Exosome-derived miRNAs regulate macrophage-colorectal cancer cell cross-talk during aggressive tumor development

Plain language summary

Colorectal cancer is one of the leading causes of death worldwide. Its incidence and mortality have significantly increased during the past few years. Colorectal cancer cells cross-talk with other cells through exosomes in their tumor microenvironment. The miRNAs containing exosomes are responsible for tumor growth, invasion, and metastasis. Multiple studies have shown that exosomal miRNAs are key players in the crosstalk between cancerous, immune, and stromal cells during colorectal cancer development. They help in the establishment of the tumorigenic microenvironment by reprogramming macrophages towards a pro-tumorigenic phenotype. In this review, we discussed various exosomal miRNAs derived both from colorectal cancer cells and macrophages that promote or inhibit cancer aggression. We also discussed various miRNA-based therapeutic approaches to inhibit cancer progression.

Colorectal cancer is the 3rd most diagnosed cancer worldwide. The rate of new diagnoses is increasing mainly due to lifestyle changes, although family history and inflammatory bowel diseases are also associated with an increased risk of colorectal cancer. Cancer cells and immune cells, including macrophages, B- and T-cells communicate with each other through various soluble molecules and cell fragments called exosomes. These exosomes contain macromolecules that can be transferred between cells thereby stimulating or inhibiting cancer growth. A crucial component of exosomes, termed small non-coding RNA (micro-RNA/miRNA), has the potential to change the function of immune cells, such as macrophages, and lead them to promote cancer growth. To counter this effect, exosomal miRNA can be targeted to develop a novel therapeutic approach.

Tweetable abstract

Non-coding micro-RNAs released via exosomes play a key role in the communication between cancer cells and macrophages and hence may serve as key therapeutic targets in colorectal cancer.

Keywords::

• colorectal cancer (CRC)
• exosome
• metastasis
• microRNAs (miRNAs)
• tumor-associated macrophages (TAM)
• tumor microenvironment (TME)

Practice points

• Colorectal cancer (CRC) is typically driven by inflammation and is a leading cause of cancer-related deaths worldwide. Macrophages in the tumor microenvironment play an essential role in CRC progression.

Aim

• Crosstalk between CRC cells and macrophages via exosomes and other inflammatory mediators promotes tumor growth. It is essential to dissect the novel molecular mechanism in macrophages and CRC cells for finding a novel therapeutic strategy for CRC.

Result

• Exosome-mediated cross-talk between CRC cells and macrophages is essential for enhancing tumor progression through the miRNA-mediated reprogramming of macrophages to alternative phenotypes.

Conclusion

• Exosomal miRNAs could be targeted through the anti-miR-based approach to suppress cancer aggressiveness.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world, accounting for 10% of all cancers, with recent estimates of 935,000 deaths/yr [1]. In addition to the aging society and food choices of a developed country, colon cancer risk is also increased by undesirable risk factors, including obesity, smoking, and less physical activity [2]. Colon cancer first appears when normal epithelium transforms into a hyperproliferative epithelium leading to advanced cancer stages [3]. Physiological and pathological knowledge has led to improved therapy for both local and advanced conditions of cancer. The tumor microenvironment (TME) is a complex network of cancer cells and immune cells, including T-cells, B-cells, Tumor-associated macrophages, and NK cells that regulate tumor survival and promotion. The TME is responsible for the more aggressive proliferation of tumor cells by recruitment of immunosuppressive cells, rejuvenating extracellular matrix, and promotion of angiogenesis ultimately promoting CRC proliferation and metastasis. In colorectal cancer, cell-to-cell communication, mediated by cytokines, chemokines, and growth factors is essential for both physiological development and pathology [4]. Cancer cells also secrete extracellular vesicles (EVs) which contain genetic information-bearing macromolecules including protein, nucleic acid mRNA, and microRNAs (miRNA) [5]. EVs are lipid bilayer-bound vesicles and are naturally secreted from most cell types and released into the extracellular space. Microvesicles (MVs), exosomes, and apoptotic bodies are three primary subgroups of EVs, and they are distinguished by their biogenesis, release mechanisms, size, composition, and action [6].

In recent times the function of exosomes to act as the primary mediator between the tumor cell and surrounding cells has inspired significant attention in cancer. Exosomes are around 40–120 nm and originate from the endosome [7]. The role of exosomes has been reported in many diseases and shows an impact during immune response maintenance. Exosomes contain bioactive cargos, that reflect their cellular origin, and can influence recipient cells and disease status, hence signifying their biological importance. They are highly enriched with tetraspanins, a family of transmembrane proteins like CD9, CD63, and CD81 which interact with many interacting proteins like MHC molecule and integrin, and reflect on their endosomal origins [8]. They also contain heat shock proteins (HSP70 and HSP90), the cytosolic proteins involved in the endocytic pathway including annexin II, RAB5/RAB7, and tumor-susceptibility protein TSG101 which are involved in endocytic pathway. Exosomes are commonly isolated using ultracentrifugation, differential centrifugation, density gradient centrifugation, precipitation, and they are identified and characterized by transmission electron microscope (TEM), flow-cytometry, and western blot analysis [9,10]. Exosome research is a newly emerging field that is rapidly advancing with descriptions of their role in post-transcriptional regulation of gene expression via mRNA targeting. The development of new isolation and manipulation approaches will make it easier to translate exosome research into therapeutic applications especially cancer cell-secreted miRNAs. To better understand how secreted exosomes interact with immune cells, it is essential to understand the role played by miRNAs during cancer progression.

MicroRNAs are a type of non-coding RNA with a short length made up of 18–25 nucleotides [11]. They are involved in post-transcriptional alterations, which play a crucial function in inflammatory response regulation. They interact toward the 3′-end of untranslated regions of messenger RNA (mRNA) to regulate gene expression. Translational repression or mRNA breakdown occurs as a result of this binding. MicroRNAs are single-stranded and highly conserved [12]. They are transcribed by RNA polymerase II or III, and their initial processing is via RNAase Drosha inside the nucleus. After that, the precursor of miRNA is transported into the cytoplasm, where it is digested by RNAase III, which is assisted by DICER. The resulting mature miRNA is integrated into the RNA-induced Silencing Complex (RISC), which further regulates protein transcription and translation [13]. Many cells express miRNAs, which regulate both pro-and anti-inflammatory responses [14]. Numerous human diseases, including cancer and inflammatory disorders, are linked to excessive miRNA expression. Controlling the expression of SOCS3 and HDAC11 at the post-transcriptional level increase miR-203 and miR-145 respectively and promote polarization of macrophages toward the M2 phenotype which suppresses the immune response and supports metastasis of colorectal cancer [15–17]. miR-25-3p, miR-425-5p, and miR-130-3p target common sites of PTEN and modulate macrophages toward anti-inflammatory type [18].

CRC-derived exosomal miRNA in the regulation of macrophage polarization

Over the past few years, convincing evidence that macrophages have a tumor-promoting function in the tumor microenvironment (TME) has been accumulating. The majority of leukocytes that infiltrate solid tumors are tumor-associated monocytes/macrophages (TAMs) mostly derived from circulating monocytes [19]. TAMs which are transformed as pro-tumorigenic, are widely distributed in both the main and metastatic locations of CRC consequential of these immune responses, and are seen throughout all cancer stages [20]. Macrophages provide cytokines, chemokines, and growth factors to the CRC, resulting in a supportive TME. Although exosomes have been firmly linked to cellular communication between CRC cells and macrophages or other TME cells, the exact mechanism is yet unknown. Exosomes produced from CRC may be responsible for the reprogramming of macrophages to differentiate into an anti-inflammatory, pro-tumorigenic M2 phenotype. Macrophages M1 function as troops that attack cancer cells by releasing the pro-inflammatory cytokines (IL-12, IL-1β, and TNF-α) while on the other side, M2 macrophages act as anti-inflammatory producing cytokines like IL-4, IL-10, and growth factors like TGF-β, PDGF, with excessive M2 phenotype resulting into immune suppression and supporting TME [21]. Consequently, the modification and migration of TAMs at the tumor site, for example through miRNAs are crucial. In this review, we discussed the exosomal miRNAs derived from both colorectal cancer cells as well as TAMs (Figure 1). The key miRNAs relevant to miRNA-mediated crosstalk between colorectal cancer cells and TAMs in TME are listed in (Table 1). Targeting major transcriptional networks which regulate the reprogramming of the M2 polarization state of macrophages to control cancer proliferation would be a novel therapeutic strategy.

Figure 1. Colorectal cancer cell secreted exosomes.

Representing exosome-mediated effect, the colorectal cancer cell secreted exosomes miR-25-3p, miR-425-5p, miR-130-3p, miR-934, miR-200a and miR-21-5p promote M2 polarization via PI3K/AKT mediated signaling, miR-203 and miR-145 mediated M2 polarization via STAT3 dimerization, and miR-21-5p and miR-29b promote M1 polarization which further increases miR-21 and leads to M1 to M2 reprogramming. The M2-polarized macrophages also secrete the exosome miR-21-5p, miR-155-5p and miR-183-5p, which mediate tumor growth, invasion, metastasis, angiogenesis and cancer progression.

Table 1. List of colorectal cancer cell and macrophage-derived exosomal miRNA involved in colorectal cancer progression.

S. No.	miRNA	Target	Function	Ref.
CRC cell derived
1	miR-203	SOCS3	Monocytes to M2 polarization, Immune suppression and metastasis	[22]
2	miR-145	HDAC11	M2 polarization and tumor growth	[16,23]
3	miR-934	PTEN	M2 polarization and liver metastasis	[24]
4	miR-1246	–	Reprogramming of TAMs	[25,26]
5	miR-25-5-p, miR-130b-3p and miR-425-5p	PTEN	M2 polarization	[18,27]
6	miR-21-5p, miR-29b and miR-200a	PTEN	SOCS1/STAT1 mediated M2 and TLR7/8 mediated M1 polarization	[28–30]
7	Let-7d	CCL7	M2 polarization	[31]
Macrophage derived
1	miR-155-5p	ZC3H1B, BRG1	Cancer proliferation and antiapoptotic	[27,32,33]
2	miR-21-5p	BRG1	CRC metastasis	[33]
3	miR-185-5p	THEM4	Cancer proliferation	[34]

TAM: Tumor-associated macrophage.

miR-203

miRNA-203 plays an essential role in the progression of colorectal cancer by supporting tumor growth, immune suppression, and metastasis [22]. miR-203 has been found to promote an M2 (alternative) macrophage phenotype in the pre-metastasis niche [35]. miRNA-203 targets SOCS3, which results in an increase in STAT3 expression and therefore promotes M2 macrophage polarization [15]. The intravenous transfer of miR-203-rich CRC cells in the xenograft liver tumor model has been shown to promote tumor growth [22].

miR-145

miRNA-145 released from the CRC cells is taken up by the macrophages, where it suppresses the expression level of the histone deacetylase HDAC11. miR-145 targets the 3′ UTR sequence of HDAC11 which promotes the acetylation of histone H3 and H4, followed by recruitment of the transcription factor sp1 and STAT3 and upregulates the IL-10 expression. To confirm the M2 polarization, the CRC cell line DLD-1 was co-cultured with the macrophage-like cell THP-1 and NOMO-1 which has shown an increase in the mRNAs level of IL-10 and VEGF, along with the increased surface markers CD11b+, CD68⁺ and CD206⁺ on the macrophage. In vivo, miR-145 promotes tumor growth and enlargement of tumor volume [16,17]. It was also found that another target of miR-145 is TLR4, which diminishes the production of pro-inflammatory cytokines and inhibits M1 polarization. TLR4 transduces its signal through several adaptor molecules including TIRAP which has been reported as the target of miR-145; by inhibiting TLR4 or TIRAP, pro-inflammatory cytokines are decreased and may be necessary for the production of anti-inflammatory cytokines in TAMs.

miR-934

miR-934 has a role in tumor growth, migration, angiogenesis, and liver metastasis by producing various growth factors, cytokines, and chemokines. This miRNA is encapsulated in the exosome with the help of hnRNPA2B1, RNA binding protein through the GGAG sequence motif binding. The hnRNPA2B1 guides the transportation and post-transcriptional regulation of miRNA [36]. As exosomes are released from tumor-derived cells, they are transported to macrophages and release their content within them. Intracellular miR-934 is released from the binding protein and subsequently binds to the 3′UTR sequence of PTEN and downregulates its expression. This leads to the activation of the PI3K/AKT signaling pathway [24], where activated PI3K initiates the conversion of PIP2 into PIP3, and the PIP3 which phosphorylates and activates AKT and thus polarization of the macrophage into the M2 (alternative) phenotype [37]. Differentiated M2 macrophages release a high amount of CXCL13 chemoattractant, which shows binding to the CXCR5 receptor present on CRC cells [38] and promotes CRLM (colorectal cancer liver metastasis). The CXCL13/CXCR5 axis promotes tumor progression by activating NF-κB/p65 signaling pathway in CRC cells and thus, provokes a positive feedback loop with the TAM [24]. Along with this, TAMs promote liver metastasis in CRC. It has been found that miR-934 targets Dickkopf-related protein 2 (DDK2) and negatively regulates the Wnt signaling pathway, and regulates the CRC cell proliferation [39].

miR-1246

Oncogenic miR-1246 was reported in colorectal cancer and involved in proliferation, invasion, and metastasis [40]. It plays a critical role in reprogramming TAM toward an anti-inflammatory phenotype and modifies the tumor microenvironment. This miRNA action is closely associated with the presence of the p53 mutant in colon cancer cells, which is the most common molecular mutation associated with cancer development in humans [25]. Mutation in p53 in cancer cells is responsible for reprogramming TAM toward tumor-supportive and anti-inflammatory phenotypes through increasing expression of TGF-β in CRC. This pathway also occurs in other cancers such as glioblastoma, wherein hypoxic glioma-derived extracellular vesicle containing miR-1246 converts macrophages toward an anti-inflammatory phenotype via activation of the STAT-3 signaling pathway and prohibits the NF-κB signaling [26]. Huang et al. found that increased expression of miR-1246 was associated with higher risk and lower survival rates in colorectal cancer patients mainly associated with KRAS and PI3K signaling cascade [41]. CRC secreted mir-1246 is taken up by endothelial cells where it activates Smad-1/5/8 signaling via directly targeting promyelocytic leukemia (PML) mRNA which results in boosting angiogenesis [42]. As miR-1246 plays a pivotal role in TME modification by targeting macrophage polarization, it may be a target for antitumor immunotherapy.

miR-25-5-p, miR-130b-3p & miR-425-5p

These cluster of miRNAs regulates macrophage polarization transferred from CRC to TAM. miR-25-3p, miR-130b-3p, and miR-425-5p are upregulated in CRC and mainly responsible for the skewing of macrophages toward an anti-inflammatory phenotype via the PTEN/PI3K/Akt mediated signaling and exosomal miR-25-3p, miR-130b-3p, and miR-425-5p facilitated the M2 polarization. PTEN controls the phosphorylation of AKT by inhibiting PI3K, which results in the reprogramming of TAM to M2 type [27]. This M2 type enhances CRC cell-secreted VEGF and EMT, ultimately resulting in angiogenesis and metastasis. These in vivo studies also reported macrophages (THP-1 or Raw264.7) transfected with miR-25-3p, miR-130b-3p, and miR-425-5p mimic mixed with CRC (HCT116 and MC38) injected into the flanks of nude mice. The tumor size rapidly increased in mice transfected with macrophages which excessively express these miRNAs upon stimulation with IL-4 expression [18]. Targeting miR-25-3p, miR-130b-3p, and miR-425-5p will be a novel approach to designing drugs to inhibit the interaction with their specific receptor molecule, which favors a more proinflammatory macrophage phenotype in colorectal cancer.

miR-21-5p, miR-29b & miR-200a

CRC-derived exosomal miRNAs have the potential to transform macrophages into M1 or M2 macrophages, depending upon the conditions. miR-21-5p (miR-21) has a significant role in CRC at the primary and secondary sites. Initially, it was known for its pro-inflammatory phenotype; a study by Ying Kuan Shao et al. has shown that miR-21-5p promotes the liver pro-inflammatory phenotype in macrophage and liver metastasis in CRC. The investigators incubated miR-21-rich exosomes derived from SW480 and SW620 cell lines with RAW264.7 and THP-1 for 24 hrs and found an increase in IL-6 and TNF-α mRNA while the anti-inflammatory cytokines showed no changes. The tail vein exosomal transfer also confirmed miR-21-5p localization predominantly in the liver. miR-21-5p has a GU motif in its nucleotide region, which plays an important role in modulating TLR-mediated signaling and activating NF-κB. Thus, in the liver, these CRC cell-derived miRNA phagocytosed by the exosome and mediated signaling through TLR7/8 leads to the M1 phenotype, which further shows the high expression of IL-6 [28]. IL-6 plays a major role in macrophage polarization and also promotes macrophages to release IL-6 and miR-21, which target STAT3 in CRC cells creating positive feedback [29,43]. Interestingly miR-21-5p with miR-29b also shows a similar effect and promotes tumor growth and invasion [29]. A recent study by Yuan Yin et al. had shown a miR-21 role in polarization toward the M2 phenotype. At the primary site of the tumor, the subpopulation of macrophages was found to be polarized into CD206⁺ macrophages. miR-21-5p and miR-200a showed the synergistic effect on specific TAM, i.e., CD206 macrophages where they target the PTEN/AKT axis and SOCS1/STAT1, and lead to polarization of macrophages toward an M2 phenotype with increased PDL-1. The expression of PDL-1 inhibits the CD8 T-cells and promotes tumor immune escape and progression [30].

miR-Let-7d

miR-Let-7d targets the C-C motif chemokine CCL7, secreted by the cancer-associated macrophages (CAFs) and downregulates its expression. CCL7 promotes macrophage polarization toward the M2 phenotype, which inhibits monocyte migration and other functions like tumor growth, progression, and metastasis [31].

Macrophage-derived exosomal miRNAs

Not only cancer cell-derived exosomal miRNAs support cancer proliferation but macrophage-derived miRNA also promotes CRC proliferation and metastasis (Table 1). miRNAs play a crucial role in cancer development, particularly in invasion, metastasis and drug resistance, and hence could be used as prominent biomarkers in cancer treatment and detection [41].

miR-155-5p & miR-21-5p

The miR-155-5p released from the M2 macrophage-derived exosomes support colon cancer proliferation and anti-apoptotic ability. Recent findings suggest the miR-155-5p target Zinc-finger-type-containing 12B (ZC3H12B) and upregulates the IL-6 expression, which results in colorectal cancer escape from the immune system [32]. ZC3H12 family proteins are mainly known for their involvement in inflammation, binding with IL-6 mRNA and downregulating the IL-6 protein expression. It can be concluded that in CRC cells, the ZC3H12B protein plays a crucial part in controlling the predominant role of IL-6 in cancer to increase tumor growth. IL-6 induces the IL-6-dependent STAT3 activation through the phosphorylation of tyrosine residues of STAT3, which result in subsequent transcription of target genes that play a crucial role in cancer development [44]. The report also suggested that TAM-secreted exosomal miR-21-5p and miR-155-5p suppress BRG-1 expression, a key molecule for cancer growth and metastasis [27]. In-vitro and in-vivo experiments showed downregulation of BRG-1 is involved in colorectal cancer metastasis through the activation of the Wnt/b-catenin signaling pathway [33]. In colorectal cancer patients, it is feasible that exosomal miR-21-5p and miR-155-3p can be transferred from TAM to CRC, perhaps via surrounding vessels. Preventing this transfer by designing therapeutic molecules may be an approach to inhibiting colorectal cancer metastasis and proliferation. Macrophages-derived exosomal miR-21-5p and miR-155-5p transfer to colon cancer cells is not a fully described mechanism, and more studies are needed in this direction.

miR-183-5p

The level of miR-183-5p was markedly elevated in M2-TAM and exosomes generated from M2-TAM, which enhanced colon cancer cell invasion and proliferation while reducing apoptosis [34]. This study also suggested that upregulation of miR-183-5p enhanced M2 polarization and mediated a booster effect on cancer cells while downregulation of miR-183-5p reversed the M2 TAM-mediated tumor-promoting effects on CRC. miR-183-5p targets the Thioesterase superfamily member 4 (THEM4) and initiated Akt and NF-κB pathways to exacerbate colon cancer formation. THEM4 is a C-terminal regulatory protein that can inhibit the phosphorylation of AKT and block downstream signaling. Western blotting determined the inhibition of p-Akt and p-NF-κB expression [34,45]. THEM4 can mediate phosphatidylinositol 3-kinase (PI3K)/Akt reversely and shows an anti-inflammatory and anti-tumor response.

miRNAs-based therapeutic approach

As previously discussed, various exosomal miRNAs are being transferred from one cell to another and promote tumor initiation, differentiation, progression and metastasis. Developing the exosomal miRNA-based therapy can open the possibility of a novel therapeutic approach in CRC which can be utilized as a new way to deal with cancer and clinical trials, where miRNA mimics or miRNA antagonists or tumor-suppressing miRNA delivered intra-tumor or systemically are being used for tumor therapy (Table 2). miRNA-based therapies are broadly used in two ways: targeting oncogenic miRNAs or using tumor-suppressive miRNAs. Both approaches have been showing effective results [46]. Meanwhile, the use of exosomal-mediated miRNA delivery is more efficient than a free miRNA delivery system [47]. Exosomes act like natural nanoparticles which have several significant features including low immunogenicity and cytotoxicity, stability in circulation, easily recognized and taken up by the specific type of cell, as well as carrying stable functional molecules such as miRNAs, siRNA or drug [48]. For such a strategy, exosomes are required to be engineered to encapsulate miRNA with suitable properties which promote uptake by the target cell [49] such as modifying the exosome membrane to differentially promote uptake by the tumor cell.

Table 2. Table of miRNA-based clinical studies in a different disease.

S. No.	Exosomal miRNA	Type	Function	Ref.
1	Anti-miR-221	CRC	Enhance anti-tumor effects	[50]
2	Let-7a	Breast cancer	Inhibits tumor growth	[51]
3	miR-335	hepatocellular carcinoma	Help in tumor shrinkage	[52]
4	miR-21-sponge	Glioma	Prevent malignancy	[53]
5	miR-494	melanoma	Inhibits tumor growth and metastasis	[54]
6	miR-126	Acute myocardial ischemia	Reduce cell injury and inflammation	[55]
7	miR-92-a-3p	Osteoarthritis	Suppress cartilage degeneration	[56]

In the case of CRC, as far as we know, there is only one study that describes the miRNA based therapeutic approach. Mesenchymal stem cell (MSC) derived exosomes were used to transport an anti-miRNA oligonucleotide (AMO) to the tumor cell. Anti-miRNA is the complementary sequence that binds and inhibits miRNA function [57]. Here, anti-miR-221 inhibits the function of the miR-221 which downregulates the expression of tumor suppressive genes including PTEN, p27kip1, TIMP3, and the Wnt signaling pathway [58]. The anti-miR-221 was incorporated into the MSCs-derived exosome through electroporation and the derived AMO-Exo showed an inhibitory effect on the NRP-1 receptor-positive tumor cell. The xenograft tumor model also confirmed that AMO-Exo targets the tumor cell and inhibits its growth with an associated decrease in the expression of miR-221 [50]. Ohno and colleagues used EXO-miRNA in breast cancer and showed encapsulated miRNA Let-7a is transported to the epidermal growth factor receptor-expressing cancerous cells and effectively inhibits tumor growth as confirmed in a xenograft tumor model [51]. Schmittgen et al. used a similar approach for neuroblastoma wherein NK-cell-derived exosomes were used to deliver the tumor-suppressing miR-186 to restore its level, inhibit tumor progression and increase survival. Similarly, the restoration of tumor-suppressive miR-335 enriched exosome led to cancer inhibition in hepatocellular carcinoma in a xenograft tumor model [52]. The exosomal miR-21 sponge construct was used to target elevated miR-21 in human glioma cells to restore the expression level of RECK and PDCD4 genes and prevent malignant properties. A similar strategy can also be used in CRC as miR-21 has a major role in cancer progression and metastasis [53].

Another way to deal with the exosomal miRNA is the inhibition of their release or uptake by the cell such as cell-secreted oncogenic miRNAs are inhibited. Such a strategy was applied in melanoma where silencing of rab27a led to blockage of the exo-miRNA cargo and regaining cellular levels of miR-494 which decreases tumor growth and metastasis [54]. This approach can also be used in the CRC to inhibit the release of exo-miRs from cancer cells so that the tumorigenic effect on the immune can be inhibited. miR-4689 has tumor suppression properties and is downregulated in the KRAS mutant colon cancer patients, so the incorporation of miR-4689 into the exosome can be used to negatively regulate the MAPK and PI3K/AKT pathways [59]. Similarly, miR-29a is also a tumor suppressive type and in the presence of its mimic shows a positive anti-tumor effect and decreases the protein expression of PI3K, AKT and GSK3-β and restricts the WNT/β-catenin signaling pathway in colon cancer. Thus, miR-29a encapsulated into the exosome can make the delivery process more effective [60].

Finally, the miRNA-based therapeutic approach has been shown to advance not only in cancer but in other diseases such as acute myocardial ischemia (AMI). miR-126 enriched exosome derived from adipose stem cells was used to treat AMI and reduce cell injury and inflammatory factors [55]. miR-92-a-3p derived from MSCs exosome has been shown to increase chondrogenesis and suppress cartilage degeneration in Osteoarthritis [56]. In the future, more results are expected that would provide evidence for the exosomal miRNA-based treatment in CRC.

Conclusion

This review summarizes the exosomal miRNAs derived from the colorectal cancer cell and macrophages that play a crucial role in developing colorectal cancer (CRC). Exosomes are small extracellular vesicles containing important cargos, including mRNAs, miRNAs, siRNAs, lncRNAs, structural and functional tetraspanins, and tetraspanin-associated proteins. Exosomes released from colorectal cancer cells and macrophages mediate their cross-talk in the tumor microenvironment (TME). These exosomes play crucial roles in CRC-related mechanisms, such as cancer growth, metastasis, and drug resistance. Exosomes can influence macrophage polarization through RNAs or proteins, leading to tumorigenesis and cancer development. After summarizing the topic, we propose a disease model of CRC and introduce the new concept of early and late-stage activation of macrophages (Figure 2). In the early stage, exosome-mediated macrophage activation recognizes a cancer cell as foreign and differentiates to pro-inflammatory phenotype (M1); in the later stage, exosomal miRNAs from CRC cells reprogram macrophages towards an alternatively activated (M2) phenotype. In support of our hypothesis, a preliminary experiment was performed on THP-1 macrophages, which shows M1 and M2 phenotypic changes in macrophages in response to COLO-205 cell-derived exosomes after 24 and 72 h of treatment, respectively (Figure 3). Greater comprehension of the function of exosome-derived miRNAs and TAM may reveal previously unknown mechanisms of cancer resistance to current therapeutic strategies and lead to new prognostic tools and targets. Dissecting the exosome-mediated molecular mechanism during colorectal cancer progression could be a promising approach to finding therapeutic targets for CRC.

Figure 2. Representation of exosome-mediated macrophage polarization into M1 phenotype and M2 phenotype.

In an early-stage colorectal cancer cell, secreted exosome promotes M1 polarization with a proinflammatory response, and in the later stage, the exosomal miRNA mediates reprogramming toward an M2 phenotype.

Figure 3. Representation of THP-1 cell morphology in normal RPMI media and 48 h of Colo 205 colorectal cancer cells condition media isolated exosome and treated conditions after 24 and 72 h.

The THP-1 cells were treated with PMA (25 ng/ml) for 48 h before the experiment for its differentiation into monocytes. The images were taken under a Bright field microscope at 20× magnification.

EXO: Exosomes; PMA: Phorbol 12-myristate 13-acetate.

Future perspective

The cross-talk through exosome-derived miRNAs between cancer cells and macrophages plays a crucial role in tumor growth, invasion, and metastasis. Colorectal cancer cells-derived exosomal miRNAs are responsible for macrophage polarization toward a pro- and anti-inflammatory phenotype. The reprogrammed anti-inflammatory macrophage also releases exosome-derived miRNA that promotes cancer aggressiveness via targeting several signaling molecules. Recently the study of noncoding miRNA has received more attention in cancer research. These exosomal miRNAs are potential biomarkers for detecting cancer conditions and offer therapeutic potential when delivered to tumor cells through various in vivo approaches. Recently in the case of colorectal cancer, an anti-miRNA (Anti-miRNA-221) based therapeutic approach has been developed with promising results. Therefore exosomal-miRNAs may represent an important future therapy for miRNA target-based immunomodulation in colorectal cancer.

Author contributions

Conceptualization and supervision: MS Baig; Writing (original draft): MS Baig, N Hirani, K Wadhonkar and N Singh; Reviewing and editing: MS Baig, N Hirani, S Parihar and FM Heralde. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

The authors acknowledge the Indian Institute of Technology Indore (IITI) for providing facilities and other support.

Funding

This work was supported by Cumulative Professional Development Allowance (CPDA) and Research Development Fund (RDF) from the Indian Institute of Technology Indore (IITI) to MSB.

Financial & competing interests disclosure

This work was supported by Cumulative Professional Development Allowance (CPDA) and Research Development Fund (RDF) from the Indian Institute of Technology Indore (IITI) to MSB. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

This work was supported by Cumulative Professional Development Allowance (CPDA) and Research Development Fund (RDF) from the Indian Institute of Technology Indore (IITI) to MSB. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.