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, 2015, 853506

Adipose-Derived Mesenchymal Stem Cell Exosomes Suppress Hepatocellular Carcinoma Growth in a Rat Model: Apparent Diffusion Coefficient, Natural Killer T-Cell Responses, and Histopathological Features

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Adipose-Derived Mesenchymal Stem Cell Exosomes Suppress Hepatocellular Carcinoma Growth in a Rat Model: Apparent Diffusion Coefficient, Natural Killer T-Cell Responses, and Histopathological Features

Sheung-Fat Ko et al. Stem Cells Int.

Abstract

We sought to evaluate the effects of adipose-derived mesenchymal stem cells (ADMSCs) exosomes on hepatocellular carcinoma (HCC) in rats using apparent diffusion coefficient (ADC), natural killer T-cell (NKT-cell) responses, and histopathological features. ADMSC-derived exosomes appeared as nanoparticles (30-90 nm) on electron microscopy and were positive for CD63, tumor susceptibility gene-101, and β-catenin on western blotting. The control (n = 8) and exosome-treated (n = 8) rats with N1S1-induced HCC underwent baseline and posttreatment day 10 and day 20 magnetic resonance imaging and measurement of ADC. Magnetic resonance imaging showed rapidly enlarged HCCs with low ADCs in the controls. The exosome-treated rats showed partial but nonsignificant tumor reduction, and significant ADC and ADC ratio increases on day 10. On day 20, the exosome-treated rats harbored significantly smaller tumors and volume ratios, higher ADC and ADC ratios, more circulating and intratumoral NKT-cells, and low-grade HCC (P < 0.05 for all comparisons) compared to the controls. The ADC and volume ratios exhibited significant inverse correlations (P < 0.001, R (2) = 0.679). ADMSC-derived exosomes promoted NKT-cell antitumor responses in rats, thereby facilitating HCC suppression, early ADC increase, and low-grade tumor differentiation. ADC may be an early biomarker of treatment response.

Figures

Figure 1
Figure 1
The flowchart shows the preparation and cultures of adipose-derived mesenchymal stem cells (ADMSCs), isolation of exosomes from culture medium, protein quantification and characterization of exosomes, and the final injection of exosomes via the penile vein for HCC treatment (min: minutes, g: gravity, PBS: phosphate buffered saline, DMEM: Dulbecco's modified Eagle's medium, FBS: fetal bovine serum, rpm: rotation per min).
Figure 2
Figure 2
The flowchart shows the timetable for blood sampling (before and 10 days after HCC induction, posttreatment day 5 and day 15), exosome treatment (after baseline and posttreatment day 10 MR), liver MR and DW imaging (baseline, posttreatment day 10 and day 20), and final killing. Please note that two rats with typical HCC features and four rats with no HCC revealed on baseline MR imaging were killed for histopathological confirmation of MR findings.
Figure 3
Figure 3
Transmission electron microscopic evaluation shows small vesicles within the expected range of exosomes (30–90 nm) in the sample isolated from the ADMSCs culture medium by ultracentrifugation.
Figure 4
Figure 4
Western blot analysis of the culture medium, conditioned medium, and exosomes probed with antibodies against CD63, tumor susceptibility gene-101 (TSG-101), and β-catenin. Please note that CD63 is present in the culture medium, conditioned medium, and exosomes. TSG101 and β-catenin are absent in DMEM (Dulbecco's modified Eagle medium) without or with 10% serum but are present in the exosome fractions (1 μg, 2 μg, 10 μg, and 50 μg), particularly the 50 μg sample.
Figure 5
Figure 5
T1-weighted ((a), (e), and (i)), enhanced T1-weighted ((b), (f), and (j)), DW image (b value = 800 sec/mm2) ((c), (g), and (k)), and ADC map ((d), (h), and (l)) of HCC at the level of greatest tumor diameter on baseline (a, b, c, d), posttreatment day 10 ((e), (f), (g), and (h)), and posttreatment day 20 ((i), (j), (k), and (l)). MR imaging of a control rat shows heterogeneously enhanced tumor with marked enlargement (whole-tumor volume ratios: D10/baseline = 1.38, D20/baseline = 1.85) whilst the ADC value (whole-tumor ADC ratios: D10/baseline = 0.92, D20/baseline = 1.04) remains low. Gross specimen (M) of the resected liver shows a large tumor in the left lobe with good correlation to MR imaging on posttreatment day 20.
Figure 6
Figure 6
T1-weighted ((a), (e), and (i)), enhanced T1-weighted ((b), (f), and (j)), DW image (b value = 800 sec/mm2) ((c), (g), and (k)), and ADC map ((d), (h), and (l)) of HCC at the level of greatest tumor diameter on baseline ((a), (b), (c), and (d)), posttreatment day 10 ((e), (f), (g), and (h)), and posttreatment day 20 ((i), (j), (k), and (l)). MR imaging of an exosome-treated rat shows partial but nonsignificant tumor reduction and significantly increased ADC ratio on posttreatment day 10 (D10/baseline whole-tumor volume ratio = 0.72 and ADC ratio = 1.29). On posttreatment day 20, the exosome-treated rat harbored significantly smaller tumor and higher ADC ratio (D20/baseline whole-tumor volume ratio = 0.29 and ADC ratio = 1.63). Gross specimen (M) of the resected liver shows a small lobulated tumor in the left lobe with good correlation to MR imaging on posttreatment day 20.
Figure 7
Figure 7
The graph shows the relationship between the whole-tumor volume ratio and ADC ratio, indicating a strong correlation (P < 0.001, R 2 = 0.679; simple linear regression analysis).
Figure 8
Figure 8
Immunohistochemical staining (200x) with CD8α in the rat in the control group (a) and the rat in the exosome-treated group (b) with focal magnification view shows significantly higher number of intratumoral CD8α+ NKT-cells (arrows) in the exosome-treated rat than in the control. Scale bar = 50 μm.

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