Role of exosomal ncRNAs released by M2 macrophages in tumor progression of gastrointestinal cancers

Summary

Macrophages (MΦs) type 2 (M2) play crucial roles in the pathogenesis of gastrointestinal cancers (GIC) by enhancing tumor progression, invasion, and metastasis. Polarized M2 has been linked to the increase of GIC tumorigenesis and drug resistance. Several studies reported that M2-derived exosomal non-coding RNAs (Exos-ncRNAs) play pivotal roles in the modulation of the GIC tumor microenvironment (TME) and mostly promote drug resistance and immunosuppression. The impact of M2-Exos-ncRNAs is attributed to altered signaling pathways, enhancement of immunoregulatory mechanisms, and post-transcriptional modulation. Recent studies described novel targets in M2-TAMs-derived Exos-ncRNAs and potential promising clinical outcomes such as inhibiting tumor formation, invasion, and metastasis. Highlighting current knowledge of M2-Exos-ncRNAs involved in GIC pathogenesis and immunomodulation would thus be a significant contribution to improving clinical outcomes. In this review, we summarize recent updates on the role of M2-TAMs-Exos-ncRNAs in GIC pathogenesis, immunosuppression, and drug resistance. A deep understanding of M2-TAMs-derived Exos-ncRNAs could help to identify potential biomarkers and therapeutic targets.

Graphical abstract

Immunology; Cancer

Introduction

Gastrointestinal cancers (GIC) are a group of malignant cancers counting for 26% of the global incidence of cancers and 35% of life-threatening cancers.^1,2 They consist of five major cancers: esophageal cancer (EC), gastric cancer (GC), liver cancer (LC) or hepatocellular carcinoma (HCC), pancreatic cancer (PaC) or pancreatic ductal adenocarcinoma (PDAC), and colorectal cancer (CRC). Usually, late diagnosis, poor prognosis, and low overall survival are mostly dominant in GIC patients. Despite advances in surgical procedures that improved outcomes, GIC malignancies continue to have high recurrence rates.^3,4,5 Thus, several studies focused on the tumor microenvironment (TME) in terms of tumor cell biology, immune cell infiltration, and the interaction between the tumor and the immune system. Macrophages (MΦs), as one of the critical myeloid immune populations in the TME,⁶ were investigated enormously in terms of subtypes, polarization, interaction with tumor cells, and immunomodulatory effects.⁷ The population of MΦs infiltrating tumors (TAMs) usually present two polarized subtypes; classical M1 and alternative M2.⁸ Adding to that non-activated phenotype is named M0.⁹ TAMs are well known as tumor enhancers by modulating immune responses in the TME; however, two clinical studies on multivariate analyses of CRC patients (46 cases in Sweden and 160 cases in China) revealed that increased CD68TF_Hotspot MΦs were associated with good survival and inhibition of tumor growth.^10,11 Importantly, both studies did not clearly distinguish the phenotype of these reported TAMs, which could be M1-TAMs as other literature linked the anti-tumor effects to M1-TAMs. It was reported that M1-TAMs show inflammatory boosting activity through activating T cell responses and tumor cell killing by releasing reactive oxygen/nitrogen species (ROS/RNS) besides the pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α), IL-6, and IL-1β.¹² In contrast, the M2-TAM phenotype was considered mostly as “bad guys” in the TME. It exerts pro-tumorigenic effects through different pathways, such as the suppression of anti-tumor immune responses, which promote tumor growth, inducing tumor proliferation, angiogenesis, metastasis, invasion, and mediating chemotherapy resistance.^13,14 Several studies demonstrated the crucial role of M2-TAMs in the pathogenesis of GIC.¹⁵ The link between the increasing number of M2-TAMs in GIC and poor prognosis has been extensively studied.^16,17,18 Furthermore, M2-polarized TAMs not only produce tumor-supporting chemokines and cytokines but also release highly effective exosomes (Exos), which in turn contribute to the promotion of tumor invasion and metastasis, in addition to the modulation of epithelial-mesenchymal transition (EMT) into cancer stem cells.^19,20

As known, Exos are a subtype of extracellular vesicles (EVs) with a diameter of ∼40–150 nm in size.²¹ It is secreted by various cell types (stromal cells, stem cells, immune cells, tumor cells, and epithelial cells) during physiological and pathophysiological conditions to facilitate cell functions.²² Various techniques have been established to investigate the biochemical and physicochemical properties of Exos despite the lack of standardized methods.²³ It is possible to characterize Exos by either their physical properties, including size, shape, surface charge, and density, which can affect their cell adhesion and also uptake into neighboring cells,²⁴ or by their biological properties, including their cargo (such as RNA species and proteins) and membrane antigens. Techniques that can be used for measuring density, size, shape, and surface charge in a suspension of vesicles (Exos), including dynamic light scattering (DLS), electron microscopy (EM), nanoparticle tracking analysis (NTA), and tunable resistive pulse sensing (TRPS).²⁵ In general, ncRNAs in Exos are more stable than free RNAs in the extracellular fluid, due to the protective surrounding lipid bilayer.²⁶ Exos-ncRNAs released by MΦs have various physical and chemical properties, depending on the type of RNAs (such as miRNA, lncRNA, and circRNA). Physically, they have spherical to cup-shaped with a small size, while their chemical properties depend on the source cells and the species of ncRNA. Furthermore, their stability depends on the specific ncRNA species and also the condition they are exposed to. These are just some of the general physical and chemical properties of Exos-ncRNAs released by MΦs.

Exos in the TME may promote cellular phenotypic and genetic changes, leading to enhance drug resistance, heterogeneity, and plasticity by releasing specific ncRNAs targeting molecular pathways, which regulate apoptosis and immune suppression.²⁷ Tumor drug resistance could be attributed to the significant development of tumor heterogeneity due to the transferred Exos-ncRNAs.²⁸ Furthermore, another study linked high levels of Exos-miR32-5p in the TME of HCC to multidrug resistance due to induction of tumor cell heterogeneity.²⁹ A recent study compared the results of high-throughput sequencing of CRC cell line (SW620) with normal colon epithelial cells (NCM460) revealed that Exos enriched with miR-2277-3p and miR-26b-3p were responsible for heterogeneous paracrine effects in CRC.³⁰ Thus, Exos-ncRNAs play a significant role in promoting a switch in tumor heterogeneity and plasticity.³¹ They induce drug resistance by creating specific signals inducing tumor heterogeneity, which enhance tumor growth and metastasis.³² Additionally, Exos-ncRNAs can promote epigenetic oncogenesis by regulating DNA methylation and histone modifications.^33,34

Recently, the focus on targeting non-coding RNAs (ncRNAs) cargo in the Exos released by M2-TAMs is increasing due to promising outcomes. The silencing of miR-21-5p in M2-Exos significantly abolished the pro-metastatic effects of these M2-Exos in renal cell carcinoma (RCC).³⁵ A recent study has shown that inhibiting lncRNA-CRNDE in M2-Exos enhances sensitivity to cisplatin (CDDP) in GC.³⁶ Another study showed that the knock-down of miR-223 M2-Exos reduced resistance to doxorubicin in GC.³⁷ Moreover, inhibition of miR-501-3p in M2-Exos inhibited tumor formation and metastasis in PDAC. This review intends to emphasize the importance of M2-TAMs-derived Exos-ncRNAs in the pathogenesis of GIC and highlights the potential opportunities in targeting M2-TAMs-Exos-ncRNAs in immunotherapies of GIC.

Interaction between GIC and M2-TAMs-Exos-ncRNAs in the TME

Previous studies suggested that TAMs accumulate in the tissue of GICs due to cancer-released mediators such as CCL-2 and CSF1, which recruit MΦs to infiltrate tumor tissues.^17,38,39 Several pieces of evidence highlighted the role of Exos released by cancer cells in the intracellular communication between TAMs and tumor cells, thus promoting M2 polarization,^40,41 as shown in Figure 1. Tumor cell-derived Exos transfer many molecules into TAMs such as oncoproteins, miRNAs, lncRNAs, circRNAs, and lipids that actively mediate polarization into M2 and thus enhancing tumor progression.⁴² A recent report highlighted the important role of Exos-ncRNAs in the polarization and function of MΦs.⁴³ As presented in Figure 1, Exos released by M2-TAMs were linked to tumor cell proliferation, invasion, and metastasis.⁴⁴ As reported, Exos-miR-934 regulates the activation of PI3K/AKT signaling, which induces M2-TAM polarization in CRC by inhibiting the expression of phosphatase and tensin homologs (PTEN).⁴⁵ Polarized M2-TAMs induced CRC liver metastasis and promoted pre-metastatic niche formation by secreting CXCL13, which activates a CXCL13/CXCR5/NF-κB/p65/miR-934 positive feedback loop in CRC cells.⁴⁵ Furthermore, Exos-miR-452-5p released by HCC induced MΦs polarization into M2 phenotype by targeting TIMP3, which promoted HCC progression. This was confirmed by the inhibition of Exos-miR-452-5p, which reduced M2 polarization and tumor progression.⁴⁶ A recent study reported on the role of Exos-miR-19b-3p released by lung adenocarcinoma in the polarization of MΦs into M2 by targeting PTPRD, which induced dephosphorylation of STAT3.⁴⁷ Polarized M2 by miR-19b-3p released Exos-linc00273, which promoted tumor metastasis. The same Exos-miR-19b was found to be elevated in the plasma of PDAC patients,⁴⁸ but its relationship with M2 polarization in PDAC remains undisclosed. Exos-circ0048117 in esophageal squamous cells (ESC) plays a significant role in the induction of TAM polarization into M2 by sponge miR-140, resulting in increased TLR4 expression.⁴⁹ M2-TAMs promote tumor cell invasion and migration by secreting TGF-β, Arg1, and IL-10.⁴⁹ Moreover, Exos-miR-301a-3p derived from PDAC showed a significant promotion of M2 polarization by inducing PTEN/PI3Kγ activation, and these M2-TAMs enhanced tumor growth and metastasis.⁵⁰ On the other hand, Exos-ncRNAs released by M2-TAMs showed a strong association with tumor progression, invasion, and metastasis. M2-Exos-miR-21-5p promoted tumor metastasis in RCC.³⁵ In the avian embryo chorioallantoic membrane in vivo tumor model as well as in vitro, tumor metastasis was prevented by miR-21-5p knockdown in M2-TMAs-Exos.³⁵ Furthermore, M2-Exos-miR-487a significantly promoted tumor proliferation and progression in vitro and in vivo in GC.⁵¹ Adding to that, Exos-miR-365 secreted by M2-TMAs was reported as a significant promoter of PDAC progression by downregulating the expression of BTG2, leading to the promotion of FAK/AKT signaling.⁵² In addition, high levels of Exos-miR-501-3p released by M2-TMAs in the TME of PDAC promoted tumor formation and metastasis by targeting the TGFBR3 gene, which increased the expression of TGF-β.⁵³ Interestingly, inhibition of M2-Exos-miR-501-3p significantly reduced PDAC formation and metastasis. Another study reported that Exos released by M2-TAMs with the increase of EC invasion and metastasis by transferring lncRNA (AFAP1-AS1) that target miR-26a, leading to the activation of transcription factor 2 (ATF2) axis. Downregulation of miR-26a and upregulation of ATF2 promoted the increase of EC invasion and metastasis.⁵⁴ X-chromosome-linked inhibitor of apoptosis protein (XIAP) has been detected in different types of tumors, which can play an important role in the inhibition of active apoptosis execution phase (caspase 3 and 7), and also apoptosis initiation phase (caspase 9) via subtypes of baculovirus IAP repeat (BIR2 and BIR3) domains of XIAP, resulting in blocking cell apoptosis, and promoting tumor progression.^55,56 Additionally, miR-122-5p has been recognized as a key biological regulator, which can play a key role in modulating cell apoptosis and tumor progression.⁵⁷ Therefore, an increased level of miR-122-5p could be related to enhanced cell apoptosis and suppression of tumor growth.⁵⁸ A recent study reported that low expression of lncRNA SBF2-AS1 in M2-TAMs-Exos leads to up-regulation of miR-122-5p and accelerates the growth of PDAC through enhancing the XIAP gene.^56,59 Thus, high levels of XIAP and low expressions of miR-122-5p play crucial roles in preventing apoptosis of PDAC cells.^56,59 Altogether, the interaction between GIC and M2-TAMs is mostly mediated via Exos-ncRNAs besides other mediators such as cytokines. The focus on the Exos-ncRNAs communication is probably the most appropriate way to provide an efficient contribution to the clinical treatment of GIC.

Figure 1.

Role of tumor cell-derived Exos-ncRNAs and M2-TAMs-Exos-ncRNAs in the polarization of TAMs in the TME of GICs and modulation of TAM function

This schematic model shows the link between Exos-ncRNAs released by tumor cells and the polarization of TAMs into M2. The figure presents the role of M2-Exos-ncRNAs in the GIC tumor progression.

Role of M2-Exos-ncRNAs in the pathogenesis of different GIC types

M2-TAM accumulation in the TME of GICs is typically associated with poor prognoses and resistance to cancer therapy. Many clinical and experimental data revealed that M2-TAMs promote tumor heterogeneity, progression, and metastasis via Exos-ncRNAs,⁶⁰ as presented in Figures 2 and 3. In the past few years, there has been an increased emphasis on the role and function of Exos-ncRNAs released by M2-TAMs, which can contribute to tumor progression. M2-TAMs-Exos-ncRNAs play crucial roles in modulating genomic imprinting, cellular differentiation, cell cycle, tumor progression, and a variety of biological activities. M2-TAMs-Exos-ncRNAs control transcription, translation, epigenetic modifications, and function of proteins.⁶¹ Different recent studies reported the functional roles of Exos-ncRNAs released by M2-TAMs in the development and progression of GIC, as summarized in Table 1. Although limited, we provide some evidence to declare the role of M2-TAMs-Exos-ncRNAs in different GICs, as described in the following.

Figure 2.

Role of Exos-ncRNAs in the TME of GICs. Exosomal ncRNAs from M2-TAMs are released into the TME and distant organs, delivering ncRNA, which can promote tumor progression

This schematic model presents the potential impact of Exos-ncRNAs released by M2-TAMs on tumor proliferation, angiogenesis, invasion, metastasis, drug resistance, and tumor immune escape.

Figure 3.

Potential biological functions of Exos-ncRNAs in the TME of GICs

This schematic diagram presents the impact of Exos-ncRNAs on cellular and molecular mechanisms involving GIC pathogenesis. Exos in GICs promote tumor progression and metastasis by transferring ncRNAs that enhance the expression of oncogenes and target tumor suppressor genes. They also induce onco-epigenetic modifications that induce heterogeneity and drug resistance in GIC by upregulating methylation of tumor suppressor genes and enhancing transcription of tumor proliferation genes. Furthermore, Exos-ncRNAs enhance proliferation and differentiation of immunosuppressive Tregs, and at the same time inhibit innate and adaptive anti-tumor immune responses. Exos can also transfer ncRNAs that induce CD8⁺ T cell exhaustion by inducing the upregulation of immune checkpoints such as PD-1 and TIM-3.

Table 1.

Summary of key studies on TAM-Exos-ncRNAs driving GIC progression

ncRNA	Expression	Tumor type	M1/M2	Target Gene	Mechanism	Cell type	Reference
miR-125a and b.	Low	HCC	TAMs	Significant decrease in CD90 expression	Promotes HCC cell proliferation and stem cell properties.	Human HCC cell lines	Wang et al.62
miR-326	High	HCC	M1-TAMs	Significant reduction in NF-κB and CD206 expression	Suppresses proliferation, migration, and invasion as well as advances apoptosis of HCC through down-regulating NF-κB expression.	Human HCC cell lines	Bai et al.63
miR-660-5p	High	HCC	M2-TAM	Low levels of KLF3	Augments the development of liver cancer.	Human cell line (HepG2).	Tian et al.64
miR-27a-3p	High	HCC	M2-TAM	Downregulation of thioredoxin-interacting protein (TXNIP).	Promotes stemness, proliferation, migration, invasion, and drug resistance of HCC cells.	Human HCC cells	Li et al.65
miR-92a-2-5p	High	HCC	M2-TAMs	Decreased level of androgen receptor (AR)	Enhances the HCC invasion via regulating the AR/PHLPP/p-AKT/β-catenin signaling pathway.	Human liver cancer cells	Liu et al.66
lncMMPA	High	HCC	M2-TAMs	Decreased level of MiR-548s and increased level of ALDH1A3	Regulates glucose metabolism and cell proliferation in HCC cells.	Human HCC cell lines.	Xu et al.67
miR-223 and miR-142	High	HCC	MΦs	Decreased levels of stathmin-1 (STMN1) and insulin-like growth factor-1 receptor (IGF-1R)	Inhibits the cellular proliferation of HCC.	Human HCC cell lines	Aucher et al.68
miR-365	High	PDAC	M2-TAMs	Significant decrease in B-cell translocation gene 2 (BTG2) and increase in the FAK/AKT.	Promote cell proliferation, migration, and invasion by activating the FAK/AKT pathway.	Human PDAC cell lines (PANC-1, BxPC-3, MIA Paca-2, and Capan-2).	Li et al.52
miR-365	High	PDAC	M2-TAMs	Increase levels of NTP/CDA	Enhance gemcitabine resistance in PDAC.	Animal PDAC cell line (PDAC K989 cell)	Binenbaum et al.69
miR-221-5p and miR-155-5p	High	PDAC	M2-TAMs	low levels of E2F2	Promote tumor growth and angiogenesis.	Mouse PDAC cell line	Yang et al.70
miR-501-3p	High	PDAC	M2-TAMs	Inhibits TGFBR3	Enhances the invasion and migration of PDAC via the activating TGF-β pathway.	Human PDAC cell lines	Yin et al.53
lncRNA SBF2-AS1	Low	PDAC	M2-TAMs	Increases miR-122-5p and restricts X-linked inhibitor of apoptosis protein (XIAP).	Inhibits the tumorigenic ability of PC cells.	Human PaC cell lines	Yin et al.59
miR-16-5p	High	GC	M1-TAMs	Decrease PD-L1	T cell immune responses are activated by miR-16-5p through regulating PD-L1.	Human GC Cell Line (AGS and NCI-N87).	Li et al.71
miR-223	High	GC	TAMs	Inhibit F box/FBXW7	Enhances doxorubicin resistance.	Human GC cell line	Gao et al.37
miR-21	High	GC	M2-TAMs	Downregulation of PTEN	Promotes chemotherapy (cisplatin) resistance and inhibits cell apoptosis.	Mouse GC MFC cell and human GC MGC-803 cell lines.	Zheng et al.72
miR-487a	High	GC	M2-TAMs	Downregulation of T cell intracellular antigen-1 (TIA1)	Enhances tumor progression of GC cells.	HS-746 T cells line (human gastric)	Yang et al.51
miR-183-5p	High	CRC	M2-TAMs	Inhibits thioesterase superfamily member 4 (THEM4)	Promotes CRC cell proliferation, invasion, and metastasis by facilitating the AKT/NF-κB -signaling pathway.	Human CC cell lines.	Zhang et al.73
miR-186-5p	High	CC	M2-TAMs	Inhibit DLC1	Promotes the proliferation and motility of CC cells.	Human CC cell lines	Guo et al.74
MiR-193b-3p	High	PaC	M2- MΦs	Low level of TRIM62	Promotes proliferation, invasion, migration, and glutamine uptake of PaC cells in vivo and in vitro.	SW1990 cell line (Human pancreatic adenocarcinoma cell line)	Zhang et al.75

Hepatocellular cancer

M2-TAMs modulate various factors in the TME of HCC to facilitate cancer progression. ncRNAs derived from M2-TAMs-Exos have been reported to upregulate immunosuppressive responses, supporting HCC initiation and progression via transferring miR-92a-2-5p.⁶⁶ This study clarified that miR-92a-2-5p can enhance HCC invasion by decreasing the expression of androgen receptors (AR) by altering PHLPP/p-AKT/β-catenin signaling.⁶⁶ It is believed that sex hormones, including androgen, interact with immune cells such as natural killer cells, monocytes, macrophages, and lymphocytes through their sex hormone receptors, which regulate chemokine and cytokine production that affect immune cell proliferation, differentiation, and maturation.⁷⁶ Further studies demonstrated that overexpression of M2-TAMs-Exos-miR-27a-3p plays an important role in enhancing HCC cell stemness, invasion, and migration by targeting thioredoxin interacting protein (TXNIP).⁶⁵ Additionally, M2-TAMs-Exos-miR-660-5p was reported to promote HCC by targeting kruppel-like factor 3 (KLF3).⁷⁷ It has been shown that lncMMPA is expressed more in TAMs than in HCC cells.⁷⁸ Interestingly, increased levels of lncMMPA play key roles in the polarization of M2-MΦs.⁶⁷ Functionally, M2-TAMs-Exos-lncMMPA regulates glucose metabolism in HCC, which promotes tumor cell proliferation.⁶⁷ Based on these studies, M2-TAMs-Exos-ncRNAs are important factors in the development of liver cancer through suppressing immune cells and accelerating cancer cell proliferation, which was linked with precancerous lesions and inhibition of tumor suppressor genes. Thus, they may have the potential to be used in the diagnosis, therapeutics, as well as management of liver cancer.

Gastric and esophageal cancers

The role of M2-TAMs-Exos-ncRNAs in the progression of GC has been well established. They play diverse roles in the initiation and progression of tumors. A recent study showed that Exos-miR-487a derived from M2-TAMs modulated gene expression of TIA1 in vivo and induced cell proliferation and progression of GC cells.⁵¹ Further to that, M2-TAMs-Exos-miR-21 inhibited GC cell apoptosis by reducing the expression of PTEN, leading to activating PI3K/AKT pathway.⁷² Another study reported that miR-21 acts as a tumor promoter by targeting the programmed cell death 4 (PDCD4) gene and preventing GC cell apoptosis by inhibiting the expression of PDCD4.⁷⁹ Evidence supports that miR-21 plays a key role in regulating tumor suppressor genes and apoptosis-associated genes through enhancing cell survival and conferring resistance to therapies. PTEN and PDCD4 are known as tumor suppressor genes, thus loss of these genes leads to the activation of anti-apoptotic signaling, which is involved in cancer progression and chemoresistance. Other studies revealed that M2-TAMs-Exos-miR-588 contributed to enhancing GC cell proliferation and inhibiting apoptosis by targeting cylindromatosis (CYLD).⁸⁰ In EC, M2-TAMs-lncRNA-AFAP1-AS1 reduced the level of miR-26a and increased the expression of transcription factor 2 (ATF2), which induces cancer cell migration, invasion, and metastasis.⁵⁴ Altogether, M2-TAMs-Exos-ncRNAs in GC mainly induce drug resistance, besides eliciting immunosuppression and tumor cell proliferation, and attempts to target them have potential to inhibit tumor growth and promote anti-GC responses.

Pancreatic ductal adenocarcinoma (PDAC)

M2-TAMs-Exos-ncRNAs were linked to the aggressive growth of PDAC. M2-TAMs-Exos-miR-501-3p promoted PDAC by inhibiting transforming growth factor β-receptor-3 (TGFBR3), which triggers tumor cell migration and metastasis.⁵³ Interestingly, TGF-β showed a role in the enhancement of TAMs-Exos-miR-501-3p, which participates in the initiation of PDAC growth by inhibiting the inflammatory immune response and eliciting immunosuppression in the TME. A recent study showed that M2-TAMs-Exos-miR-365 promoted PDAC progression by inhibiting the expression of B-cell translocation gene 2 (BTG2), leading to activating the FAK/AKT signaling pathway.⁵² Another study reported that M2-TAMs-Exos-miR-155-5p and miR-221-5p contributed to the promotion of tumor growth and angiogenesis of PDAC by targeting E2F2.⁷⁰ E2F2 is a member of the E2F family of transcription factors, that play an important role in the regulation of the cell cycle progression,⁸¹ and loss of E2F2 expression accelerates tumor growth and metastasis.⁸² Moreover, M2-TAM-Exos-miR-21-5p was highly expressed in PaC and promoted tumor progression through enhancing PaC stem cell activity and differentiation via down-regulation of KLF3.⁸³ MiR-21 is considered as an oncogenic effect in different tumors, and its overexpression can promote solid tumor development.⁸⁴ Thus, miR-21 inhibition may reduce fibrosis and suppress populations of immunosuppressive cells simultaneously, thereby slowing premalignant progression and shrinking the tumors.⁸⁵ Overall, M2-TAMs-Exos-ncRNAs are essential for tumor growth, development, and in drug resistance. Thus, new findings and insights on M2-TAMs-Exos-ncRNAs could improve clinical responses in PDAC.

Colorectal cancer (CRC)

Studies on the M2-TAMs-Exos-ncRNAs in CRC are few. Although limited, many findings highlighted the key role of TAMs in the TME of CRC. An in vivo study revealed that M2-TAMs-Exos-miR-186-5p promoted CRC development and progression through targeting DLC1.⁷⁴ Furthermore, M2-TAMs-Exos-microRNA-155-5p was linked to the growth of CRC due to enhancing immune escape by downregulating the expression of ZC3H12B.⁸⁶ ncRNAs also play a key role in regulating the expression of proteins, which are required for tumor development. For instance, in CRC, primary cancer cells release miR-21-containing Exos, which can be transferred into the liver via circulation and engulfed by MΦs.⁸⁷ A recent study reported that M2-TAMs-Exos-miR-155-5p and M2-TAMs-Exos-miR-21-5p promoted CRC invasion and migration by targeting the transcription activator BRG1.⁴⁴ Furthermore, miR-183-5p was highly expressed in M2-TAMs-Exos.⁷³ This miR-183-5p is transferred into CRC cells through Exo and promotes tumor cell proliferation and metastasis by activating AKT/NF-κB pathway through targeting thioesterase superfamily member 4 (THEM4).⁷³ Overall, M2-TAMs-Exos-ncRNAs are a cluster of tumorigenic enhancers in CRC, suggesting the need for further studies exploring their potentials as molecular targets.

Immunosuppressive impact of M2-Exos-ncRNAs in the TME of GIC

The immunosuppressive role of M2 in the TME has been recognized by several studies, which is due to different factors including mitochondrial metabolism, release of anti-inflammatory agents such as CCL3, CCL4, arginase 1, TGF-β, and IL-10, and Exos carrying immunomodulatory ncRNA,^15,88,89,90 as explained in Figures 2 and 3. Importantly, there is little literature describing the impact of released Exos-ncRNAs that mediate the immunosuppressive function of M2-TAMs in GIC. In CRC, Exos-miR-203 drives M2-TAMs tumoral functions.⁹¹ Exos-miR-203 was reported as a regulator of DC immune responses by upregulating the expression of TLR4.⁹² Thus, M2-TAM-miR-203 may not only regulate tumor cell progression but also induces immunosuppression through regulating DC functions in the TME.⁹³ In addition, levels of miR-29a-3p were downregulated in some GIC types like laryngocarcinoma, GC, and HCC,^94,95,96 while expression of miR-21-5p was upregulated.⁹⁷ However, immune modulatory effects of these miRNAs have not been elucidated in GIC. However, in epithelial ovarian cancer, microarray analysis of M2-Exos-ncRNAs revealed that M2-Exos are enriched with miR-29a-3p and miR-21-5p, which increase the ratio of Treg to Th17 by suppressing STAT3 signaling, leading to enhance tumor progression.⁹⁸ Furthermore, transferring miR-146a from HCC cells to TAM induces M2 phenotype that suppresses T cell activation by inducing exhaustion through upregulating the expression of PD-1.⁹⁹ Further studies reported that expression of Exos-miR-130b-3p serves as an inhibitor of extracellular cold-inducible RNA-binding protein (eCIRP), which can be released during sepsis .¹⁰⁰ Adding to that, a recent microarray study linked the expression of miR-223, miR-124, miR-34a, miR-132, miR-125a-5p, let-7c, and miR-146a to M2 polarization and immunosuppression function.¹⁰¹ Most of these miRNAs are associated with GIC progressions such as miR-125a-5p and miR-124 in GC,^102,103 and miR-132 in PDAC.¹⁰⁴ Moreover, inhibition of miR-223 showed significant association with increased pro-inflammatory cytokines such as IL-1β and IL-6.¹⁰⁵ In colon cancer, M2-TAM-Exos enriched with miR-183-5p were suggested to promote regulatory effects that support tumor growth by targeting THEM4 and activated Akt and NF-κB pathways.⁷³ Further studies showed that M2-TAMs-Exos carrying miR-21 enhance the immune escape of tumor cells by modulating paternally expressed gene 3 (PEG3).¹⁰⁶ However, studies revealing mechanisms of M2-Exos-ncRNAs in promoting immunosuppression in GIC are very limited and the available studies are not fully clear. Therefore, a better understanding of M2-Exos-ncRNA immunosuppressive functions is needed to develop new therapeutic targets using modulation of the immunosuppressive role of M2-TAMs in the TME of GIC.

Role of M2-Exos-ncRNAs in drug resistance of GIC

M2-TAMs-Exos induce chemotherapy resistance by modulating the expression of special genes such as mitogen-activated protein kinase 8 interacting protein 3 (MAPK8IP3), or by increasing the level of competitive endogenous RNAs (ceRNAs) such as MSTRG.292666.16 that sponge miR-6386-5p and thus induce MAPK8IP3 overexpression, leading to chemotherapy resistance.¹⁰⁷ Recent studies reported that M2-Exos-ncRNAs can increase tumor cell chemotherapy resistance. It has been shown that M2-TAM-miR-223 increased resistance to doxorubicin and oxaliplatin by inhibiting F box and WD repeat domain-containing-7 (FBXW7) pathway in GC.³⁷ Further to that, M2-TAMs-Exos-miR-21 promoted cisplatin resistance in GC by inhibiting apoptosis by activating PTEN/PI3K/AKT pathway.⁷² In addition, M2-TAMs-miR-588 promoted cisplatin resistance via targeting cylindromatosis (CYLD).⁸⁰ lncRNAs have been demonstrated to be associated with chemotherapy sensitivity through modulating autophagy.¹⁰⁸ Decrease in lncRNA-CRNDE expression in M2-Exos was associated with chemotherapy resistance in GC, whereas its increase revealed the opposite effects. A recent study has shown that the high level of Exos-lncRNA-CRNDE enhanced cell proliferation and tumor progression by targeting the expression of PTEN, and inhibiting CRNDE in M2-Exos enhanced the sensitivity to cisplatin in GC.³⁶ Another study showed that the knock-down of M2-Exos-miR-223 reduced resistance to doxorubicin in GC.³⁷ In an in vitro study, M2-TAM-miR-365 increased PDAC cell resistance to gemcitabine by increasing the triphospho-nucleotide pool and inducing the expression of cytidine deaminase (CDA), which is capable of inactivating intracellular gemcitabine and decreasing PDAC cell sensitivity to this therapy.⁶⁹ Thus, further investigations of M2-TAMs-Exos-ncRNAs could identify potential predictive biomarkers for the response to anti-tumor drugs, as well as discover promising targets for improving sensitivity to anti-cancer drugs.

Role of Exos-ncRNAs in T cell recruitment and function in the TME of GICs

Exos-ncRNAs play important roles in regulating the recruitment and function of T cells into the TME of GICs. In recent studies, the low expression of Exos-miR-140 in GICs was significantly linked to tumor progression, metastasis, and immunosuppression.^109,110 Interestingly, upregulation of PD-L1 and decreased CD8⁺ T cells in the TME of GC might be promoted by reduced expression of miR-140.¹¹¹ Overexpression of miR-140 inhibited PD-L1 and Tregs in GC mice model, while it increased the recruitment of CD8⁺ T cells, leading to inhibition of GC growth.¹¹¹ Another study investigating the relationship between Exos-miR-16-5p and anti-tumor immune responses in GC revealed that Exos-miR-16-5p targets PD-L1 and triggers T cell immune responses, which inhibit tumor formation.⁷¹ The investigation of Exos-miR-206 function in GC and HCC revealed that miR-206 has a key suppressor role in the TME of these tumors.^112,113 Interestingly, overexpression of miR-206 promoted the recruitment of CD8⁺ T cells to the TME by inducing M1 polarization through increasing CCL2.¹¹⁴ In nasopharyngeal carcinoma (NPC), there was a significant upregulation of miR-24-3p in the serum that inhibited proliferation of Th1 and Th17 but induced differentiation of Tregs by inhibiting fibroblast growth factor 11 (FGF11) gene in T cells.¹¹⁵ A study investigated YAP1 transcription by lncRP11-323N12.5 in GC and T cells by binding to c-MYC in the YAP1 promoter region concluded that Exos-lncRP11-323N12.5 upregulates YAP1, which enhances Treg differentiation and immunosuppression in GC.¹¹⁶ Furthermore, upregulation of miR-448 in CRC suppresses apoptosis of CD8⁺ T cells by inhibiting the expression of IDO1.¹¹⁷ Overall, Exos-ncRNAs orchestrate several tumorigenesis and immunomodulation activities in the TME of GICs, as summarized in Table 2.

Table 2.

Biological functions of exosomal ncRNAs in GIC

Exosomal ncRNAs	Biological and Mechanism function	Target cells or molecules	Source of exosome	Cancer type	Reference
miR-223	Enhances chemo-resistance to doxorubicin	GC cells	MΦs	GC	Gao et al.37
circSHKBP	Promotes tumor progression (via proliferation, angiogenesis, migration, and invasion)	GC cells	GC cells	GC	Xie et al.118
lncRNA-PCGEM1	Promotes invasion and migration of other GC cells	GC cells (normoxic cells)	GC cells (hypoxic cells)	GC	Piao et al.119
miR-519a-3p	Promotes liver metastasis	HCC	GC cells	GC	Qiu et al.120
miR-934	Promotes liver metastasis	HCC	CRC cells	CRC	Zhao et al.45
CircRNA-100338	Promotes HCC metastasis by enhancing angiogenesis and invasiveness	HCC	HCC cells	HCC	Huang et al.121
miR-106b-3p	Promotes invasiveness, EMT, and metastasis	Deleted in liver cancer-1 (DLC-1)	CRC cells	CRC	Liu et al.122
miR-431-5p	Promotes tumor progression	CRC cells	Human umbilical cord mesenchymal stem cells (hUCMSCs)	CRC	Qu et al.123
LncR-RPPH1	Promotes metastasis	TUBB3 and TAM-M2 polarization	CRC cells	CRC	Liang et al.124
miR-30b-5p	Promotes angiogenesis	Glypican-1 (GJA1)	Hypoxic PaC cells	PDAC	Chen et al.125
miR-21	Promotes tumor progression	Hepatic stellate cells	HCC cells	HCC	Zhou et al.126
miR-19a	Involved in pancreatic cancer pathogenesis, which is associated with diabetes	Adenylyl cyclase-1 ADCY1 and exchange protein directly activated by Camp-2 EPAC2	PaC cells	PaC	Pang et al.127
miR-21-5p	Promotes liver metastasis through inducing liver M1- MΦs polarization	HCC	CRC cells	CRC	Shao et al.87
miR-320a	Enhances tumor progression by promoting M2-MΦs polarization.	Regulates PTEN/PI3Kγ	Cancer-associated fibroblast	PaC	Zhao et al.128
miR-34	Suppresses growth and invasion of gastric cancer	Gastric cancer	Cancer-associated fibroblast	GC	Shi et al.129
miR-139	Suppresses gastric cancer progression	Matrix metalloproteinase-11 (MMP11)	Cancer-associated fibroblast	GC	Xu et al.130
miR-4454	Enhances tumor growth and vascularization of HepG2 cells, as well as accelerating cycle arrest, apoptosis, and oxidative stress of HepG2 cells	Vps4A and Rab27A	HCC	HCC	Lin et al.131
miR-4800-3P	Promotes HCC progression by regulating the Hippo pathway via targeting STK25	Targeting STK25	HCC cells	HCC	Lin et al.132
lncR-H19	Promotes HCC progression after propofol treatment by upregulating the microRNA-520a-3p/LIMK1 axis	Targeting the microRNA-520a-3p/LIMK1	HCC cells	HCC	Yu et al.133
CircUHRF1	Contributes to immunosuppression through inhibiting NK cell function and may also cause resistance to anti-PD-1 immunotherapy	NK cells	HCC cells	HCC	Zhang et al.134
Circ-002136	Promotes progression of HCC	Targeting miR-19a-3p/RAB1A pathway.	HCC cells	HCC	Yuan et al.135
miR-203	Promotes tumor metastasis by promoting differentiation of monocytes to M2-MΦs	Monocytes	CRC cells	CRC	Takano et al.91
miR-203	Contributes to dysfunction of DCs through regulating TLR4	Targeting TLR4 in DCs	PaC cells	PaC	Zhou et al.92
lncRNA ENST00000560647	Promotes tumor progression and immune escape	DCs	PaC cells	PaC	Chen et al.136
miR-301a-3p	Promotes pancreatic cancer via polarizing MΦs	MΦs	Hypoxic PaC cells	PaC	Wang et al.50
miR-107	Induces expansion of MDSCs and ARG1 expression by inhibiting the DICER1 and PTEN expression	MDSC	GC cells	GC	Ren et al.137
miR-10a-5p	Promotes tumor progression via inducing PD1+ MΦs population	Monocyte’s differentiation into MΦ	GC cells	GC	Zhu et al.138

Conclusions and future directions

Several studies reported the elevation of TAM levels in GIC, which was usually linked to tumor progression and lower survival rates. TAMs are essential in GIC growth, invasion, immune regulation, angiogenesis, metastasis, and progression. TAM polarization into M2 is linked to poor prognoses and drug resistance in GIC. Polarization of M2-TAMs is mainly mediated by tumor cells via released Exos and other immunomodulators that induce the secretion of M-CSF. The polarized TAMs then promote tumor progression and induce most of the resident TAMs into M2 phenotype, leading to immune suppression, drug resistance, and tumor progression. As TAMs have the capability to switch polarization into different functional phenotypes, inducing M1 phenotype could modulate the released Exos with anti-tumor functional ncRNAs, which can activate immune responses, inhibit tumor growth, and improve response to chemotherapeutic interventions. However, further explorations of the differences between Exos-ncRNAs released by M2-MΦs and M1-MΦs are still needed.

Little information is available about the molecular mechanisms mediated by M2-Exos-ncRNAs in the TME of GIC, which are supposed to orchestrate immunomodulation and tumor progression. Recently, the importance of Exos-ncRNA as targets for modulating the TME and immune responses in GIC was demonstrated by several studies. M2-TAMs release Exos-ncRNAs, which are linked to the underlying mechanisms of tumorigenesis, including tumor angiogenesis, invasion, immunosuppression, tumor cell proliferation, and drug resistance. The role of Exos-ncRNAs in molecular mechanisms supporting GIC progression has been the subject of numerous recent studies. M2-TAMs-Exos-ncRNAs demonstrated functional pathological effects in GIC by regulating tumor cell stemness, invasion, and metastasis. Further to that, recent studies reported a link between M2-TAMs-Exos-ncRNAs and modulation of tumor cells and immune cells, resulting in the promotion of immunosuppression and tumorigenesis. Moreover, M2-Exos-ncRNAs can induce tumor immune evasion and enhance resistance to chemotherapy through alteration of gene expression in cancer and immune cells, which can be used as diagnostic biomarkers and/or therapeutic targets in GICs. Further elucidation of the immunosuppressive roles of M2-Exos-ncRNAs is ultimately needed to provide a clear understanding and new therapeutic targets, relying on modulation of the immunosuppressive role of M2-TAMs in the TME of GIC. Furthermore, it is possible to reverse Exos-ncRNA effects with antagonists; therefore, developing effective and clinically valuable antagonists in GIC has important potential.

The focus on M2-TAMs-Exos-ncRNAs is very important to explore critical mechanisms in GICs. We propose that studying molecular mechanisms underlying M2-TAMs-Exos-ncRNAs functions in the TME of GIC would uncover novel targets that could lead to promising clinical interventions. Exos from M2-MΦs carry many signaling molecules, such as ncRNAs, which regulate target cell biology and cellular functions through unknown mechanisms. Thus, it may be possible to investigate how target cells take up Exos-ncRNAs, how they modulate gene expression, and how they affect cell behavior; these could serve as potential diagnostic and/or prognostic biomarkers, and to respond to anti-cancer treatments, in addition to discovering novel targets to improve anti-tumor drug sensitivity. Overall, further investigation of Exos-ncRNAs released by M2-TAMs is a promising area of research with many potential future directions.