CD90 determined two subpopulations of glioma-associated mesenchymal stem cells with different roles in tumour progression

Abstract

Human glioma-associated mesenchymal stem cells (gbMSCs) are the stromal cell components that contribute to the tumourigenesis of malignant gliomas. Recent studies have shown that gbMSCs consist of two distinct subpopulations (CD90⁺ and CD90^- gbMSCs). However, the different roles in glioma progression have not been expounded. In this study, we found that the different roles of gbMSCs in glioma progression were associated with CD90 expression. CD90^high gbMSCs significantly drove glioma progression mainly by increasing proliferation, migration and adhesion, where as CD90^low gbMSCs contributed to glioma progression chiefly through the transition to pericytes and stimulation of vascular formation via vascular endothelial cells. Furthermore, discrepancies in long non-coding RNAs and mRNAs expression were verified in these two gbMSC subpopulations, and the potential underlying molecular mechanism was discussed. Our data confirm for the first time that CD90^high and CD90^low gbMSCs play different roles in human glioma progression. These results provide new insights into the possible future use of strategies targeting gbMSC subpopulations in glioma patients.

Fig. 1. Isolation and characterization of gbMSCs derived from human high-grade glioma tissues.

a Adherent growth pattern of gbMSCs cultured in 10% DMEM (×40, scale bars = 200 μm). b FACS analysis of typical gbMSCs (n ≥ 3) in culture. c Tri-lineage differentiation of gbMSCs: gbMSCs were treated with specific conditions for osteogenic differentiation (upper left), adipogenic differentiation (upper middle), and chondrogenic differentiation (upper right) (×200, scale bars = 50 μm). The lower panels show staining of cells grown in MSC medium as a control

Fig. 2. Characteristics of CD90^highand CD90^low gbMSCs cultured in vitro.

a, b Adherent growth patterns of CD90^high and CD90^low gbMSCs cultured in 10% DMEM (×40, scale bars = 200 µm). c FACS analysis of sorted CD90^high gbMSCs (n ≥ 3). d FACS analysis of sorted CD90^high gbMSCs (n ≥ 3). e Growth of CD90^high and CD90^low gbMSCs cultured in 10% DMEM (n ≥ 3). *P < 0.05, **P < 0.01. f Wound-healing assay of CD90^high and CD90^low gbMSCs (n ≥ 3) for 8 h with different media (×40, scale bars = 200 µm). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

Fig. 3. The migration, proliferation and adhesion capacities of U87 cells incubated with different media in vitro.

a Transwell assay of U87 cells cultured for 24 h in different media (n ≥ 3) (serum-free medium, CD90^low CM and CD90^high CM). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Serum-free medium was used as a control. b CCK8 assay of U87 cells (n ≥ 3) to evaluate proliferation in different media in vitro. U87 cells were incubated in 0%DMEM, CD90^high CM and CD90^low CM. *P < 0.05, **P < 0.01, ***P < 0.001. Serum-free medium was used as a control. c Adhesion assay to estimate the effect of 0%DMEM, CD90^high CM and CD90^low CM on U87 cell adhesion. (n ≥ 3) *P < 0.05, **P < 0.01, ***P < 0.001. Serum-free medium were used as a control

Fig. 4. Tube formation capacity of gbMSCs and HUVECs incubated in different media.

a Angiogenic capacity of CD90^high and CD90^low gbMSCs cultured in 0%DMEM, 10%DMEM, 0%gb-CM and S-gb-CM for 6 h on Matrigel (×100, scale bars = 100 µm). (n ≥ 3) *P < 0.05, **P < 0.01, ***P < 0.001. b Angiogenic capacity of HUVECs cultured in 0%DMEM, CD90^high CM and CD90^low CM for 6 h on Matrigel (×100, scale bars = 100 µm). (n ≥ 3) *P < 0.05, **P < 0.01. c Attachment capacity of DiO-labelled CD90^low and CD90^high gbMSCs onto vascular structures formed by HUVECs in 0%gb-CM (×100, scale bars = 100 µm). (n ≥ 3) *P < 0.05 and **P < 0.01

Fig. 5. Conditioned media from CD90^high and CD90^low gbMSCs have different functions in vivo.

a Representative mice from intracranial xenograft experiments in which U87 cells with CD90^high CM (left) or U87 cells with CD90^low CM (right) were injected into the right frontal lobes of nude mice. Obviously, the sizes of the CD90^high CM group tumours were greater than those of their CD90^low CM counterparts. *P < 0.05. b Both the CD90^high CM and CD90^low CM tumour sections were stained with HE (×200, scale bars = 50 μm). In the CD90^high CM and CD90^low CM tumour tissues, IHC was employed to detect CD31 and Ki-67 expression (×400, scale bars = 25 µm). (n ≥ 3) *P < 0.05, **P < 0.01. c Survival curves of glioma-bearing mice. The survival times of mice implanted with U87 cells cultured with CD90^high CM were not significantly shorter than those of mice implanted with U87 cells cultured in CD90^low CM. d Double staining for CD105 (green) and CD90 (red) revealed that CD105⁺CD90⁻ cells were located in the vessel walls, whereas CD105⁺CD90⁺ cells were located around the tumour parenchyma. (×400, scale bars = 25 µm). e Kaplan–Meier survival curves for patients with low and high CD90 expression. The survival of glioma patients with different CD90 expression levels in TCGA database was not significantly different

Fig. 6. The VEGF, IL-6, bFGF, MMP9, CCL5 and SDF-1αlevels in different treatment media by ELISA.

a The VEGF (n ≥ 3) levels were significantly higher in CD90^low CM compared to those in CD90^high CM and 0%DMEM. b The bFGF (n ≥ 3) levels were significantly higher in CD90^low CM compared to those in CD90^high CM and 0%DMEM. c The IL-6 (n ≥ 3) levels were significantly higher in CD90^low CM compared to those in CD90^high CM and 0%DMEM. * d The SDF-1α (n ≥ 3) levels were higher in CD90^high CM compared to those in CD90^low CM and 0%DMEM. e The CCL5 (n ≥ 3) levels were higher in CD90^high CM compared to those in CD90^low CM and 0%DMEM. f The MMP9 (n ≥ 3) levels were significantly higher in CD90^high CM compared to those in CD90^low CM and 0%DMEM. P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

Fig. 7. Clariom D expression profiles of CD90^high and CD90^low gbMSCs (n = 3).

a Heatmap of differentially expressed lncRNAs from a microarray assay performed on CD90^high and CD90^low gbMSCs. ‘Red’ indicates high relative expression, and ‘green’ indicates low relative expression. b GO terms for the predicted targeted genes. P < 0.05 using the two-sided Fisher’s exact test was defined as statistically significant