Pro-tumoral functions of tumor-associated macrophage EV-miRNA

Abstract

MicroRNAs (miRNAs) are central players in cancer biology. Their relevance in cancer development, progression and resistance to therapy has been further emphasized by the discovery that they are important cargo component of extracellular vesicles (EVs), which represent a prominent means of inter-cellular communication within the tumor microenvironment (TME). This review article focuses on the interaction between cancer cells and tumor-associated macrophages (TAMs) and in particular on the pro-tumoral phenotype elicited by EV-contained miRNAs released by TAMs and transferred to cancer cells. All main hallmarks of the malignant phenotype are affected by TAM-derived vesicular miRNAs, paving the road to the identification of such miRNAs as promising upcoming novel anti-cancer agents.

Introduction

When tumors arise, neoplastic cells recruit cells from the surrounding tissue. This supportive network of cells, matrix and vessels are fundamental to the growth and survival of tumors, and is typically referred to as the tumor microenvironment (TME). Understanding the relationships between cells of the TME is helping to identify new therapeutic targets in cancer patients (1). One cell type which is significant component of the TME is a specialized macrophage, known as the tumor-associated macrophage (TAM). A number of pro-tumoral effects exhibited by TAMs have been demonstrated, making these cells a popular target for the development of immunotherapeutic agents (2). In addition, many of the cancer promoting actions exerted by TAMs in the TME are carried out by their extracellular vesicles (EVs).

EVs have emerged over the past two decades as a means of communication between cells within the TME (3). Transfer of molecules between cells of the TME via EVs is one mechanism by which EVs mediate cancer progression. MicroRNAs (miRNAs) are small non-coding strands of RNA, they are an important player in post-transcriptional regulation of genes (4), and their presence in EVs is well established (5, 6). Within the TME, miRNAs delivered from one cell to another via EVs (here referred to as EV-miRNA) are now understood to be relevant factors in the progression of cancer (7).

There is a great interest now in studying miRNA derived from TAM EVs, to elucidate the role of TAM EVs in cancer, as this will both provide new avenues for therapy and the enhance the potential of these EVs as cancer biomarkers. This review will focus on the role of EV-miRNAs secreted by TAMs in the promotion of cancer growth and survival.

Tumor-associated macrophages

In regular non-cancerous tissue, resident macrophages are responsible for maintaining homeostasis, prominently during infection and wound healing, through phagocytosis and inflammation regulation (8). In the TME however, TAMs are highly abundant, and are predictive of outcome in cancer patients, with high TAM levels being linked to poor prognosis (9). These particular macrophages are distinct from regular tissue macrophages in their expression of surface proteins. Macrophages in the TME typically are enriched in CD163, CD206, consistent with the often considered pro-tumoral “M2” macrophages (10). As well as the expression of specific markers, these macrophages distinguish themselves in that they secrete many factors which aid tumor development, such as interleukin 8 (11), and interleukin 10 (12). Moreover, TAMs are implicated in multiple tumor supporting processes, including migration and metastasis (13), angiogenesis (14), and chemoresistance (15).

TAMs are believed to arise from infiltrating monocytes, partly driven by tumor EV activation of the monocytes (16). These infiltrating monocytes are also differentiated into myeloid derived suppressor cells (MDSCs) (17). In the tumor microenvironment, MDSCs play a major role in the generation of immunosuppressive signals. Traditionally defined as CD11b+CD33+HLA-DR- in humans, MDSCs are considered as a mixed group of immature myeloid cells able to inhibit T-cell activation (18). Tumor-derived EVs elicit profound modifications in the immune response (as revised in (19)). Huber et al showed that melanoma EVs were able to trigger MDSC generation from monocytes by transferring a defined set of miRNAs (20). Higher levels of these miRNAs in the plasma of melanoma patients were correlated to reduced clinical efficacy of anti-PD-1 therapy. Evidently, tumor EVs are able to drive differentiation of myeloid cells to inhibit the immune system, furthermore, tumor EVs can also cause differentiation of MDSCs themselves into TAMs (21, 22).

Extracellular vesicle miRNA

Most cells release various types of lipid-bilayer enclosed vesicles of distinct size and origin, collectively referred to as EVs (23). The term EVs encompasses small vesicles of around 100nm in size, often called exosomes, larger vesicles (microvesicles) up to 1000nm, and large apoptotic bodies larger than 1000nm. Nomenclature used to name EVs in the literature can be confusing, since characterisation data is often insufficient to use a specific identifying name, and terms are not used consistently, therefore the term EV will be used exclusively in this review to refer to all types of vesicles discussed.

Cells produce EVs primarily through intraluminal vesicle formation in multivesicular bodies and through the outwards budding of the plasma membrane (24). EVs express proteins on their membrane reflective of their parent cell, and they also contain a multitude of molecules within their lumen. Various RNA species have been identified in EVs, with miRNA often representing a large portion of the RNA identified in a vesicle population (25). Higher miRNA expression within a cell will mean greater loading of that miRNA into EVs, whilst high levels of the miRNA target RNA will reduce miRNA loading (26). There are also a number of miRNA motifs that enable miRNAs containing these motifs to be selectively loaded into EVs (27). Furthermore, a number of RNA-binding proteins (RBPs) have been shown to direct miRNA loading into EVs (28–30). Interestingly, EVs are also loaded with pre-miRNAs, and carry the machinery involved in miRNA maturation, meaning these pre-miRNAs can be processed, becoming mature miRNAs within the EV itself (31). Secretion of EVs, containing miRNAs, is variable, depending on the state of the secreting cells. In cancer, changes in the local microenvironment can alter the quantity of EVs secreted. Hypoxia leads to increased secretion of EVs in breast cancer cells (32); increased acidity in the TME also leads to increased release of EVs regardless of the histology of the cancer (33), with the overall cargo makeup of the EVs also being altered (34). Tumor microenvironment acidosis is a hallmark of cancer and creates an evolutionary pressure to foster the malignant phenotype. This metabolic feature can be exploited to develop new therapeutic anticancer approaches, as brilliantly described by Pillai et al. (35). An acidic microenvironment can alter the miRNA content of EVs, Tian et al describe upregulation of pro-tumorigenic miRNAs in hepatocellular carcinoma EVs when parent cells are subjected to acidic conditions (36).

Following secretion from their parent cell, EVs, carrying these aforementioned cargos, contribute to cell-cell communication through the delivery of their contents to the surface and cytoplasm of recipient cells. In cancer, there is a vast amount of data describing EV-mediated cell-cell communication with the TME (3), and now miRNAs from these EVs are understood to be involved in this process. Once miRNAs are delivered by EVs to cells they can silence genes via typical post-transcriptional regulation (37), and they can also function atypically, through activation of toll-like receptors (38). The delivery of EV-miRNA to recipient cells and their mechanisms of action has been reviewed by our group previously.

TAMs are known to secrete EVs that are tumor-promoting in nature (39). In the following section, we will provide an overview of the miRNAs that TAMs secrete in their EVs, and the pro-tumoral processes these miRNAs drive. The miRNAs known to be involved in these processes are summarized in table 1 and represented visually in figure 1.

Table 1.

List of miRNAs enriched in TAMs-derived EVs and their functions.

microRNA	Cancer Type	Overall effect	Type of study	Reference
miR-223	Breast cancer Gastric Cancer Epithelial Ovarian Cancer	Pro-invasive Drug Resistance Drug Resistance	In vitro In vitro + human tissues In vitro + in vivo human tissues	(Yang et al. 2011) (Gao et al. 2020) (Zhu et al. 2019)
miR-21	Gastric Cancer Colorectal cancer	Drug Resistance Metastasis promotion	In vitro + in vivo In vitro + in vivo	(Zheng et al. 2017) (Lan et al.2019)
miR-365	Pancreatic Adenocarcinoma	Drug Resistance, Tumor progression	In vitro + in vivo In vitro + in vivo	(Binenbaum et al. 2018; (Li et al. 2021b)
miR-5100	Breast cancer	EMT, invasion and metastasis suppression	In vitro + in vivo	(Yue et al. 2021)
miR-7	Epithelial Ovarian Cancer	Metastasis suppression	In vitro + in vivo	(Hu et al. 2017)
miR-29a-3p and miR-21–5p	Epithelial Ovarian Cancer	Immune escape Metastasis promotion	In vitro + in vivo	(Zhou et al. 2018)
miR-16–5p	Gastric Cancer	Immune response stimulation Inhibition of Tumor Formation	In vitro + in vivo Human tissues	(Li et al. 2020)
miR-130b-3p	Gastric Cancer	Survival, Metastasis and Angiogenesis	In vitro + in vivo Human tissues	(Zhang et al. 2020)
miR-155 and miR-196a-5p	Non-small-cell Lung Cancer	Metastasis promotion	In vitro + in vivo	(Li et al. 2021a)
miR-155–5p	Colon Cancer	Immune Escape Metastasis promotion	In vitro + in vivo In vitro + in vivo	(Ma et al. 2021) (Lan et al.2019)
miR-501–3p	Pancreatic adenocarcinoma Lung Cancer	Invasion and metastasis promotion Tumor Progression	In vitro + in vivo In vitro + human tissues	(Yin et al. 2019) (Lei et al. 2021)
miR-95	Prostate cancer	Tumor progression	In vitro + in vivo Human tissues	(Guan et al. 2020)
miR-221–3p	Epithelial Ovarian Cancer	Tumor cells proliferation	In vitro + in vivo Human tissues	(Li et al. 2020)

Figure 1. TAM EV-miRNAs promote tumor growth and survival.

EVs secreted by TAMs deliver miRNAs to cells of the TME. These specific miRNAs contribute to the following pro-tumoral processes: instructing cancer cells to be more proliferative, migratory and invasive, regulation of immune cells to enable immune escape for cancer cells, and increasing resistance of cancer cells to anti-cancer drug treatment.

Roles of miRNAs from TAM-EVs

Migration and invasion

One of the major effects produced by TAMs on tumor cells behavior is their capacity to profoundly alter tumor invasiveness and metastasis. This key feature is extremely important for proliferating tumor cells which need to take advantage of this support to maximize their chances of survival. Acting as allies, TAMs often lend a hand by releasing EVs containing particular miRNAs that are able to boost the invasive potential of the recipient cancer cells. In colorectal cancer (CRC), TAMs have been positively associated with increased metastasis. Lan et al. described the transfer of two microRNAs, miR-21–5p and miR-155–5p, from M2-macrophage exosomes to CRC cells (40). The interaction resulted in increased migratory and invasive abilities by the cancer cells after co-cultures due to BRG1 downregulation. These effects were proven to be dependent from miR-21–5p and miR-155–5p because the use of M2-macrophages depleted of the two microRNAs led to a reduction of their levels in the cancer cells accompanied by a significant impairment of their invasive capabilities. Another study revealed that M2-macrophage-derived exosomal miR-155 and miR-196a-5p promote EMT and metastasis of non-small-cell lung cancer (NSCLC) through targeting RASSF4 (41).

The direct relationship between M2-macrophages EVs and cancer metastasis is also evident in pancreatic ductal adenocarcinoma (PDAC). Exosomes derived from M2 macrophages were shown to shuttle miR-501–3p into PDAC cell lines causing enhanced invasiveness. Similar effects were found in vivo where the injection of miR-501–3p-enriched exosomes from M2-macrophages produced more tumor formation and metastasis in mice (42). Mechanistically, the transfer of miR-501–3p inhibited the expression of Transforming growth factor-beta receptor 3 (TGFBR3), an important tumor suppressor and key regulator of metastatic PDAC.

Another miRNA linked to invasion-potentiating effects is miR-223. This miR was found to be highly expressed in exosomes produced by interleukin-4 (IL-4)-activated macrophages, i.e. polarized M2 macrophages endowed with pro-tumorigenic potential (43). After being exposed to exosomes secreted by IL-4 activated macrophages, miR-223 levels increased in breast cancer cells (44). MiR-223 acted as an enhancer of β-catenin accumulation in the cancer cells nuclei, promoting the development of a more invasive behavior.

The miRNA content of TAM-derived EVs can be influenced by the effect of cytokines present in the inflamed tumor microenvironment. Also, the combination of anti/proinflammatory molecules might profoundly alter the functional effects exerted from EVs released by TAMs. It’s the case of progranulin (PGRN), an inflammatory protein often overexpressed in cancerous tissues (45). Wild-type TAMs normally express PGRN which contributed to their pro-tumorigenic properties. On the contrary, TAMs carrying PGRN deletions in their exosomes were associated with inhibited invasion and epithelial-to-mesenchymal transition (EMT) in breast cancer cells after co-cultures. When injected in breast cancer xenograft mouse models, PRGN−/− TAMs-exosomes inhibited lung metastases (46). Mir-5100, up-regulated in PRGN−/− TAMs-exosomes, was found to alter C-X-C motif chemokine 12 (CXC-12) expression resulting in a reduced activation of the CXC-12/CXCR4 pathway which translated into a less invasive tumor phenotype. The TNF-related weak inducer of apoptosis (TWEAK) protein is another example of regulator of TAMs exosomes effects. Hu et al. conducted an interesting study describing how TWEAK-stimulated macrophages transferred exosomes directly to ovarian cancer cells with an ultimate effect of metastasis inhibition in vitro and in vivo (47). The authors registered high levels of miR-7 in TWEAK-treated macrophages, their exosomes and recipient cancer cells suggesting how this miR might be the main responsible for the observed phenotypic change. Indeed, by using a specific antagomir, miR-7 levels sensibly decreased in macrophages and in the recipient cancer cells which recovered their invasiveness.

Immune regulation

EVs from TAMs demonstrated to significantly contribute to immune evasion. Even though few in numbers, new experimental evidence has been published on this subject. In a work by Ma et al., M2-macrophages-derived exosomes were delivered to CRC cells leading to enhanced proliferation and anti-apoptotic signaling (48). The authors evidenced the central role played by miR-155–5p in the downregulation of Zinc-finger-type-containing 12B (ZC3H12B), a protein involved in the regulation of the inflammatory process (49). The internalization of miR-155–5p in CRC cells caused a drastic reduction of ZC3H12B levels followed by sustained activation of interleukin-6 (IL-6). ZC3H12B is known to decrease the stability of IL-6 mRNA; hence, reduced ZC3H12B expression determines a subsequent increase of IL-6 signaling towards T-cells, impairing their proliferation and recognition capabilities.

TAMs EVs could also interfere with immune surveillance by acting directly on the immune cells. Zhou et al. proposed a mechanism where M2-exosomes are able to alter Treg/Th17 ratio in epithelial ovarian cancer (EOC) tissues transferring their contents to the T-cells in order to deliver immune-suppressive signals and favor EOC metastasis (50). Specifically, M2-exosomes were shown to transfer miR-29a-3p and miR-21–5p to CD4+ T cells. Once taken up by the immune cells, these microRNAs appeared to bind Signal transducer and activator of transcription 3 (STAT3) shifting the Treg/Th17 ratio towards more Treg-polarized cells. A higher Treg/Th17 was related to increased tumor progression and metastasis development in vitro and in vivo.

On the other hand, exosomes released by M1-macrophages revealed opposite effects regarding T-cell response. In a paper by Li et al, miR-16–5p-containing exosomes from M1 TAMs exerted an inhibitory effect on tumor formation in a series of in vitro and in vivo experiments. Mir-16–5p proved to target Programmed Death Ligand-1 (PD-L1) in gastric cancer (GC) cells, decreasing its expression and boosting anti-tumor immune response (51).

Chemoresistance

Exosomal microRNAs released from TAMs have recently been shown to promote drug resistance in a variety of cancer types. In gastric cancer, macrophage-derived exosomal miR-223 induced drug resistance to doxorubicin by inhibiting F-box and WD repeat domain-containing 7 (FBXW7) (52). Hypoxia-induced enrichment of miR-223 in M2-macrophage-derived exosomes caused enhanced drug resistance to cisplatin (cDDP) in EOC cells via the miR-223/PTEN/PI3K/AKT axis (53). Interestingly, the authors evidenced a direct link in clinical data where EOC patients characterized by elevated levels of hypoxia-inducible factor 1 (HIF-1a) showed marked CD163+ cells infiltration and corresponding high miR-223 expression.

M2-macrophage-derived exosomal miR-21 also caused drug resistance to cisplatin in gastric cancer by inhibiting apoptosis and activating the PI3K/AKT signaling pathway (54). Cisplatin resistance by M2-exosomes is also reported in lung cancer. Here, miR-3679 played a pivotal role down-regulating the expression of Neural precursor cell expressed developmentally downregulated gene 4-like (NEDD4L). This E3 ligase is responsible for c-MYC ubiquitination; hence, NEDD4L decreased expression resulted in c-MYC stabilization, subsequent glycolysis, and ultimately drug resistance. In PDAC, exosomal transfer of miR-365 TAMs to the cancer cells induced drug resistance to gemcitabine (55). Molecularly, the increased uptake of miR-365 in PDAC cells led to higher amount of nucleotide triphosphate pools, ready to compete with the intermediate metabolites of gemcitabine for DNA incorporation. Moreover, miR-365 proved to be capable of enhancing cytidine de-aminase, an enzyme dedicated to the conversion of gemcitabine in de-aminated byproducts which are promptly excreted out of the tumor cells, thereby promoting chemoresistance.

Tumor proliferation

TAMs can shuttle microRNAs in extracellular vesicles to cancer cells in order to promote tumor progression via the regulation of specific target proteins. For example, exosomal miR-130b-3p from M2 macrophages regulates the expression of Grainyhead Like Transcription Factor 2 (GRHL2) by downregulating mixed-lineage leukemia protein 3 (MLL3) in the recipient gastric cancer cells. This alteration reduces H3K4me1 and H3K27ac enrichment in the GRHL2 enhancer which ultimately promotes the survival and progression of the tumor cells (56). Proliferation and EMT are common effects exhibited by cancer cells after uptake of TAMs-exosomes. Guan et al. propose that elevated miR-95 levels in TAMs-exosomes might partly explain the aberrant progression observed in prostate cancer (PCa) cells (57). Using gain and loss-of-function assay coupled with in vivo models, the authors discovered miR-95 transfer from TAMs to PCa cells where it directly binds to its target JunB to potentiate cancer cells proliferation. In PDAC, M2-EV-miR-365 has been shown to promote proliferation, migration and invasion by targeting and downregulating BGT2 and therefore activating the focal adhesion kinase (F/ATP)-dependent tyrosine kinase (AKT) pathway (58). Another microRNA to be enriched in TAMs-derived EVs that plays a significant role in promoting tumor progression is miR-501–3p. miR-501–3p was found to promote tumor progression via downregulation of WD repeat domain 82 (WDR82) in lung cancer (LC) cells. LC cells treatment with M2 exosomes increased levels of miR-501–3p, suppressed apoptosis and enhanced proliferation, migration and invasion in the cancer cells (59). Furthermore, TAMs-exosomal miRNAs could target key cell cycle regulators to trigger tumor cell proliferation. In their paper, Li et al. asserted the decisive role of miR-221–3p in EOC progression (60). Specifically, mir-221–3p is loaded into M2 exosomes and shuttled inside EOC cells where it targets cyclin-dependent kinase inhibitor 1B (CDKN1B) reducing its expression and facilitating G1/S transition.

Future directions

It has become clear that EV-delivered miRNAs from TAMs play a key role in TAM driven tumor promotion. Understanding these EV-miRNA-directed mechanisms in TAM-tumor crosstalk has uncovered potential therapeutic targets and identifies possible diagnostic markers. EV-miRNA has shown promise as a biomarker for identifying cancer in patient blood (61, 62). Since the abundance of TAMs in tumors is a strong prognostic marker, their associated EV-miRNAs could serve a similar purpose, or enable early detection of TAM presence in neoplastic tissue. Furthermore, EVs have the advantage over tissue biopsy in that they are less invasive, blood samples may be sufficient in detecting the relevant EV-miRNA.

There are currently anti-miRNA therapies being studied, such as the use of anti-miRNA oligonucleotides (AMOs) (63). These nucleotide sequences have regions complementary to specific miRNAs, allowing them to sequester miRNAs within a cell, preventing the miRNA from carrying out its function. Indeed, sequences complimentary to tumor-promoting miRNAs are now being presented as decoys, or “sponges”, to block the action of the miRNA they specifically target (64). Elucidating the miRNAs most important in TAM-tumor crosstalk could enable development of specific anti-miRNAs to target miRNAs. Since it is clear that TAM-derived EVs aid tumors, abrogating EV release from TAMs could also be a desirable method for inhibiting TAM-tumor crosstalk. Many inhibitors of EV release have been reported (65), although how inhibitors such as these could be used to precisely target a specific cell type like the TAM remains unclear. Alternatively, our understanding that EVs effectively deliver miRNAs to cells, impacting cell behavior, is opening the door for the possibility of manipulating EVs themselves to deliver desirable miRNAs to achieve a therapeutic outcome (66). It is worth noting that macrophage removal of therapeutic agents through EV secretion is a possible mechanism of resistance to treatment. Cancer cells are known to secrete chemotherapeutic molecules encapsulated in EVs, thus protecting themselves from the harmful effects of the therapy (67, 68). Furthermore, gold nanoparticles engulfed by macrophages have been shown to be ejected from the cells within their EVs (69), revealing how macrophages might resist treatment with small molecules. Innovative therapeutic approaches include the use of natural EVs and nanoparticles to deliver chemotherapeutic agents or photodynamic molecules to cancer cells (70). While EVs are excellent vehicles of therapeutic cargoes, selectivity and specificity of its uptake by cancer cells or other cells of interest within the TME remains an important challenge. A better understanding of the mechanisms involved in EV organotropism and cargo delivery is currently ongoing, and will lead to the development of effective strategies to achieve this important goal.

We have presented an overview of the various EV-miRNAs secreted by TAMs, and we see that there are diverse mechanisms by which tumor growth and survival is impacted. Uncovering the EV-miRNAs involved in TAM activity gives us a greater understanding of how TAMs function, hopefully leading to the discovery of new cancer biomarkers or targets for drug development.