p in exosomes is released and may inhibit the CELF2/WWOX pathway after entering CRC cells, thereby promoting the migration, invasion, EMT and chemotherapy resistance of CRC cells [119].CAFs-derived exosomes upregulate microRNA-135b-5p to promote CRC cell growth and angiogenesis by inhibiting TXNIP [120].After entering CRC cells, WEE2-AS1 in the exosomes of CAFs can promote the degradation of MOB1A, thereby inhibiting the Hippo pathway and promoting the occurrence and progression of CRC tumors [121].Hypoxia can lead to the loss of miR-200b-3p in the exosomes of CAFs, thereby reducing the sensitivity of CRC cells to 5-FU [122].CAFs-exosomes show higher expression of miR-17-5p than normal fibroblasts-exosomes and can deliver exosomal miR-17-5p from parental CAFs to CRC cells.Further studies confirm that that miR-17-5p directly target RUNX3 3'-UTR to affect CRC metastasis.RUNX3 interacts with the MYC proto-oncogene, and both RUNX3 and MYC bind to the promoter of TGF-β1 on 1005-1296 base pairs, thereby activating the TGF-β signaling pathway and promoting tumor progression [123].LncRNA CCAL is transferred from CAFs to the cancer cells via exosomes and promotes oxaliplatin resistance of CRC cells.Mechanistically, CCAL interacts directly with mRNA stabilizing protein HuR and promoting the expression of β-catenin [124].Exosomes from the normal fibroblasts also influence CRC cells.For example, Exosomal Wnts derived from fibroblasts can induce the differentiation of cancer cells to promote chemoresistance in CRC [125].
Exosomes from mesenchymal stem cells (MSCs) are closely related to the therapeutic efficacy of MSCs.After being ingested by CRC cells, MSC exosomes can exert their effects through the miR-100/mTOR/miR-143 axis in CRC cells, inhibiting the proliferation, migration, invasion, and metastasis of CRC cells and inducing CRC cell apoptosis.It suggests that MSC-exosome treatment and miR-100 restoration might be considered potential therapeutic strategies for CRC [126].MSC-exosomal miR-3940-5p inhibits the growth, metastasis, invasion, and EMT of CRC cells by targeting ITGA6 and the following TGF-β1 inactivation [127].The exosomes produced by bone marrow mesenchymal stem cells (BMSCs) contain abundant miR-4461.After being taken up by CRC cells, miR-4461 can directly target COPB2, leading to COPB2 downregulation and inhibiting the migration and invasion of CRC cells [128].Inhibited exosomal miR-424 from BMSCs inhibited malignant behaviors of CRC cells by targeting TGFBR3, thus suppressing the progression of CRC [129].BMSCs exosomes contain microRNA-16-5p, and exosomes containing microRNA-16-5p are swallowed by CRC cells, which inhibit the proliferation, migration, and invasion of CRC cells and promote apoptosis of CRC cells [130].Exosomes derived from MSCs are rich in miR-22-3p, which inhibits CRC cell proliferation and invasion by regulating RAP2B and PI3K/AKT pathways, suggesting the potential of hBMSCs-exo-miR-22-3p for the treatment of CRC in the future [131].
Some other cells may also affect the fate of CRC cells.For example, Granulocytic Myeloid-derived suppressor cells (G-MDSCs) promote the stemness of CRC cells through exosomal S100A9 [132].Adipocyte-derived exosomal MTTP could suppress ferroptosis and promote chemoresistance in CRC [133].MiR-203a-3p derived from hepatocyte exosomes increases the expression of Ecadherin in CRC cells and inhibits Src expression, which in turn leads to a decrease in the invasion rate of CRC cells [134].

The Potential Clinical Value of Exosomes in CRC
Due to their unique properties, exosomes contribute to many aspects of precise tumor diagnosis and treatment, including predicting prognosis and drug efficacy, dynamic monitoring, and precisely targeted drug delivery [14].

Exosomes in CRC Diagnosis and Prognosis
Because of the various cargos in the exosomes and the typical features on the surface of exosomes, they can reflect the status of their parent cells.Thus, exosomes have emerged as a platform with potentially broader and complementary applications to be used in the field of liquid biopsy for the diagnosis, prognostic analysis, and monitoring of cancer [135].In this manuscript, we summarized the exosomes used in the diagnosis and prognosis of CRC (Table 3, Ref. [55, 58,72,75,).
Exosomal LncRNA RPPH1 levels in blood plasma are higher in untreated CRC patients but lower after tumor resection.It displayed a better diagnostic value (AUC = 0.86) compared to CEA and CA199 and could serve as a potential target for therapy and diagnosis in CRC [55].The miR-208b derived from exosomes of CRC cells can promote Treg amplification by regulating PDCD4, and it has the ability to reduce the sensitivity of oxaliplatin-based chemotherapy in CRC.These research results suggest that the exosomal miR-208b can serve as a biomarker for predicting oxaliplatin treatment response and may become a new target for immunotherapy [72].Exosomal miR-221/222 promotes CRC progression and may serve as a novel prognostic marker and therapeutic target for CRC with liver metastasis [58].By deep sequencing, it has been demonstrated that miR-7641 has the potential to be a candidate for the non-invasive and specific molecular markers for CRC diagnosis and prognosis [167].Exosome-delivered FZD10 increases Ki-67 expression via Phospho-ERK1/2 and may be a promising novel prognostic and diagnostic biomarker for CRC [168].Elevated plasma GPC1 + exosomes and decreased plasma miR-96-5p and miR-149 expression may be specific markers for the diagnosis of CRC and targets for the treatment of CRC.Compared with the healthy control group, the percentage of GPC1 + exosomes in tumor tissue and plasma and the expression of GPC1 protein in exosomes in CRC patients before surgery were significantly increased.However, miR-96-5p and miR-149 expression in tumor tissue and plasma and GPC1 + exosomes in CRC patients were significantly reduced compared to healthy controls [169].The expression of exosomal miR-377-3p and miR-381-3p are decreased in CRC patients; they can serve as circulating biomarkers of diagnosis for CRC [136].CircCOG2 is associated with poor prognosis and can be used as a therapeutic target for CRC [137].Circulating or tissue-based miR-1539 derived from CRC cells may be used as a novel potential biomarker for CRC screening, as well as a predictor of poor clinicopathological behavior in tumors [138].Circulating exosomal miR-150-5p and miR-99b-5p derived from CRC cells may be considered diagnostic biomarkers for CRC [139].Hsa-miR-3937, contained in tumor-originated exosomes, is a potential and effective liquid biopsy marker for CRC detection and therapy [140].The downregulation of exosomal miR-548c-5p in serum predicts poor prognosis in patients with CRC, and thus it may be a critical biomarker for CRC diagnosis and prognosis [141].Circulating exosomal CPNE3 may be a diagnostic and prognostic biomarker for CRC [142].Plasma exosomal miRNA-139-3p is decreased in CRC and may act as a novel biomarker for early diagnosis and metastasis monitoring in CRC [143].CRC cell-derived exosomes are rich in miR-424-5p, and miR-424-5p is secreted into peripheral blood through exosomes.The circulating exosomes miR-424-5p can be used as a marker for early diagnosis of CRC [75].Exosomal miR-320d could significantly distinguish metastatic from nonmetastatic CRC patients and be a biomarker for metastatic CRC [144].The expression of LncRNA GAS5 and miR-221 in CRC tissue, patient plasma, and exosomes can be independent prognostic factors for CRC [145].Studies have revealed that the expression of hsa-circ-0004771 in circulating exosomes in CRC patients is significantly upregulated, which is expected to become a new biomarker for colorectal cancer diagnosis [146].After the circ_0006174-rich ex- Wnts Fibroblasts Induce the dedifferentiation of cancer cells to promote chemoresistance [125] osomes enter CRC cells, the DOX resistance of CRCs is improved through the miR-1205/CCND2 axis.Therefore, exosome circ_0006174 is expected to become a biomarker for the diagnosis of chemotherapy resistance in CRC [147].Serum exosomal miR-874 is significantly downregulated in CRC patients and has been negatively associated with distant metastasis, lymph node metastasis, differentiation, and advanced TNM stage.Thus, serum exosomal miR-874 may be a statistically significant independent prognostic factor for CRC patients [148].It is also demonstrated that exosomal FGB and β2-GP1 can be used as significant diagnostic efficacy for early CRC by using comprehensive proteomics analyses [149].Circulating exosomes miR-17-5p and miR-92a-3p may be non-invasive prognostic markers in patients with CRC, being either primary or metastatic CRC [150].Exosome lncRNAs FOXD2-AS1, NRIR, and XLOC_009459 are promising biomarkers for diagnosing CRC, especially early-stage CRC [151].Plasma exosomal miR-21 levels are a useful biomarker for the prediction of recurrence and poor prognosis in CRC patients with TNM stage II, III, or IV [152].MiR-6869-5p may play a cancersuppressing role in CRC, and serum exosome miR-6869-5p is a promising circulating biomarker for predicting the prognosis of CRC [153].Serum exosomal miR-122 is a novel potential diagnostic and prognostic biomarker in CRC patients with liver metastasis (LM) [154].Serum exosome circ-PNN may be a new biological marker for CRC detection, with non-invasive characteristics, and may play an important role in the tumorigenesis of CRC [155].Exosomal UCA1 is detectable and stable in the serum of CRC patients.Moreover, circulating exosomes containing UCA1 can predict the clinical outcome of treatment with cetuximab in CRC patients [156].Exosomal miR-150 can be used as a marker for the prognosis of CRC patients after surgical resection, and its low expression predicts poor prognosis after surgery.Therefore, exosome miR-150 may be a prognostic factor and potential therapeutic target for CRC [157].Exosome-derived QSOX1 is a new promising marker for early diagnosis and non-invasive risk stratification of CRC [158].Serum exosomal miR-150-5p is expressed differently between healthy people and CRC patients and is expected to be a potential molecular marker for the diagnosis and prognosis of CRC [159].KRAS mutations are very common in CRC, KRAS mutations are manifested in exosomes, and plasma exosome KRAS mutation status predicts the prognosis of patients with metastatic CRC [160].
The higher the expression of miR-125b-5p in plasma exosomes, the stronger the CRC aggressiveness, suggesting that miR-125b-5p in plasma exosomes may be related to the phenotype of CRC aggressiveness [161].Exosomederived miR-92b is down-regulated in the plasma of CRC patients and can be used as a molecular marker for early diagnosis of CRC [162].Circulating CCAT2 is present in exosomes, protected by exosomes, and can serve as a potential predictor of CRC [163].In CRC cells, miRNAs can be encapsulated into exosomes and secreted outside the cell, regardless of intracellular miRNA expression.Among these miRNAs, serum exosomes miR-126, miR-1290, miR-23a, and miR-940 are novel potential molecular markers for early diagnosis of CRC [164].In patients with advanced and recurrent CRC, plasma exosome microRNA-125b can be used as a molecular marker for detecting mFOLFOX6based chemotherapy resistance [165].A group of miRNAs which includes miR-181b, miR-193b, miR-195, and miR-411, is able to detect lymph node metastasis in the exosomal vs. cell-free component.This suggests that exosomal miRNA-based liquid biopsy features can strongly identify invasive submucosal CRC (T1 CRC) patients at risk of lymph node metastasis in the preoperative setting [166].

Exosomes in CRC Clinical Transformation
Because of the properties and functions of exosomes, they can be designed for drug or functional nucleic acid delivery [170] and have the therapeutic potential to be used in the treatment of CRC.For example, engineered exosomes have been used to simultaneously deliver the anticancer drug 5-FU and the miR-21 inhibitor oligonucleotide (miR-21i) to Her2-positive cancer cells.The results revealed that systematic administration of exosomes loaded with 5-FU and miR-21i in tumor-bearing mice showed significant anti-tumor effects in colon cancer [171].Studies have shown that the expression of PGM5 antisense RNA 1 (PGM5-AS1) in colon cancer is induced by GFI1B.PGM5-AS1 prevents colon cancer cells from proliferating, migrating, and acquiring oxaliplatin tolerance, and engineered exosomes that co-deliver PGM5-AS1 and oxaliplatin can reverse colon cancer resistance [172].MiR-506-3p delivered to CRC cells via exosomes reduces CRC proliferation and induces apoptosis, suggesting that delivery of miR-506-3p to CRC cells via exosomes may become a novel diagnosis and treatment method for CRC [173].Tumor-derived exosomes (TEXs) may induce beneficial anti-tumor immune responses, and TEXs have shown certain beneficial antitumor properties in addition to miRNA delivery functions, suggesting that the introduction of TEX-miR-34a may be a new promising approach for combination therapy for CRC [174].Studies have shown that dendritic cells are loaded with exosomes from cancer stem cell-rich spheroids, which may be a new potential immunotherapy approach [50].It has been reported that the ascites-derived exosomes (Aex), in combination with the granulocyte-macrophage colony-stimulating factor (GM-CSF), could be used in the immunotherapy of CRC.The phase I clinical trial suggested that AEX combined with GM-CSF immunotherapy for advanced CRC is feasible and safe and may be an alternative to immunotherapy for advanced CRC [175].
Although exosomes have been considered promising emerging therapeutic options and tumor neo-antigen drug delivery tools and carriers in tumor precision therapy, there are still many issues waiting to be resolved.Nowadays, exosomes can be isolated and purified by ultracentrifugation, density gradient centrifugation, polymer precipitation, ultrafiltration, and size exclusion chromatography; however, flexible and fast detection platforms are still lacking.Moreover, how to load exosomes with specific cargo still need to be explored.Because few studies have focused on the fusion-based loading methods present, their detrimental effects on recipient cells remain unclear [14].

Conclusions and Prospects
Exosomes are derived from the endosomal pathway of nearly all cells and may be considered a cell-to-cell system in the human body that can mediate near-and long-distance intercellular communication and affect various aspects of cell biology.They are involved in different physiological and pathological processes, such as the communication of the nervous system, reproduction and development, inflammatory responses, cardiovascular diseases, and cancer development.
CRC is one of the most highly malignant tumors worldwide.Numerous studies have revealed that exosomes play important roles in tumor growth, invasion, metastasis, angiogenesis, immune evasion, TME remodeling, metabolic reprogramming, therapy resistance, and some other processes of CRC.Because of these features and functions of exosomes, they may be used in the fields of diagnosis, prognosis, and treatment of CRC.We have reviewed the current state of studies focused on the exosomes in CRC.We have compared the effects of tumor-derived and nontumor-derived exosomes on the processes of CRC, summarized the clinical value of various types of exosomal cargos, and presented the potential of exosomes to be used in the clinical transformation process.
However, there are still many issues waiting to be resolved before exosome-based therapy can be used in the clinical treatment of CRCs.First, it is still a problem to obtain a large number of exosomes quickly so far.The isolation and purification of exosomes can be realized by the following methods, such as separation based on the size of density or particle, sedimentation or phase, affinity, microfluidic systems, or thermophoretic enrichment.However, the efficiency, quantity, and quality of exosomes separated by different separation methods are different, and there is still a lack of unified methods that can quickly and effectively obtain exosomes.Moreover, how to equip exosomes with specific drugs is also a problem.There are two methods for loading exosomes with drug, endogenous and exogenous.The two methods both have their own disad-vantages; the endogenous method is not accurate in quantifying the specific substances in the exosomes, while the loading efficiency is not high using the exogenous methods.Thus, how to equip exosomes with specific drugs is also a question that needs to be resolved.Furthermore, the longterm efficacy and safety of using exosomes in the treatment of CRC remain unclear.Therefore, there is a need for comprehensive clinical studies or trials of exosomes used in CRC.It is possible that with technological developments and further clinical trials, exosomes will be a real emerging tool for the "cell-free" treatment of CRC.

Fig. 1 .
Fig. 1.Biogenesis and intercellular communications of exosomes.The plasma membrane is double invaginated, forming the intracellular multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs).MVBs fuse to the plasma membrane, and ILVs are ultimately secreted as exosomes through exocytosis.The intercellular communications of exosomes depend on the interactions between ligands and receptors.When the exosomes meet the proper recipient cells, they can directly fuse with cell membranes or be endocytosed by recipient cells and release the cargo into target cells.

Fig. 2 .
Fig. 2. Exosomes play a role in physiological and pathological processes.Exosomes are involved in basic physiological processes such as immune response, neural communication, gamete maturation, fertilization, embryo implantation, organ development, cell differentiation and matrix synthesis, melanin synthesis, cell proliferation, homeostasis, and maturation.They are also involved in some pathological processes, such as immune disturbance, tumorigenesis, cancer cell migration and metastasis, angiogenesis, metabolic reprogramming, tumor microenvironment (TME) formation, pathogen infection, neurodegenerative diseases, and cardiovascular disease.
Tumor-derived exosomes (TDEs) were first named in 1981, and it has attracted much attention in the past years[42].Exosomes act as signal messengers or transducers for communications between cells[43].Once the recipient cells take up TDEs, the exosomes are internalized, and exosomal contents (such as microRNA, lncRNA, and cir-cRNA) are released.The recipient cells respond to these exosomal contents by changing their phenotypes.This process is finely regulated and specifically determined by complex surface molecules on the extracellular vesicles and the recipient cell membrane [44].This paper will summarize the functions and mechanisms of tumor-derived exosomes in CRC (Fig.3;

Fig. 3 .
Fig. 3. Mechanism of tumor-derived exosomes in colorectal cancer (CRC).Exosomes derived from CRC cells can transport their cargo to other cells and play an important role in the occurrence and progression of CRC, including tumor growth, invasion and metastasis, angiogenesis, immune evasion, TME remodeling, metabolic reprogramming, and therapy resistance.

3. 1 . 1
Tumor Growth One of the typical hallmarks of cancer is sustaining proliferation signaling, ultimately causing the rapid growth of the tumor.CRC tumor-derived exosomes (CTDEs) can regulate the growth of CRC cells through a variety of pathways, such as involvement in cell proliferation, cell cycle, and apoptosis.For example, circLPAR1 is encapsulated by CRC cell exosomes, internalized by CRC cells, and inhibits tumor growth.The investigation of underlying mechanisms showed that exosome circLPAR1 might bind to eIF3h directly and inhibit METTL3-eIF3h interaction, ultimately reducing the translation of BRD4 [45].Exosome-carried circ_0094343 derived from CRC cells plays an inhibitory role in the proliferation and clone formation of HCT116 cells via the miR-766-5p/TRIM67 axis [46].Exosomal circ_PTPRA can function as a competing endogenous RNA (ceRNA) and increase the expression of SMAD4 by binding to miR-671-5p, ultimately inducing CRC cell cycle arrest and inhibiting cell proliferation [47].It has also been demonstrated that a hypoxic microenvironment in CRC may promote tumor cells to release exosomes rich in miR-410-3p.These exosomes may transfer to normoxic cells and enhance the proliferation of normoxic CRC cells [93].Exosomal circEPB41L2 derived from CRC cells could suppress CRC cell proliferation and promote apoptosis by serving as a sponge for miR-21-5p and miR-942-5p and regulating the PTEN/AKT signaling pathway [48].CRC cell exosome miR-224-5p promotes cell proliferation by downregulating CMTM4, thus promoting the malignant transformation of human normal colonic epithelial cells [49].Oncogenic mutant β-catenin contained in the extracellular vesicles can activate the Wnt signaling pathway in the recipient cells and increase tumor burden in vivo [50].
have shown that CTDEs are associated with the invasion and metastasis of CRC.For example, tumor-derived exosomes miR-934 can target PTEN, resulting in downregulation of the PTEN expression, which in turn activates the PI3K/AKT signaling pathway to induce M2 macrophage polarization, ultimately promoting CRC liver metastasis [51].Cancer-derived exosomal circPACRGL can absorb miR-142-3p/miR-506-3p as a sponge and facilitate the TGF-β1 expression, thus promoting CRC cell migration and invasion [52].The exosomes of CRC cells which have undergone EMT contain miR-106b, and miR-106b in exosomes can directly inhibit PDCD4 post-transcriptionally, activate the PI3Kγ/AKT/mTOR signaling pathway, and promote the M2 polarization of macrophages.Activated M2 macrophages promote EMT-mediated CRC cell migration, invasion, and metastasis in a positive feedback manner [53].The hypoxic microenvironment in the CRC primary lesion promotes exosome release, selectively initiating the formation of a favorable premetastatic niche in the liver.Molecular mechanism exploration has found that Kupffer cells (KCs) can engulf exosomes containing miR-135a-5p, which enter the liver from the blood circulation.Exosomal miR-135a-5p initiates the large tumor suppressor kinase 2-yes-associated protein-MMP7 axis and promotes CRC liver metastasis [54].Exosome-encapsulated miR-NAs, which are from CRC cells, can enhance the M2 polarization of macrophages and contribute to CXCL12/CXCR4induced liver metastasis of CRC [95].LncRNA RPPH1 is significantly upregulated in CRC tissues, and it could be encapsulated in the exosomes of CRC cells.Then, exosomes rich in RPPH1 are transported into macrophages and mediate macrophage M2 polarization, thereby promoting metastasis and proliferation of CRC cells [55].Hypoxia can induce the secretion of circ-133-rich exosomes, which are transported into normoxic cancer cells.Circ-133 contained in the exosomes promotes cell migration via the miR-133a/GEF-H1/RhoA axis [56].Exosomal miR-27b-3p secreted after epithelial-mesenchymal transformation of CRC cells increases vascular permeability and promotes the production of circulating tumor cells (CTCs), thus promoting the metastasis of CRC[96].HuR, which is an RNAbinding protein contained in CRC-derived exosomes, can stabilize c-Myc mRNA and promote lung cell proliferation[97].Fusobacterium nucleatum infection may stimulate tumor cells to generate exosomes rich in miR-1246/92b-3p/27a-3p and CXCL16/RhoA/IL-8 that are delivered to uninfected cells to promote metastatic behaviors in CRC[57].Exosomal miR-221/222 targets SPINT1 and plays a key role in forming a favorable premetastatic niche (PMN), thus leading to the metastasis of CRC[58].Exosometransmitted miRNA-335-5p derived from metastatic CRC cells promotes CRC migration, invasion, and metastasis which may be due to EMT caused by RASA1[59].MiR-200c-3p in exosomes derived from CRC cells influences the exosomal expression of ZEB-1 mRNA, and further alters ZEB-1 protein expression in CRC cells, ultimately inhibiting CRC migration and invasion after LPS stimulation[60].

Fig. 4 .
Fig. 4. The Mechanism of non-tumor-derived exosomes function in CRC.Exosomes derived from non-CRC cells can transport their cargo to CRC cells and affect most pathological processes in CRC, including stemness, EMT, migration, invasion, metastasis, angiogenesis, apoptosis, tumor growth, and therapy resistance.