Adult diffuse low-grade gliomas (LGG) are classified and graded based on histological and molecular parameters according to the World Health Organization (WHO) classification of brain tumors [1
]. Although glioblastoma (WHO grade IV) remains the most aggressive and deadliest tumor of the central nervous system, with a median survival time of 12.2–18.2 months [2
], WHO grade II gliomas also represent a clinical challenge, due to their highly variable behavior [3
]. Indeed, some LGG remain indolent for years, while others rapidly progress to glioblastoma, therefore, the survival of LGG patients ranges from 15 years to 1 year [3
]. Comprehensive genome studies have contributed to a better prognostic stratification of lower grade glioma [5
]. Specifically, diffuse gliomas isocitrate dehydrogenase (IDH) wildtype are characterized by the worst prognosis, oligodendrogliomas, IDH mutant, and 1p/19q co-deleted, by the best while diffuse astrocytoma IDH mutant by an intermediate prognosis [5
]. However, even inside the same molecular group, there are differences in both risk and timing of malignant transformation. This makes it difficult to choose the therapeutic strategy, aimed at aggressively treating patients with a potentially more malignant neoplasm and avoiding adjuvant therapies to patients who could only progress after many years [3
]. In fact, chemotherapy and especially radiotherapy, the only available adjuvant treatments, are burdened by severe side effects, such as the risk of late cognitive defects following radiotherapy. To refine the therapeutic approach towards personalized medicine, it is increasingly important to understand the molecular mechanisms driving the malignant evolution of LGG. To this aim, Bai et at. compared the genomic landscape of 41 WHO grade II IDH mutant astrocytomas to their higher-grade, progressed counterpart. Integrated genomic analyses, including whole-exome sequencing and copy number, gene expression, and DNA methylation profiling, showed, in the progressed sample, the activation of the MYC and RTK-RAS-PI3K pathways and upregulation of genes involved in the cell cycle transition, such as FOXM1 and E2F2, and the epigenetic silencing of genes related to Polycomb repressive complex 2 [10
However, increasing evidence shows that cancer progression is driven, not only by genetic alterations, but also by the crosstalk established by tumor cells and the surrounding microenvironment [11
In gliomas, the tumor microenvironment (TME) consists of non-tumor cells, such as endothelial cells, tumor-associated macrophages (TAMs), microglia, resident astrocytes, extracellular matrix components, proteins, and secreted molecules, all acting in the intercellular communication with tumor cells, thus modulating disease progression [15
Important players of the potent cross talk between cancer cells and the TME are exosomes, small extracellular vesicles, with a diameter ranging between 50 to 150 nm [18
]. They originate in the multivesicular body compartment of the endoplasmic reticulum and are released into the extracellular space and in the body fluids from many cell types [21
]. After release, exosomes are internalized by neighboring or distant cells delivering their content in form of biologically active molecules (e.g., proteins, mRNAs, miRNAs, and lncRNAs), thus modifying the phenotype of target cells [22
]. Indeed, there are increasing evidences that tumor cells release exosomes to create an environment supporting angiogenesis, invasion, tumor proliferation, and the premetastatic niche [24
]. Moreover, it has also been shown that tumor-associated fibroblasts, through the release of exosomes, could induce in breast tumor cells the acquisition of a metastatic phenotype [27
In glioblastoma, exosomes released by tumor cells, containing mRNA, miRNA, and angiogenic proteins, are able to act on endothelial cells, favoring the development of a tumor-permissive microenvironment [28
]. Exosome miRNAs were associated with gliomagenesis by modulation of several signaling pathways [29
]. miRNAs are non-coding single stranded RNAs of 18–27 nucleotides that influence various cellular processes decreasing the level of translation of their target mRNAs [32
The packaging of miRNAs and proteins inside the exosomes is a selective process, as their content can be modulated at different stages of cancer development, mirroring the physiological state of the cell of origin, thus representing prognostic/predictive biomarkers [33
Altogether, this knowledge contributes to increased attention on the TME as a novel source of therapeutic targets and markers of disease progression [36
Cells residing in the TME would be less prone to develop drug-resistance, being devoid of mutations unlike cancer cells. Indeed, multiple TME-directed therapies are now under evaluation in clinical trials [38
To deepen the comprehension of the role played by the TME on the malignant progression of LGG, we have taken advantage of our previously established in vitro model of glioma microenvironment, represented by glioma-associated stem cells and their released exosomes [41
Glioma-associated stem cells (GASC), isolated both from human low- and high-grade glioma samples, are characterized by an undifferentiated mesenchymal phenotype, clonogenicity, and multipotency [41
]. This cell population does not show genetic aberrations of the tumor of origin, and it is devoid of tumor-initiating properties, in vivo; nevertheless, GASC are characterized by the ability to grow in an anchorage-independent way, in vitro. Moreover, GASC from LGG were endowed with prognostic potential when comparing low-grade and high-grade gliomas’ specific surface markers. We also demonstrated that GASC could prognostically stratify LGG based on the expression of the NF-κB-related transcriptional program exerting their tumor-supporting properties through the stimulation of cytokines such as TNFα, IL1β, and IL-6, constituting new targets for novel adjuvant therapies.
In our in vitro model, GASC, from both HGG and LGG, were able to increase the in vitro aggressiveness of glioblastoma, through the release of exosomes, although those from LGG at a significant lower extent [41
]. These data suggest that the degree of the tumor-supporting ability was proportional to the grade of malignancy of the tumor of origin. We have also identified a pro-migratory protein (SEMA-7A) able to promote the motility of glioma stem cells (GSC) in vitro, exposed on the surface of HGG-GASC exosomes [44
As we have already demonstrated the prognostic value of GASC and assessed the importance of exosomes in sustaining the bidirectional crosstalk with tumor cells through the release of bioactive molecules, in this paper, we aimed at dissecting the miRNA content of exosomes derived from either GASC isolated from LGG characterized by a good prognosis or GASC isolated from LGG characterized by a rapid anaplastic transformation, regardless of WHO classification status. Taking advantage of deep sequencing and bioinformatic tools, we identified putative miRNAs possibly responsible for the heterogeneity in prognosis that characterizes LGG.
WHO grade II gliomas, here defined as LGG, represent a group of usually slow-growing primary malignant brain tumors, which, however, in 70% of cases undergo recurrence and/or transformation into tumors with greater malignancy [4
]. Thus, LGG outcome is heterogeneous, with an overall survival ranging from 2 to over 12 years. Predicting the outcome and distinguishing low-risk and high-risk patients is fundamental to assist clinicians in the decision-making process aimed at not either overtreating or undertreating patients [3
]. Although age (>40 years), incomplete extent of resection, radiological progression, and/or neurological symptoms contribute to recognizing high-risk patients, a better prognostic stratification is required [3
]. The molecular classification of LGG into three classes (IDH wildtype, IDH mutant only, and IDH mutant and 1p/19q co-deleted), according to WHO criteria [1
], has further contributed to recognize patients at risk of quick progression [5
]. Beside prognostic stratification, the choice of adjuvant treatments also remains challenging. Available therapies, radiotherapy and chemotherapy, are burdened by important side effects (e.g., late cognitive defects after radiotherapy), which limit their use in patients with a long life expectancy.
For this reason, there is an increasing interest of the scientific community in the TME [12
]. The TME consists of various types of non-tumor cells, such as endothelial cells, stromal cells, pericytes, immune cells, and components of the extracellular matrix. The bidirectional crosstalk between tumor cells and TME contributes to tumor development and progression by promoting tumor cell proliferation, invasion, and angiogenesis; reprogramming of energy metabolism, and suppressing cell death and immune response, well-known hallmarks of cancer [12
]. It has been shown that the TME can predict prognosis, and it is considered an interesting therapeutic target [51
]. It has been suggested that more than ablation of the TME, it might be useful to interrupt the bidirectional crosstalk with tumor cells or to reverse its phenotype by re-establishing a microenvironment capable of counteracting tumor growth [51
]. It has been shown that, in the tumor, both cancer cells and tumor-associated stromal cells promote tumor-induced immune suppression, angiogenesis, and metastasis through the release of exosomes [18
]. The latter, through their content in biologically active molecules, including miRNAs, can act as a potent intercellular communication system [18
To better investigate the role of the TME in glioma, we have isolated from low-grade and high-grade gliomas a population of stem cells, named glioma-associated stem cells (GASC). GASC are a population of tumor derived stem cells representative of the TME: although devoid of the genetic alterations of the tumor cells, they are able to support, in vitro, the tumor growth [41
]. Additionally, we demonstrated that GASC derived from LGG can independently predict patient prognosis [41
], and, from the transcriptomic analysis of GASC derived from LGG with different prognosis, it was possible to identify a prognostic gene signature further validated in 530 newly diagnosed diffuse LGG patients comprised within The Cancer Genome Atlas (TCGA) database [42
]. Importantly, GASC exert their tumor-supporting function through the release of exosomes [41
]. Specifically, GASC-derived exosomes are able to increase proliferation, motility, and anchorage-independent growth of both commercially available glioblastoma cell lines and patient-derived glioma stem cells (GSC) [41
To obtain insights into the TME-related mechanisms, possibly responsible of the more aggressive behavior of some LGG, we isolated exosomes from the supernatant of GASC derived from both LGG patients undergoing a rapid malignant transformation (EXO_BAD) and LGG patients experiencing malignant transformation after at least 7 years (EXO_GOOD).
EXO_BAD and EXO_GOOD were produced with the same yield from GASC_GOOD and GASC_BAD and shared similar size, concentration, and markers that ascribed them as exosomes. However, some differences could be detected in their miRNA content. It is important to notice that, although ultrafiltration using 100 kDa molecular weight cutoffs could remove some RNPs, we cannot exclude the presence of ribonucleoprotein particles complexes or high-density lipoproteins. Still, the samples were isolated in the same conditions resulting in a shared medium-derived contaminant background throughout the experiment.
By taking advantage of deep sequencing technologies, we analyzed the miRNA profiles of both exosomes and their producing cells. With respect to their cellular counterpart, exosomes were either enriched in some miRNAs or depleted in others. This is consistent with what previously published: as shown for mRNA [28
], miRNAs also seem not to be randomly selected and loaded into exosomes [57
Comparing the miRNAs enriched or depleted in EXO_GOOD and EXO_BAD, with respect to their cellular counterparts, we identified: 1. a large group of miRNAs similarly regulated in the two groups and 2. miRNAs differently regulated in EXO_GOOD and EXO_BAD.
Focusing our attention on the miRNA signature shared by GASC exosomes, independently from patient prognosis, we observed either upregulation or down regulation of both oncosuppressive and oncogenic miRNAs. For example, with respect to their cellular counterpart, GASC exosomes resulted in the depletion of many members of the let-7 family. This latter exerts a tumor suppressive function by inhibiting proliferation and malignancy of glioblastoma cells by targeting both NRAS and KRAS [60
] and by modulating microglia activation through TLR7 [62
]. On the other hand, GASC exosomes resulted in the depletion of miR-21 and miR-221, two miRNAs whose oncogenic role in glioblastomas has been widely demonstrated [63
]. Similarly, GASC exosomes were enriched in miR-451 and miR-150, which are known to inhibit GSC growth [67
] and glioma cell proliferation, invasion and apoptosis through the Akt pathway [68
] and to suppress glioma cell proliferation and migration by targeting MMP1, respectively [69
]. Conversely, GASC exosomes were enriched in miR-1246, responsible for glioma immune escape by targeting TERF2IP [70
], and in miR-4516, known to exert a tumor promoting function by regulating the Hippo pathway in glioblastoma [71
]. It would be extremely interesting to compare this upregulated and downregulated “baseline” miRNA signature with non-transformed glial cells, to assess if this signature could be representative of glial cells.
Looking instead to the two miRNAs specifically enriched in EXO_GOOD, miR-320c has been shown to exert a tumor suppressive function in glioma, by acting through the modulation of either RAF1/MAPK pathway or E2F2, a key transcription factor involved in the regulation of the G1/S phase [72
]. Li et al. reported that miR-320c could increase radiosensitivity of glioma cell by inhibiting SIRT1 through FOXM1 modulation [74
]. No information is instead available on a possible role of miR-4532 in glioma. Regarding the miRNAs selectively upregulated in GASC_BAD, an oncogenic role, in glioma, has been described for miR-223-3p [75
], miR-10b-5p [76
], and miR-182-5p [78
], while miR-338-3p [80
], miR-204-5p [82
], and miR-126-3p [83
] were described as oncosuppressive. Instead, few or no information on the role of miR-4492 [84
], miR-4508, miR-7704, miR-4488 [85
], and miR-151b on glioma are available.
To better understand these data of non-univocal interpretation, we decided to verify the possible clinical significance of the identified exosome signatures in 477 newly diagnosed diffuse LGG patients comprised within the TCGA database [42
]. We considered two signatures corresponding to the miRNA upregulated in EXO_GOOD and in EXO_BAD and two signatures corresponding to the miRNA downregulated in EXO_GOOD and in EXO_BAD. Interestingly, all four signatures, if upregulated, had negative prognostic significance. This suggests that miRNA signatures, although composed of oncogenic and oncosuppressive miRNAs, are globally the representation of a more aggressive tumor phenotype. Additionally, the fact that both upregulated and downregulated miRNAs were associated with a poor prognosis suggests that the tumor supportive function of exosomes is likely mediated by the delivery of over-expressed miRNAs rather than by the reduced transfer of tumor suppressor miRNAs. In fact, even miRNAs whose expression is reduced in exosomes were oncogenic. We can therefore hypothesize that they represent a class of oncogenic miRNA mainly retained at intracellular level.
Consistently, when we proceeded with the functional annotation of miRNAs upregulated and downregulated in EXO_GOOD and EXO_BAD, we noticed that, independently from the GASC of origin, upregulated and downregulated miRNAs were targeting different KEGG pathways. In fact, downregulated miRNAs, either in EXO_GOOD or EXO_BAD, were mainly acting on cell metabolism, cell growth, and cell–cell matrix interaction. This supports the hypothesis that an intracellular enrichment of these miRNAs could confer a more aggressive tumor phenotype. Upregulated miRNAs were instead modulating KEGG pathways associated with different cancers, including glioma, chronic myeloid leukemia, prostate carcinoma, non-small cell carcinoma, renal cell carcinoma, and pancreatic carcinoma. This suggests that exosomes can transfer miRNAs able to modulate a more general “tumor-related mechanism” globally shared by different cancers.
As mentioned, when we compared the KEGG pathways modulated by upregulated EXO_BAD and EXO_GOOD, no major differences were detected. Same observation could be done for downregulated miRNAs in EXO_GOOD and EXO_BAD.
Therefore, to get insights into the mechanisms by which EXO_BAD could exert a stronger tumor-supporting function, we focused our attention on two KEGG pathways shared by all the four exosome-related signatures: “Glioma” and “Proteoglycan in cancer”. The first one was selected because it strictly related to our tumor setting. The second one because it is known that proteoglycans can regulate cell signaling and migration, as well as immune response, by interacting with both extracellular (extracellular ligands, growth factor receptors, and extracellular matrix) and intracellular (enzymes and structural proteins) elements [86
]. Therefore, proteoglycans are key players in intracellular oncogenic pathways as well as in tumor–TME crosstalk [86
]. Additionally, in glioma, it has been demonstrated that the expression of heparan sulfate proteoglycans is significantly increased in high grade gliomas, with respect to LGG [87
], and a similar pattern has been demonstrated for the dermatan sulfate proteoglycan endocan [88
] and the cell surface chondroitin sulfate proteoglycan CD44 [89
Restricting our analysis to EXO_BAD, we assessed that, while some miRNAs were similarly upregulated or downregulated in EXO_GOOD, few others were specific of EXO_BAD. We named the first ones EXO-SHARED miRNAs and the latter BAD-SPECIFIC miRNAs. In particular, miR-126-3p and miR-182-5p were BAD-SPECIFIC miRNAs upregulated in both “Glioma” and “Proteoglycans in cancer” KEGG pathways, while miR-223-3p was exclusively upregulated in the second one. Concurrently, three BAD-SPECIFIC miRNAs (miR-29b-3p, miR-34a-5p, and miR-497-5p) were downregulated in both “Glioma” and “Proteoglycans in cancer” pathways, and one, miR-582-3p, exclusively in the “Proteoglycans in cancer” pathway.
miR-182-5p is a well-described oncomiR that, in HGG, is known to promote the proliferative and invasive capacities of tumor cells through the modulation of either STAT3 or NF-κB signaling pathways [78
]. Interestingly, analyzing the target genes comprised in the KEGG pathways, miR-182-5p modulates all the validated targets present in the “Glioma” pathway and most of the targets present in the “Proteoglycans in cancer” pathway. miR-223 is another well-recognized oncogenic miRNA, which, in glioblastoma, promotes growth and invasion of tumor cells by targeting the tumor suppressor PAX6, exerting its function by inhibiting the expression of metalloproteases MMP2 and MMP9 [91
]. As mentioned above, the role of miR-126-3p is more controversial, since it has been recognized as an oncosuppressive miRNA [83
]. However, more recently, its role in retinal angiogenesis has been demonstrated, both in vitro and in vivo, by protecting endothelial cells from apoptosis [92
Concerning miRNAs downregulated in the EXO_BAD, miR-34a-5p along with miR-497-5p were the most involved in the modulation of all predicted targets in both “Glioma” and “Proteoglycans in cancer” pathways. miR-34a is a p53 transcriptional target poorly expressed in HGG compared to normal brain tissues [93
], and it is known to suppress in vitro and in vivo tumor growth by targeting c-MET and NOTCH, thus directly modulating glioma cell cycle [94
]. Moreover, miR34a has been reported to modulate glioma cells apoptosis by targeting BCL2 [95
] and stimulating senescence in glioma cells by inducing DNA damage [96
]. On the other hand, the clinical significance of miR-497 has not been fully elucidated yet, but studies described a significant decrease in this miRNA in HGG with respect to LGG and demonstrated its role in suppressing angiogenesis, by targeting VEGFA [97
] and glioma proliferation and epithelial-to-mesenchymal transition, by modulating WNT3A [98
]. Moreover, decreased expression of miR-497 was associated, in glioma, with a worse prognosis [97
Therefore, our study suggests that targets specific for the aggressive phenotype in LGG are modulated by miR-182-5p, miR-223-3p, miR-34a-5p, and miR-497-5p. It could be extremely interesting to further investigate these four miRNAs and the role they play in the evolution of LGG.
It is also worth noting that most of the gene targets in the “Glioma” pathway are included in the “Proteoglycans in cancer” pathway, thus confirming the importance of proteoglycans in glioma progression [86
Finally, considering the genes targeted by either BAD-SPECIFIC or EXO-SHARED miRNAs for each KEGG pathway analyzed, three were the main observations. First, BAD-SPECIFIC miRNAs, even when fewer than the EXO-SHARED ones, were able to modulate most of the target genes included in the considered KEGG pathway. Second, BAD-SPECIFIC miRNAs and EXO-SHARED miRNA presented a certain number of common gene targets, suggesting that there was an increased modulation of these genes in cells capable of internalizing EXO_BAD. Third, BAD-SPECIFIC miRNAs could hit an additional number of gene targets, compared to EXO-SHARED miRNAs. This leads us to hypothesize that the greater tumor-supporting function of EXO_BADs could be linked to their increased ability to modulate target pathways. Several issues remain to be elucidated. The presence of an increased expression or downregulation of both oncosuppressive and oncogenic miRNAs can be due to the fact that LGG are a low-grade tumor anyway, and some miRNAs can be the manifestation of a baseline tumor suppressive signal, which could explain the slower malignant evolution of this neoplasm.
The confirmation that miR-182-5p, miR-223-5p, miR-34a-5p, and miR-497-5p could be putative miRNAs involved in the aggressive phenotype of the tumor requires further in vitro and in vivo study.
Additionally, the mechanisms responsible for the selection of the miRNA cargo remain an interesting field of study; understanding them could get insights into the biology of cancer and the possibility to hijack this intracellular communication system to deliver tumor-suppressive messages able to revert the tumor-supporting function of the TME [51
In conclusion, the widely variable LGG prognosis and the lack of specific adjuvant treatment requires the identification of novel prognostic markers and therapeutic targets. In this regard, our work suggested that patient-derived GASC, stem cells representative of the tumor microenvironment, and their released exosomes could give important insights. We have demonstrated that exosomes released by GASC isolated from LGG characterized by different prognosis possess a miRNA content that can modulate key pathways in tumor progression and aggressiveness. We could identify miRNA signatures endowed with clinical significance, being able to predict prognosis in 477 LGG patients. Additionally, exosomes derived from GASC of patients with a bad prognosis possess a unique repertoire of BAD-SPECIFIC miRNAs, possibly responsible for the more aggressive function. Further studies are required to demonstrate whether this information could lead to novel therapies aimed at targeting the crosstalk between tumor cells and their tumor-supporting microenvironment.