CD133-containing microvesicles promote colorectal cancer progression by inducing tumor angiogenesis

Abstract

Angiogenesis is an indispensable mechanism in cancer progression, as cancer cells need to establish blood vessels to supply oxygen and nutrients. Extracellular vesicles (EVs) derived from cancer cells act as messengers in the tumor microenvironment and induce resistance to anti-angiogenic cancer treatment. EVs can be classified into two categories: exosomes and microvesicles (MVs). Although exosomes are involved in angiogenesis, the role of MVs in angiogenesis and cancer progression remains unclear. CD133 plays a key role in MV formation and oncoprotein trafficking. In this study, we investigated the role of CD133-containing MVs derived from colorectal cancer (CRC) in angiogenesis and cancer progression. CRC-derived MVs were incorporated into endothelial cells and increased the mesh area and tube length of endothelial cells. CD133-containing MVs also stimulate vessel sprouting in endothelial cell spheroids and mouse thoracic aortas. However, MVs derived from CD133-knockdown CRC cells exerted a limited effect on tube formation and vessel sprouting. CD133-containing MVs induced angiogenesis through p38 activation and angiogenesis induced by CD133-containing MVs was insensitive to the anti-vascular endothelial growth factor antibody bevacizumab. Survival analysis revealed that high expression level of CD133 correlated with poor prognosis in patients with metastatic CRC. These findings suggest that CD133-containing MVs act as key regulators of angiogenesis and are related to the prognosis of CRC patients.

Keywords: Angiogenesis; CD133; Colorectal cancer; Microvesicle; p38.

Fig. 1

CD133-containing MVs are incorporated into endothelial cells. (A) Stable shMock and shCD133 HCT116 cells were generated. MVs were isolated from shMock and shCD133 HCT116 cells. Protein level was determined using western blotting. Full-length blot images are shown in Fig. S1 (B) shMock and shCD133 MVs were stained with WGA-488. EA.hy926 cells were incubated with stained MVs for 16 h. Images were captured by confocal laser scanning microscopy (green, WGA-488; blue, DAPI). Scale bar = 10 μm. The number of MVs were counted by imageJ. All p values were obtained using unpaired two-tailed Student's t-test. *p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

CD133-containing MVs promote tube formation and vessel sprouting in endothelial cells. (A) EA.hy926 cells were treated with MVs isolated from shMock and shCD133 HCT116 cells for 16 h. Scale bar = 100 μm. (B) Meshes and tubes were measured from five random fields in each well. (C) EA.hy926 cell spheroids embedded in 3D collagen gels were treated with shMock or shCD133 MVs for 48 h. Representative images of endothelial spheroids were shown. Scale bar = 100 μm. (D) Five random spheroids were measured in each well. (E) Mouse aortic rings were embedded in 3D collagen gels and stimulated with 2% FBS media supplemented with VEGF (30 ng/mL). After 48 h, rings were washed twice, and media were changed with 2% FBS containing shMock or shCD133 MVs and incubated for 3 days. Representative images of mouse aortic rings were shown. Scale bar = 200 μm. (F) All the sprouts of each ring were measured. Mean ± SEM of n = 3 independent experiments. All p values were obtained using unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01 ***p < 0.001.

Fig. 3

CD133-containing MVs induce angiogenesis through the p38 MAPK signaling pathway. (A) EA.hy926 cells were treated with shMock and shCD133 MVs for 16 h. Protein level was determined using western blotting. p-p38, phosphorylated p38; pAKT, phosphorylated AKT; pERK, phosphorylated ERK. Full-length blot images are shown in Fig. S2 (B) Quantification of the p-p38 level. (C) EA.hy926 cells were pre-treated with SB203580 (20 μM) for 2 h and treated with shMock MVs in the presence of SB203580 for 13 h. Representative images of tube formation were shown. Scale bar = 100 μm. (D) Meshes and tubes were measured from five random fields in each well. Mean ± SEM of n = 3 independent experiments. All p values were obtained using an unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4

CD133-containing MV-induced angiogenesis is not mediated by the canonical angiogenesis pathways. (A) Angiogenesis antibody array was performed using EA.hy926 cells treated with shMock MVs or shCD133 MVs. Red represents high protein expression levels, and blue represents low protein expression levels. (B) Protein expression was determined using western blot analysis. Human recombinant VEGF165 protein was used as a control. Full-length blot images are shown in Fig. S3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Angiogenesis induced by CD133-containing MVs is resistant to bevacizumab and CD133 overexpression predicts poor prognosis in metastatic CRC patients. (A) EA.hy926 cells were treated with VEGF (200 ng/mL) or shMock MVs in the presence or absence of bevacizumab (100 μg/mL) for 16 h. Representative images of tube formation. Scale bar = 100 μm. (B) Meshes and tubes were measured from five random fields in each well. Mean ± SEM of n = 3 independent experiments. All p values were obtained using an unpaired two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001. (C) The correlation between CD133 mRNA expression level and patient survival rate was analyzed. Kaplan-Meier plots were obtained from the Human Protein Atlas database. The metastatic CRC survival rate represents patients in stages Ⅲ and Ⅳ. The overall survival rate represents CRC patients in all stages.

Fig. 6

Schematic diagram regarding the role of CD133-containing MVs derived from CRC cells in angiogenesis and CRC progression.