Biomolecular characterization of exosomes released from cancer stem cells: Possible implications for biomarker and treatment of cancer

Abstract

Cancer recognized as one of the leading irrepressible health issues is contributing to increasing mortality-rate day-by-day. The tumor microenvironment is an important field of cancer to understand the detection, treatment and prevention of cancer. Recently, cancer stem cell (CSC) research has shown promising results aiming towards cancer diagnostics and treatment. Here, we found that prostate and breast cancer stem cells secreted vesicles of endosomal origin, called exosomes showed strong connection between autophagy and exosomes released from CSCs. Exosomes may serve as vesicles to communicate with neoplastic cells (autocrine and paracrine manner) and normal cells (paracrine and endocrine manner) and thereby suppress immune systems and regulate neoplastic growth, and metastasis. They can also be used as biomarkers for various cancers. We detected tetraspanin proteins (CD9, CD63, CD81), Alix and tumor susceptibility gene-101 (TSG101) of exosomal markers from rotenone treated CSCs. We have also detected the induction of autophagy genes, Atg7 and conversion of autophagy marker (LC3-I to LC3-II), and tetraspanin proteins (CD9, CD63, CD81) in rotenone treated CSCs by western blotting. The mRNA expression of CD9, CD63, CD81 and TSG101 analyzed by qRT-PCR showed that the rotenone induced the expression of CD9, CD63, CD81 and TSG101 in CSCs. Electron microscopy of rotenone treated CSCs showed the mitochondrial damage of CSCs as confirmed by the release of exosomes from CSCs. The constituents of exosomes may be useful to understand the mechanism of exosomes formation, release and function, and also serve as a useful biomarker and provide novel therapeutic strategies for the treatment and prevention of cancer.

Figure 1. Detection of exosomal markers CD9 and CD63 in prostate and breast CSCs

(A) Rotenone (40 μM) treated prostate and breast CSCs released CD9 marker of exosomes (scale bar, 10 μm). (B) Quantification of CD9 exosomes release from the prostate and breast CSCs. (C) Rotenone (40 μM) treated prostate and breast CSCs released CD63 marker of exosomes (scale bar, 10 μm). (D) Quantification of CD63 exosomes release from the prostate and breast CSCs. Data represent mean ± s. e. m. from at least three independent experiments. *P<0.05 compared with rotenone-treated and control.

Figure 2. Detection of exosomal markers CD81 and Alix in prostate and breast CSCs

(A) Rotenone (40 μM) treated prostate and breast CSCs released CD81 marker of exosomes (scale bar, 10 μm). (B) Quantification of CD81 exosomes release from the prostate and breast CSCs. (C) Rotenone (40 μM) treated prostate and breast CSCs released Alix marker of exosomes (scale bar, 10 μm). (D) Quantification of Alix exosomes release from the prostate and breast CSCs. Data represent mean ± s. e. m. from at least three independent experiments. *P<0.05 compared with rotenone-treated and control.

Figure 3. Exosomal release in rotenone treated prostate and breast CSCs

(A) Rotenone (40 μM) treated prostate and breast CSCs released exosomes (scale bar, 200 nm). (B) Quantification of exosomes release from the prostate and breast CSCs. (C) Rotenone (40 μM) treated prostate and breast CSCs released CD9 marker of exosomes (scale bar, 30 nm). (D) Quantification of CD9 exosomes release from the prostate and breast CSCs. Data represent mean ± s. e. m. from at least three independent experiments. *P<0.05 compared with rotenone-treated and control.

Figure 4. Exosomal release in rotenone treated prostate and breast CSCs

(A) Rotenone (40 μM) treated prostate and breast CSCs released CD81 marker of exosomes (scale bar, 30 nm). (B) Quantification of CD81 exosomes release from the prostate and breast CSCs. (C) Rotenone (40 μM) treated prostate and breast CSCs released Alix marker of exosomes (scale bar, 30 nm). (D) Quantification of CD9 exosomes release from the prostate and breast CSCs. Data represent mean ± s. e. m. from at least three independent experiments. *P<0.05 compared with rotenone-treated and control.

Figure 5. Molecular mechanism of exosomal release from the rotenone (40 μM) treated prostate and breast CSCs

(A) Chemical structure of rotenone. (B) Electron microscopy of mitochondrial damaged in prostate and breast CSCs by rotenone (40 μM) (scale bar, 500 nm). (C) Quantification of mitochondrial damage in prostate and breast CSCs. (D) Activation of autophagy (LC3 and Atg7) in rotenone (40 μM)-treated prostate and breast CSCs. (E) Detection of exosomal markers CD9, CD63 and CD81 in exosomes isolated from rotenone-treated prostate CSCs by western blotting. (F) Detection of exosomal markers CD9, CD63 and CD81 in exosomes isolated from rotenone-treated breast CSCs by western blotting. (G) mRNA expression of exosomal markers (CD9, CD63, CD81 and TSG101) in exosomes released from the prostate CSCs. (H) mRNA expression of exosomal markers (CD9, CD63, CD81 and TSG101) in exosomes released from the breast CSCs. Data represent mean ± s. e. m. from at least three independent experiments. *P<0.05 compared with rotenone-treated and control.

Figure 6. Model represents the activation of autophagy, formation of autophagosomes and release of exosomes from the rotenone treated CSCs

Rotenone induces autophagic vacuolation in CSCs which are associated with the mitochondrial damage. These processes induce the formation of autophagosomes and autophagy in CSCs. Autophagosomes combine with the protein complexes, ribosomes, endoplasmic reticulum and peroxisomes, and form multi-vesicular endosomes. The multi-vesicular endosomes release exosomes.