The miRNA-21-5p Payload in Exosomes from M2 Macrophages Drives Tumor Cell Aggression via PTEN/Akt Signaling in Renal Cell Carcinoma

Abstract

M2 macrophages in the tumor microenvironment are important drivers of cancer metastasis. Exosomes play a critical role in the crosstalk between different cells by delivering microRNAs or other cargos. Whether exosomes derived from pro-tumorigenic M2 macrophages (M2-Exos) could modulate the metastatic behavior of renal cell carcinoma (RCC) is unclear. This study found that M2-Exos promotes migration and invasion in RCC cells. Inhibiting miR-21-5p in M2-Exos significantly reversed their pro-metastatic effects on RCC cells in vitro and in the avian embryo chorioallantoic membrane in vivo tumor model. We further found that the pro-metastatic mechanism of miR-21-5p in M2-Exos is by targeting PTEN-3'UTR to regulate PTEN/Akt signaling. Taken together, our results demonstrate that M2-Exos carries miR-21-5p promote metastatic features of RCC cells through PTEN/Akt signaling. Reversing this could serve as a novel approach to control RCC metastasis.

Keywords: M2 macrophages; PTEN/Akt; exosomes; invasion; miRNA-21-5p; migration; renal cell carcinoma.

Figure 1

M2 macrophages promote RCC migration and invasion. (A) H&E and CD163 IHC staining (red) of four representative clinical RCC samples. Scale bar: 50 μm. (B) Macrophage marker mRNA expression was assayed in THP-1-Mφ and THP-1-M2 cells by RT-qPCR. (C) IF staining of nuclei (DAPI), CD206, and F-actin in THP-1-M2 cells. Scale bar: 50 μm. (D–G) Cellular migration and invasion of 786-O and ACHN cells treated with M2-CM or control media were assessed by transwell and wound-healing assays. Scale bar: 100 μm. ** p < 0.01, *** p < 0.001, ns: not significant. pRCC: papillary renal cell carcinoma; CT: control; RT-qPCR: reverse transcription-quantitative PCR.

Figure 2

Characterization and internalization of M2-Exos. (A) TEM image of M2-Exos. Scale bar: 200 nm. (B) DLS measurement of M2-Exos size. (C) Western blotting assay of exosomal markers in THP-1-M2 cellular lysate and M2-Exos preparation. (D,F) Fluorescence images of 786-O and ACHN cells treated with or without PKH67-labeled M2-Exos (green). Scale bar: 50 μm. (E,G) Three-dimensional confocal reconstruction of 786-O and ACHN cells treated with PKH67-labeled M2-Exos (green). (H) Fluorescence staining analyzing the internalization of M2-Exos by 786-O cells over 12 h. Scale bar: 10 μm. CT: control.

Figure 3

M2-Exos promote RCC migration and invasion. 786-O and ACHN cells treated with or without M2-Exos. (A–D) The impact of the M2-Exos treatment on the migration and invasion capabilities of RCC cells was analyzed by transwell (A,C) and wound-healing assays (B,D). Scale bar: 100 μm. (E,F) The impact of M2-Exos treatment on RCC cell: mRNA (E,G) and protein expression (F,H) of MMP-9 and vimentin were assayed by RT-qPCR and western blotting, respectively. (I,J) The impact of M2-Exos treatment on cell proliferation was analyzed by MTT assay. ** p < 0.01, *** p < 0.001. CT: control.

Figure 4

MiR-21-5p in M2-Exos promotes RCC migration and invasion in vitro. (A) The expression of three miRNAs in THP-1-M2 cells was analyzed by RT-qPCR. (B) The level of miR-21-5p expression in M2-Exos, M2-miR-21-Inh-Exos, and M2-miR-21-NC-Exos was assessed by RT-qPCR. (C,D) The intracellular content of miR-21-5p in 786-O and ACHN cells treated with control media, miR-21-Inh-Exos, or miR-21-NC-Exos was assessed by RT-qPCR. (E,F) The level of MMP-9 and vimentin in treated cells was assessed by western blotting. (G–J) The migration and invasion of treated cells were assessed by transwell (G,I) and wound-healing assays (H,J). Scale bar: 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.

Figure 5

MiR-21-5p in M2-Exos directs RCC aggressive behaviors in vivo. (A) Schematic diagram of the timeline of CAM xenograft implantation. Three groups of 786-O tumor cells were implanted on CAMs following treatment with exosomes-free media (the control group), miR-21-Inh-Exos, or miR-21-NC-Exos. (B) H&E stains of three representative CAM-derived tumors from each group are shown. Magnification: 40×, 200×, for each tumor sample. (C) The tumor weights of harvested CAM tumors from each group. (D) qPCR for human ALB in embryonic livers normalized to avian ACTB. ** p < 0.01, **** p < 0.0001, ns: not significant.

Figure 6

MiR-21-5p in M2-Exos regulates PTEN/AKT signaling to promote RCC metastasis. (A) The sequences of the putative target site for miR-21-5p in the PTEN-3′UTR (PTEN-3′UTR-WT) and a mutated version of this target (PTEN-3′UTR-Mut) were shown. (B,C) 786-O and ACHN cells were co-transfected psiCHECK2-PTEN-3′UTR-WT or psiCHECK2-PTEN-3′UTR-Mut, with miR-21-Inh or miR-21-NC. The relative luciferase activities in the cells were assayed. (D–G) The expression of PTEN mRNA, and protein level of PTEN, total Akt, and p-AktS473 were assessed in 786-O and ACHN cells treated with miR-21-Inh-Exos or miR-21-NC-Exos. (H–K) The migration and invasive capabilities of cells treated with miR-21-Inh-Exos or miR-21-Inh-Exos + PTEN inhibitor (PTEN-Inh) were analyzed by transwell and wound-healing assays. Scale bar, 100 μm. ** p < 0.01, *** p < 0.001, ns: not significant.

Figure 7

Schematic diagram of the proposed mechanism of M2-Exos mediated enhancement of RCC aggression via miR-21-5p.