Glioblastoma-Associated Microglia Reprogramming Is Mediated by Functional Transfer of Extracellular miR-21
Open accessAbstract
Gliomas are primary, diffusely infiltrating brain tumors. Microglia are innate immune cells in the CNS and make up a substantial portion of the tumor mass. Glioma cells shape their microenvironment, communicating with and reprogramming surrounding cells, resulting in enhanced angiogenesis, immune suppression, and remodeling of the extracellular matrix. Glioma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Mouse glioma cells stably expressing a palmitoylated GFP to label EVs were implanted intracranially into syngeneic miR-21-null mice. Here, we demonstrate functional delivery of miR-21, regulating specific downstream mRNA targets in microglia after uptake of tumor-derived EVs. These findings attest to EV-dependent microRNA delivery as studied in an in vivo-based model and provide insight into the reprograming of microglial cells by tumor cells to create a favorable microenvironment for cancer progression.
AI snapshot
Written from the paper’s full text.
TL;DR
Glioma-derived extracellular vesicles (EVs) transfer functional miR-21 to microglia in vivo, where it downregulates specific target mRNAs and promotes microglial proliferation. Using miR-21-null mice and fluorescently labeled GL261 EVs, the study shows EV-mediated miRNA delivery can reprogram tumor-associated microglia.
Key findings
- GL261-derived EVs contain high levels of miR-21, with significantly higher miR-21 in the 100,000 × g EV fraction than in cellular levels; EVs ranged from 100 to 500 nm by nanoparticle tracking analysis.
- After intracranial implantation of GL261.pGHR cells into miR-21-null mice, microglia and monocytes/macrophages that took up EV-GFP had significantly higher miR-21 than GFP-negative cells; transfer was most efficient in microglia compared with monocytes/macrophages.
- RNA sequencing of EV-GFPpos versus EV-GFPneg microglia found 441 upregulated and 359 downregulated genes; among 59 curated miR-21 targets, 25 had lowest expression in EV-GFPpos microglia, with significant reduction of Nfat5, Bmpr2, Btg2, Rhob, and Kbtbd2 (adjusted p < 0.05), while FasL and Tgfbi were significantly higher.
- In vitro, primary miR-21-null microglia exposed to GL261 EVs (2.43 × 10^10 particles) showed increased miR-21 and downregulation of Bmpr2, Btg2, Kbtbd2, Pdcd4, Pten, and Rhob, with Gapdh unaffected; intracranial injection of GL261 EVs (1.26 × 10^9 particles) led to EV uptake in about 0.3% of all microglia and significant Btg2 reduction. miR-21 mimic or Btg2 siRNA increased Ki67-positive microglia.
Methods
- GL261 glioma cells stably expressing palmitoylated GFP and H2B.mRFP (GL261.pGHR) were implanted intracranially (1 × 10^5 cells) into the left striatum of adult syngeneic miR-21-null mice; after three weeks, brains were digested and cells sorted by FACS for microglia (CD11b/CD45, RFP-negative) and monocytes/macrophages, with ImageStream used to visualize EV-sized GFP uptake.
- EVs were isolated from GL261 conditioned medium by differential ultracentrifugation (300 × g, 2,000 × g, 100,000 × g), characterized by nanoparticle tracking analysis, western blot, and iodixanol density gradient; primary neonatal miR-21-null or wild-type microglia were exposed to EVs or transfected with miR-21 mimic, scrambled control, Btg2 siRNA, or siRNA control.
- RNA sequencing used DESeq2 with Benjamini-Hochberg adjusted p values; qRT-PCR and droplet digital PCR measured miRNA/mRNA targets; proliferation was quantified as Ki67-positive cells per DAPI-positive cells; statistics used unpaired t tests, multiple t tests, and one-way ANOVA with Tukey’s multiple comparisons, with significance at p < 0.05.
Limitations
- The paper states that the palmitoylated GFP reporter is not specific to one EV subtype (exosomes, ectosomes, or microvesicles) but tracks all naturally formed membrane-bound vesicles.
- The authors state they do not exclude additional involvement of other miR-21 carriers, such as large EVs or non-floating non-EV components; high-density fractions containing high-density lipoproteins may also contribute to miR-21 transfer and could explain miR-21 in GFP-negative microglia.
- The paper notes that incomplete Pdcd4 downregulation after intracranial EV injection may reflect relatively low miR-21 levels in that experiment; evident from the text, the main in vivo model uses miR-21-null recipient mice, and RNA sequencing and EV injection experiments used 3 mice per group.
Model-written summary. Check it against the paper before relying on it.
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Claims the paper makes
5 of 7 cited sources checked · 2 supported · 1 flagged · accuracy not yet measured
- Premisereasoning
The presence of GFP in microglia indicates tumor-derived EV uptake.
“The presence of GFP in microglia thus indicates tumor-derived EV uptake (Figure 2C).”
Quote in the paperSubstantial claim2 of 2 sources checked- cites Lai et al., 2015 source
The Results section, “Visualizing tumour-derived EVs in vivo,” identifies membrane-targeted PalmGFP as a label for tumour-derived vesicles, supporting the use of that signal to track EVs. But the Discussion says the identity of non-tumour cells taking up EVs remains to be established; the paper does not show uptake by microglia specifically. It also distinguishes membrane-targeted PalmGFP from soluble GFP, so GFP presence alone is not established as proof of EV uptake. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites van der Vos et al., 2016 source
In the in vivo uptake section (Figs. 5–6), GFP labels microglia and monocyte-derived macrophages, while tumor-derived EVs carry a red fluorescent label. The paper identifies red puncta within GFP-positive cells—not GFP in microglia alone—as evidence of EV uptake. (LLM, conf=0.90)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measured
- Study design and conductown data
Heterogeneity of EV fractions was confirmed by probing for different vesicular protein markers.
“Heterogeneity of these different fractions was confirmed by probing for different vesicular protein markers that are present in all types of EVs and larger vesicle fractions (Flotillin-1) (Kowal et al., 2016), those typically associated with exosomes and absent in 2,000 × g (ALIX and TSG101), and GAPDH found to be enriched in the 2,000 × g and cellular fraction (McNamara et al., 2018).”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites Kowal et al., 2016 source
- cites McNamara et al., 2018 source
The Materials and Methods section (“Protein analysis”; page unavailable) says the authors immunoblotted EV and control fractions using an antibody to GAPDH. It does not report GAPDH enrichment in a 2,000 × g fraction or show that this marker confirmed heterogeneity among the fractions in the citing sentence. The “EV isolation” section instead describes centrifugation at 800 × g and 16,000 × g. (LLM, conf=0.84)
Experimental — accuracy not yet measured
- Findingown data
In miR-21-null microglia exposed to GL261-derived EVs, miR-21 expression was significantly increased compared with UcM.
“Twenty-four hours later, the expression level of miR-21 was significantly increased in microglia exposed to GL261-derived EVs, compared with UcM (Figure 5D).”
Quote in the paperSubstantial claim - Findingown data
Among 59 selected miR-21 target genes, 25 genes showed lowest expression in EV-GFPpos microglia.
“Relative gene expression of the selected 59 target genes showed 25 genes with lowest expression in EV-GFPpos microglia (Figure 4C).”
Quote in the paperSubstantial claim - Findingown data
Intracranial injection of GL261-derived EVs into miR-21-null mice results in about 0.3% of all microglia taking up EVs.
“Sixteen hours after injection of the EVs into the left striatum (using similar coordinates as tumor implantation) of miR-21-null mice, about 0.3% of all microglia in the brain (blue circle) that had taken up EVs (green box) were detected (Figure 6B).”
Quote in the paperNot a substantial claim
- Findingown data
Pathway analysis showed that pathways involved in cell cycle control were upregulated in EV-GFPpos compared with EV-GFPneg microglia, indicating a higher degree of proliferation after EV uptake.
“Pathway analysis of genes differentially expressed at higher levels in EV-GFPpos compared with EV-GFPneg microglia indeed showed that a number of pathways involved in cell cycle control were upregulated, indicating a higher degree of proliferation in microglia after EV uptake (Figure S6A).”
Quote in the paperSubstantial claim - Findingown data
Genes described to be involved in microglial proliferation are expressed at higher levels in microglia from tumor-bearing brains, with highest expression in EV-GFPpos microglia.
“All of these genes are expressed at higher levels in microglia from tumor-bearing brains, with the highest expression in EV-GFPpos microglia (Figure 7A).”
Quote in the paperSubstantial claim - Findingown data
GL261-derived EVs contain significantly higher levels of miR-21 compared with GL261 cellular levels.
“Importantly, miR-21 was present in GL261 cells, 2,000 × g fraction, and GL261-derived EVs, with significantly higher levels of miR-21 in the EVs compared with cellular levels (Figures 1D and 1E).”
Quote in the paperSubstantial claim - Findingown data
FasL and Tgfbi, two genes regulated by miR-21, are expressed at significantly higher levels in EV-GFPpos compared with EV-GFPneg microglia (p-adj < 0.05).
“Interestingly, two genes (FasL and Tgfbi) regulated by miR-21 are expressed at a significantly higher level in EV-GFPpos compared with EV-GFPneg microglia (p-adj < 0.05) (Figure 4C).”
Quote in the paperNot a substantial claim - Findingown data
ImageStream analysis confirmed the presence of subcellular GFP-positive particles in microglial cells and MOs/macrophages.
“This analysis, as expected, did confirm the presence of subcellular GFP-positive particles in the microglial cells and MOs and/or macrophages cells (Figures 2E and 3D).”
Quote in the paperSubstantial claim - Findingown data
Downregulation of Btg2 through transfection with miR-21 mimic or siRNA specific to Btg2 increased proliferation in microglia in culture.
“By downregulation of Btg2 through transfection with miR-21 mimic or siRNA specific to this message, we showed an increased level of proliferation in microglia in culture.”
Quote in the paperSubstantial claim - Findingown data
miR-21 levels are significantly higher in microglia that have taken up tumor EVs (EV-GFPpos) compared with GFP-negative microglia.
“The expression level of miR-21 was detected at significantly higher levels in microglia, which had taken up tumor EVs compared with GFP-negative cells (Figure 2D).”
Quote in the paperSubstantial claim - Findingown data
Genes Nfat5, Bmpr2, Btg2, Rhob, and Kbtbd2 have significantly reduced expression in EV-GFPpos compared with EV-GFPneg microglia (p-adj < 0.05).
“The genes with significantly reduced expression in the EV-GFPpos compared with EV-GFPneg microglia (adjusted p [p-adj] < 0.05) include Nfat5, Bmpr2, Btg2, Rhob, and Kbtbd2 (Figure 4C, significantly changed genes in bold).”
Quote in the paperSubstantial claim - Findingown data
The gene most strikingly altered by EV-mediated miR-21 transfer was Btg2.
“The gene that was most strikingly altered by EV-mediated miR-21 transfer was Btg2 (synonyms, Pc3 and Tis21).”
Quote in the paperSubstantial claim - Findingown data
In miR-21-expressing mice with tumors, similar patterns of downregulation of miR-21 targets were found compared with miR-21-null mice.
“We found similar patterns of downregulation of miR-21 targets in the miR-21 wild-type mice compared with miR-21-null mice (Table S1; Figure S5B).”
Quote in the paperNot a substantial claim - Findingown data
In wild-type microglia exposed to glioma-derived EVs, similar trends of downregulation of miR-21 target mRNAs were observed.
“Here we found similar trends of downregulation of the mRNA targets (Figure S5A).”
Quote in the paperSubstantial claim - Findingown data
EV-GFP signal was no longer detectable 40 h after injection, indicating degradation of EV-GFP.
“This EV uptake occurs soon after injection, as the EV-GFP signal was no longer detectable 40 h after injection of EVs (data not shown), indicating the degradation of EV-GFP.”
Quote in the paperNot a substantial claim - Findingown data
miR-21 levels are increased in EV-GFPpos MOs/macrophages compared with EV-GFPneg cells.
“Similar to the microglial cells, an increased level of miR-21 was detected in EV-GFPpos MOs and/or macrophages in comparison with EV-GFPneg cells (Figure 3C).”
Quote in the paperSubstantial claim - Findingown data
Transfection of miR-21-KO microglia with miR-21 mimic results in a larger number of microglia expressing Ki67 proliferation marker after 24 h.
“Using a miR-21 mimic, a larger number of microglia express the Ki67 proliferation marker after 24 h of transfection (Figures 7B and 7C).”
Quote in the paperSubstantial claim - Findingown data
siRNA-mediated downregulation of Btg2 results in a larger number of microglia expressing Ki67.
“The downregulation of Btg2 after siRNA transfection also yielded a larger number of microglia expressing Ki67 (Figures 7D–7F).”
Quote in the paperSubstantial claim - Study design and conductreasoning
The use of miR-21-null mice allowed differentiation between endogenous upregulation and transfer of exogenous miR-21.
“By using miR-21-null mice, we were able to differentiate between endogenous upregulation of miRNAs following tumor implantation and the transfer of exogenous miR-21 from the tumor to stromal cells.”
Quote in the paperSubstantial claim - Interpretationown data
EV-mediated transfer of miRNA from tumor cells to microglia results in downregulation of specific target genes.
“EV-mediated transfer of miRNA from tumor cells to microglia results in downregulation of specific target genes.”
Quote in the paperSubstantial claim - Interpretationown data
Glioma cells can reprogram microglia in part by transferring miR-21 through EVs.
“In conclusion, the results of this study demonstrate that glioma cells can reprogram microglia in part by transferring miR-21 through EVs.”
Quote in the paperSubstantial claim - Interpretationown data
Functional delivery of miR-21 from glioma cells to microglia via tumor-derived EVs downregulates specific downstream miR-21 target mRNAs in microglia.
“Here, we demonstrate functional delivery of miR-21, regulating specific downstream mRNA targets in microglia after uptake of tumor-derived EVs.”
- Also stated in: § INTRODUCTION · boosted
“Here we demonstrate functional delivery of miR-21 from glioma cells to the surrounding innate immune cells subsequently leading to downregulation of specific miR-21 mRNA targets.”
- Also stated in: § DISCUSSION · plain
“EV-mediated miRNA transfer downregulates specific mRNAs in vivo.”
Quote in the paperSubstantial claim - Also stated in: § INTRODUCTION · boosted
- Interpretationown data
miR-21, either introduced by transfection or delivered through EVs, can downregulate specific mRNAs in miR-21-null and wild-type microglia that were downregulated in vitro and in vivo.
“Taken together, these data demonstrate that miR-21, either introduced by transfection or delivered through EVs, can downregulate specific mRNAs in miR-21-null and wild-type microglia that were downregulated in vitro and in vivo.”
Quote in the paperSubstantial claim - Interpretationown data
Transfer of miR-21 by EVs in vivo results in downregulation of some miR-21 target mRNAs in microglia.
“Overall, these results demonstrate that the transfer of miR-21 by EVs in vivo results in downregulation of some of the miR-21 target mRNAs.”
Quote in the paperSubstantial claim - Study design and conductown data
By using miR-21-null mice, the study was able to dissect the effects of transfer of this single miRNA.
“Although other EV components, such as different small and large RNAs as well as proteins and lipids, are also anticipated to affect microglia, we have, by making use of the miR-21-null mouse, been able to dissect the effects of the transfer of this single miRNA.”
Quote in the paperSubstantial claim - Study design and conductcitation
The palmitoylated GFP reporter is not specific to one type of vesicle but follows uptake of all naturally formed membrane-bound vesicles.
“This reporter is not specific to one type of vesicles (e.g., exosomes, ectosomes, or microvesicles) (Cocucci and Meldolesi, 2015) but follows the uptake of all naturally formed membrane-bound vesicles.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Cocucci and Meldolesi, 2015 source
- Interpretationown data
miRNAs can be transferred by EVs to cells in the brain in vivo with functional consequences.
“It is proof that miRNAs can be transferred by EVs to cells in the brain in vivo with functional consequences.”
Quote in the paperSubstantial claim - Interpretationown data
The downregulation of target mRNAs supports the notion that miR-21 is released into the cytosol after uptake of miR-21+ tumor EVs.
“The downregulation of target mRNAs as we detected supports the notion that miR-21 is release into the cytosol after uptake of miR-21+ tumor EVs.”
Quote in the paperSubstantial claim - Interpretationown data
This study demonstrates in vivo functional transfer of miRNA by spontaneously released EVs.
“Although EV-mediated miRNA transfer was previously either shown in vitro or after injection of arbitrary amounts of highly subselected EVs in vivo, this study demonstrates in vivo functional transfer of miRNA by spontaneously released EVs.”
Quote in the paperSubstantial claim- Rests on an assumption: EV-mediated miRNA transfer had not previously been demonstrated in vivo using spontaneously released EVs.
- Interpretationown data
EVs secreted by tumor cells function as miRNA carriers that deliver molecules changing the transcriptome and subsequent proliferative capacities of microglia.
“Reviewing our data, we propose a model in which EVs secreted by tumor cells function as miRNA carriers, which deliver molecules that change the transcriptome and subsequent proliferative capacities of microglia.”
Quote in the paperSubstantial claim
- May combine two claims. 16
- Classified as probably not a substantial claim. 19
- The quote only partly states this claim. 1
May combine two claims.
FindingcitationmiR-21 co-localizes with GFP in EVs, but is also present in high-density fractions possibly associated with high-density lipoproteins.
“Overall, miR-21 was found to co-localize with GFP, but miR-21 was also present in the high-density fractions, possibly associated with high-density lipoproteins (Figures S2B and S2C) (Vickers et al., 2011).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites Vickers et al., 2011 source
May combine two claims.
FindingreasoningmiR-21 transfer is most efficient in microglia compared with MOs/macrophages, although percentage of EV-GFP uptake is highest in MOs/macrophages.
“Analysis of the EV-GFP uptake and the subsequent miR-21 levels in miR-21-null mice showed that although the percentage of cells positive for EV-GFP uptake was highest in the MO and/or macrophage population, the miR-21 transfer was most efficient in microglia.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn miR-21-null microglia exposed to GL261-derived EVs, transcripts Bmpr2, Btg2, Kbtbd2, Pdcd4, Pten, and Rhob were significantly downregulated, while Gapdh was unaffected.
“We also observed significant downregulation of the in vivo EV downregulated miR-21 targets Bmpr2, Btg2, Kbtbd2, Pdcd4, Pten, and Rhob transcripts, while Gapdh expression appeared unaffected (Figures 5E and 5F).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn EV-GFPpos versus EV-GFPneg microglia, 441 genes were significantly upregulated and 359 genes were downregulated.
“Four hundred forty-one genes were significantly upregulated and 359 genes were downregulated in EV-GFPneg versus EV-GFPpos (Figure 4B, significantly changed genes in red).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe difference in miR-21 levels between EV-GFPneg and EV-GFPpos is smaller in MOs/macrophages, and relative levels in EV-GFPpos MOs/macrophages are lower than in EV-GFPpos microglia.
“Surprisingly the difference between EV-GFPneg and GFPpos in MOs and/or macrophages was smaller, and the relative levels in EV-GFPpos MOs and/or macrophages were lower compared with EV-GFPpos microglia (Figures 2D and 3C).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe heterogeneous population of EVs shed by GL261 tumor cells is labeled with membrane-bound GFP and contains high levels of miR-21.
“Taken together, the heterogeneous population of EVs shed by tumor cells is labeled with membrane-bound GFP and contains high levels of miR-21.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataGapdh, a control gene not targeted by miR-21, was not affected, indicating that mRNA target gene downregulation is miR-21 specific.
“A control gene not targeted by miR-21, Gapdh, was not affected, an indication that mRNA target gene downregulation is miR-21 specific (Figure 5B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataBtg2, Nfat5, and Pdcd4 expression levels were significantly downregulated after EV uptake in vivo and by miR-21 mimic in vitro.
“From the selected genes, expression levels for Btg2 and Nfat5 as well as Pdcd4 were significantly downregulated after EV uptake in vivo and were shown to be significantly downregulated by the miR-21 mimic (Figure 5A).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataNTA revealed a broad size distribution of EVs ranging from 100 to 500 nm, confirming their heterogeneity.
“Nanoparticle tracking analysis (NTA) of the EVs isolated by 100,000 × g ultracentrifugation revealed a broad size distribution of EVs ranging from 100 to 500 nm, further confirming their heterogeneity (Figure S1A).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataIn the intracranial EV injection experiment, both Pdcd4 and Btg2 transcript levels were reduced in microglia that took up EV-GFP, but reduction reached significance only for Btg2.
“In this experiment, both transcript levels were reduced in microglia that took up EV-GFP, but this reduction reached significance only for the Btg2 replicates (Figure 6D).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataA similar trend of higher miR-10b expression is seen in EV-GFPpos microglia compared with EV-GFPneg microglia.
“A similar trend between EV-GFPneg and EV-GFPpos microglia was seen in glioma-specific miR-10b, in which the expression level was higher than in EV-GFPneg microglia (Figure S1D).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown datamiR-21 is detected in EV-GFPpos microglia but not in EV-GFPneg microglia after intracranial EV injection, demonstrating effective transfer of miR-21 via EVs derived from in vitro cultured tumor cells to microglia in vivo.
“Interestingly, miR-21 was detected in the EV-GFPpos microglia, compared with being at non-detectable levels in EV-GFPneg microglia, demonstrating that miR-21 was effectively transferred via EVs derived from in vitro cultured tumor cells and delivered to microglia in vivo (Figure 6C).”
Quote in the paperSubstantial claimThe quote only partly states this claim.
Findingown dataA smaller percentage of CD11blowCD45high lymphocytes have taken up EV-GFP compared with other immune cells.
“In this population of non-phagocytic cells, we found a smaller percentage of cells that have taken up EV-GFP (Figures S3A and S3B).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenMale and female mice were randomly assigned to experimental groups.
“Male and female mice ranging from 12 – 14 weeks in age were randomly assigned to experimental groups.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Study design and conductnone givenStatistical significance was defined as p < 0.05.
“Significance was calculated using unpaired t test, multiple t tests and One-way ANOVA with Tukey’s multiple comparisons test, with statistical significance defined as p < 0.05.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAnimal experiments were conducted under oversight of the Massachusetts General Hospital Institution Animal Care and Use Committee.
“All animal experiments were conducted under the oversight of the Massachusetts General Hospital Institution Animal Care and Use Committee.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenLibraries were diluted and denatured with the addition of 1% PhiX Sequencing Control V3.
“Finally, libraries were diluted and denatured with the addition of 1% PhiX Sequencing Control V3 (Illumina).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataEVs were characterized by size distribution analysis using nanoparticle-tracking analysis (NTA 3.2; Malvern).
“Final EV pellet was resuspended in DPBS and characterization of EVs was performed by size distribution analysis using nanoparticle-tracking analysis (NTA 3.2; Malvern), with screen gain set at 3.0 and camera level at 13.0.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataThe combination of CD11b and CD45 can be used to isolate microglia and MOs/macrophages, and GFP-positive EVs released by tumor cells in vivo can transfer miR-21 to microglia.
“Taken together, this demonstrates that the combination of CD11b and CD45 can be used to isolate microglia and MOs and/or macrophages cells and that GFP-positive EVs released by tumor cells in vivo can transfer miR-21 to microglia.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll qPCR reactions were done in triplicate.
“All qPCR reactions were done in triplicate and normalized to β-Actin mRNA levels.”
Quote in the paperNot a substantial claimMay combine two claims.
Study design and conductown dataGFP protein was detected in both cellular and extracellular fractions, confirming that this marker can be used to track the fate of all different subtypes of EVs.
“Importantly, GFP protein was detected in both cellular and extracellular fractions, confirming that this marker can be used to track the fate of all different subtypes of EVs (Figure 1C).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenmiRNA expression was normalized to U6 or UniSP6 spike-in RNA.
“miRNA expression was normalized to U6 or UniSP6 spike-in RNA as listed in figure legends.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataqPCR data were normalized to β-Actin mRNA levels.
“All qPCR reactions were done in triplicate and normalized to β-Actin mRNA levels.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataIn the absence of a tumor, microglia can take up tumor cell-derived EVs injected into the striatum, leading to miR-21-dependent downregulation of Btg2.
“However, these results suggest that in the absence of a tumor, microglia can take up tumor cell-derived EVs injected into the striatum, leading to miR-21-dependent downregulation of Btg2.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataRNA libraries included ERCC RNA Spike-In Mix.
“RNA libraries were prepared with poly(A) selection using 3′-SMART CDS Primer II A within the SMARTer Ultra Low Input RNA Kit for Sequencing–v3 (Clontech Takara) following manufacturer’s protocol including ERCC RNA Spike-In Mix (Life Technologies).”
Quote in the paperNot a substantial claimMay combine two claims.
PremisereasoningIn miR-21-null mice, endogenous miR-21 is not expressed in stromal cells, so presence of miR-21 in stromal cells after tumor implantation is derived exclusively from implanted tumor cells.
“In this model, endogenous miR-21 is not expressed in stromal cells, so the presence of miR-21 should be derived exclusively from the implanted tumor cells.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Ma et al., 2011
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Classified as probably not a substantial claim.
Study design and conductown dataEqual amounts of protein were loaded for western blot analysis.
“Protein concentration was determined using Pierce BCA protein assay (Thermo Fisher) and equal amounts were loaded and resolved on 10% SDS-PAGE gel (Thermo Fisher).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenDifferential expression analysis was subjected to statistical significance using Benjamini and Hochberg multiple testing adjusted p values.
“Differential expression analysis as performed in DESeq2 was subjected to statistical significance using Benjamini and Hochberg multiple testing adjusted p values.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataMicroglial proliferation is increased upon delivery of miR-21 and after downregulation of Btg2.
“Overall, these results show that microglial proliferation is increased upon the delivery of miR-21 and after the downregulation of Btg2.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenA cut-off value of significance of multiple testing adjusted p value < 0.05 was used to include differentially expressed genes.
“A cut-off value of significance of multiple testing adjusted p value < 0.05 was used to include differentially expressed genes.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenNon-specific binding of immunoglobulin to Fc receptors was blocked.
“Non-specific binding of the immunoglobulin to the Fc receptors was blocked by incubating cells 10 min on ice in 0.5% BSA in DPBS (without Ca2+ or Mg2+) with 2 mM EDTA supplemented with TruStain fcX (anti-mouse Cd16/32, BioLegend, #101319, clone 93, 1:100).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataLive cells were sorted for analysis.
“DAPI was added to cells at final concentration of 1 μg/ml. Live cells were sorted using a BD FACSAria II SORP Cell Sorter.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenEqual molar individual libraries were pooled.
“Equal molar individual libraries were pooled, and the pool concentration was determined using the KAPA SYBR® FAST Universal qPCR Kit.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenRNA concentration and integrity (RIN score) were determined using the Agilent 2100 Bioanalyzer Pico-chips.
“The RNA concentration and integrity (RIN score) were determined using the Agilent 2100 Bioanalyzer Pico-chips (Agilent Technologies) following manufacturing protocol.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataGL261 cells were tested for mycoplasma contamination and found negative.
“Cells were tested for mycoplasma contamination (Mycoplasma PCR Detection Kit, abm G238) and found negative.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Study design and conductreasoningThe use of EVs isolated from GL261 grown in vitro allows study of uptake and effect without confounding action of other factors released by tumor cells.
“By using EVs isolated from GL261, grown in vitro, we can study the uptake and effect of these EVs without confounding action of other factors released by the tumor cells.”
Quote in the paperSubstantial claim
The paper relies on this without support.
EV-mediated miRNA transfer had not previously been demonstrated in vivo using spontaneously released EVs.
“Although EV-mediated miRNA transfer was previously either shown in vitro or after injection of arbitrary amounts of highly subselected EVs in vivo, this study demonstrates in vivo functional transfer of miRNA by spontaneously released EVs.”
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Pdcd4 is both included and excluded among the initially downregulated in vivo targets
MinorThe paper first says Pdcd4 was significantly downregulated after EV uptake in vivo. It later says that, of Pdcd4 and Btg2, only Btg2 was among the initially downregulated miR-21 targets in vivo.
“From the selected genes, expression levels for Btg2 and Nfat5 as well as Pdcd4 were significantly downregulated after EV uptake in vivo and were shown to be significantly downregulated by the miR-21 mimic (Figure 5A).”
“Interestingly, of the two targets, only Btg2 was among the initially downregulated miR-21 targets in vivo.”
Cited works that were retracted
Works this paper cites that were later retracted, from the retraction notices publishers deposit. Major when the notice came out before this paper was published.
Cites a retracted work: 10.1074/jbc.m116.772392
InfoThe cited work was retracted on 2020-05-01 (notice 10.1074/jbc.w120.013725), not before this paper was published (2019-09).
Cited work 10.1074/jbc.m116.772392 · retracted 2020-05-01 · notice 10.1074/jbc.w120.013725 · this paper published 2019-09
“Hu, 2017, miR-21-mediated radioresistance occurs via promoting repair of DNA double strand breaks, J. Biol. Chem., 292, 3531, 10.1074/jbc.M116.772392”
Cites
1 retracted
miR-21-mediated Radioresistance Occurs via Promoting Repair of DNA Double Strand Breaks
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RetractedModerated estimation of fold change and dispersion for RNA-seq data with DESeq2
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MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins
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Many roads to maturity: microRNA biogenesis pathways and their regulation
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MicroRNA-21 Is an Antiapoptotic Factor in Human Glioblastoma Cells
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Self-citationMicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts
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New tools for studying microglia in the mouse and human CNS
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The role of microglia and macrophages in glioma maintenance and progression
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Ectosomes and exosomes: shedding the confusion between extracellular vesicles
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“In addition, studies evaluating GB-derived EVs in CSF indicated that elevated miR-21 levels are associated with worse prognosis (Akers et al., 2013; Shi et al., 2015).”
BackgroundExtracellular Vesicles: Unique Intercellular Delivery Vehicles
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Self-citationMicroRNA 21 Promotes Glioma Invasion by Targeting Matrix Metalloproteinase Regulators
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“Interference with miR-21 reduces the malignant potential, as downregulation of miR-21 has been shown to inhibit cell proliferation and invasion in vitro and tumor progression in vivo (Belter et al., 2016; Corsten et al., 2007; Gabriely et al., 2008; Põlajeva et al., 2012).”
BackgroundSelf-citationSelective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis
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Self-citationMicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene
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Modulation of K-Ras-Dependent Lung Tumorigenesis by MicroRNA-21
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Regulation and function of miRNA-21 in health and disease
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Self-citationMultidimensional communication in the microenvirons of glioblastoma
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Self-citationMicroRNA-21 Is Up-Regulated in Allergic Airway Inflammation and Regulates IL-12p35 Expression
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Coding and noncoding landscape of extracellular RNA released by human glioma stem cells
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Self-citationmiR-21 Gene Expression Triggered by AP-1 Is Sustained through a Double-Negative Feedback Mechanism
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Self-citationMicroRNA-21 targets Sprouty2 and promotes cellular outgrowths
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Loss of the miR-21 allele elevates the expression of its target genes and reduces tumorigenesis
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Therapeutic miR-21 Silencing Ameliorates Diabetic Kidney Disease in Mice
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Micrornas Control Hepatocyte Proliferation During Liver Regeneration
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Functional Roles for Fatty Acylated Amino-terminal Domains in Subcellular Localization
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Glioblastoma‐derived extracellular vesicles modify the phenotype of monocytic cells
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MicroRNA miR-21 Regulates the Metastatic Behavior of B16 Melanoma Cells
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A novel reciprocal loop between microRNA-21 and TGFβRIII is involved in cardiac fibrosis
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“Increased levels of proliferation in microglia have been reported in neuro-inflammation and traumatic brain injury, indicating that disruption of CNS homeostasis as is also occurring during tumor growth can result in proliferation of microglia (Febinger et al., 2015; Pepe et al., 2017).”
BackgroundInductive microRNA‐21 impairs anti‐mycobacterial responses by targeting IL‐12 and Bcl‐2
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miRNA-21 ablation protects against liver injury and necroptosis in cholestasis
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MicroRNA-21 is an important downstream component of BMP signalling in epidermal keratinocytes
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Genome Editing Reveals Glioblastoma Addiction to MicroRNA-10b
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Self-citationMicroRNA-21 regulates T-cell apoptosis by directly targeting the tumor suppressor gene Tipe2
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The Bcl6 target gene microRNA-21 promotes Th2 differentiation by a T cell intrinsic pathway
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Regulation of TIMP3 in diabetic nephropathy: a role for microRNAs
Fiorentino, L., Cavalera, M., Mavilio, M., Conserva, F., Menghini, R., Gesualdo, L., & Federici, M. (2013) · Acta Diabetologica · doi:10.1007/s00592-013-0492-8 · Cited by 88 · see citing works
McDonald, R. A., White, K. M., Wu, J., Cooley, B. C., Robertson, K. E., Halliday, C. A., McClure, J., Francis, S., Lu, R., Kennedy, S., et al. (2013) · European Heart Journal · doi:10.1093/eurheartj/eht105 · Cited by 70 · see citing works
Rapid atrial pacing induces myocardial fibrosis by down-regulating Smad7 via microRNA-21 in rabbit
He, X., Zhang, K., Gao, X., Li, L., Tan, H. K., Chen, J., & Zhou, Y. (2016) · Heart and Vessels · doi:10.1007/s00380-016-0808-z · Cited by 70 · see citing works
Vecchioli, S. F., Tanori, M., Micheli, L., Mancuso, M., Leonardi, L., Saran, A., Ciotti, M. T., Ferretti, E., Gulino, A., Pazzaglia, S., et al. (2007) · The FASEB Journal · doi:10.1096/fj.06-7548com · Cited by 64 · see citing works
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Wu, T., Liu, Y. F., Fan, Z., Xu, J., Jin, L., Gao, Z., Wu, Z., Hu, L., Wang, J., Zhang, C., et al. (2015) · Stem Cells · doi:10.1002/stem.2081 · Cited by 57 · see citing works
Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue
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miRNA-21 is developmentally regulated in mouse brain and is co-expressed with SOX2 in glioma
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MicroRNA-21 Contributes to Liver Regeneration by Targeting PTEN
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Self-citationREST–miR-21–SOX2 axis maintains pluripotency in E14Tg2a.4 embryonic stem cells
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Flagged first, then by citations
Cited by
Sorted by citations
RNA delivery by extracellular vesicles in mammalian cells and its applications
O’Brien, K., Breyne, K., Ughetto, S., Laurent, L. C., & Breakefield, X. O. (2020) · Nature Reviews Molecular Cell Biology · doi:10.1038/s41580-020-0251-y · Cited by 1,811 · see citing works
Self-citationGhosh, S., & Ghosh, S. (2022) · Frontiers in Pharmacology · doi:10.3389/fphar.2022.878058 · Cited by 13 · see citing works
Shanthi, K., Fischer, D., Sharma, A., Kiviniemi, A., Kaakinen, M., Vainio, S., & Bart, G. (2023) · Genes · doi:10.3390/genes14040853 · Cited by 5 · see citing works
- Authors
- Abels, E. R., Maas, S. L. N., Nieland, L., Wei, Z., Cheah, P. S., Tai, E., Kolsteeg, C.-J., Dusoswa, S. A., Ting, D. T., Hickman, S., El Khoury, J., Krichevsky, A. M., Broekman, M. L. D., & Breakefield, X. O.
- Affiliations
- Harvard University (US)
- Utrecht University (NL)
- Brigham and Women's Hospital (US)
- Universiti Putra Malaysia (MY)
- Amsterdam University Medical Centers (NL)
- Leiden University Medical Center (NL)
- Corresponding author
- Abels, Erik R.
- Breakefield, Xandra O.
- ORCID
- Venue
- Cell Reports
- Publisher
- Elsevier BV
- Published
- 2019-09
- Volume
- 28
- Issue
- 12
- Pages
- 3105-3119.e7
- ISSN
- 2211-1247 (ISSN-L)
- PMC
- PMC6817978
- OpenAlex
- W2974646711
- Semantic Scholar
- 023efccb5b74d12f75092f0aea6024ea7a7e5386
- Cited by
- 199
- References
- 108
- Citations by year
- 2026: 14 · 2025: 13 · 2024: 28 · 2023: 26 · 2022: 36 · 2021: 44 · 2020: 35 · 2019: 3
- FWCI
- 8.59
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Cell Reports (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Pure Amsterdam UMC (publishedVersion, other-oa)
- Amsterdam UMC (VU Amsterdam) - Institutional Repository (publishedVersion, other-oa)
- Leiden Repository (Leiden University) (submittedVersion, cc-by)
- PubMed Central (submittedVersion, cc-by-nc-nd)
- Universiti Putra Malaysia Institutional Repository (Universiti Putra Malaysia) (submittedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion)
- Leiden Repository (Leiden University) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
- Utrecht University Repository (Utrecht University) (submittedVersion, cc-by)
- Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
- Funders
- National Institutes of Health
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
- Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
- Keywords
- glioblastoma, microglia, miR-21, extracellular vesicles, microglial reprogramming, tumor microenvironment, microRNA delivery, glioma
- MeSH terms
- Animals, Cell Communication, Glioblastoma, Mice, Transgenic, RNA, Neoplasm, Microglia, MicroRNAs, Cell Line, Tumor, Mice, Tumor Microenvironment, Cellular Reprogramming
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-21.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-21.
Cited sources are checked by gpt-6-sol.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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