Focused ultrasound-induced blood-brain barrier opening to enhance temozolomide delivery for glioblastoma treatment: a preclinical study

Kuo-Chen Wei, Po-Chun Chu, Hay-Yan Jack Wang, Chiung-Yin Huang, Pin-Yuan Chen, Hong-Chieh Tsai, Yu-Jen Lu, Pei-Yun Lee, I-Chou Tseng, Li-Ying Feng, Peng-Wei Hsu, Tzu-Chen Yen, Hao-Li Liu, Kuo-Chen Wei, Po-Chun Chu, Hay-Yan Jack Wang, Chiung-Yin Huang, Pin-Yuan Chen, Hong-Chieh Tsai, Yu-Jen Lu, Pei-Yun Lee, I-Chou Tseng, Li-Ying Feng, Peng-Wei Hsu, Tzu-Chen Yen, Hao-Li Liu

Abstract

The purpose of this study is to assess the preclinical therapeutic efficacy of magnetic resonance imaging (MRI)-monitored focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption to enhance Temozolomide (TMZ) delivery for improving Glioblastoma Multiforme (GBM) treatment. MRI-monitored FUS with microbubbles was used to transcranially disrupt the BBB in brains of Fisher rats implanted with 9L glioma cells. FUS-BBB opening was spectrophotometrically determined by leakage of dyes into the brain, and TMZ was quantitated in cerebrospinal fluid (CSF) and plasma by LC-MS\MS. The effects of treatment on tumor progression (by MRI), animal survival and brain tissue histology were investigated. Results demonstrated that FUS-BBB opening increased the local accumulation of dyes in brain parenchyma by 3.8-/2.1-fold in normal/tumor tissues. Compared to TMZ alone, combined FUS treatment increased the TMZ CSF/plasma ratio from 22.7% to 38.6%, reduced the 7-day tumor progression ratio from 24.03 to 5.06, and extended the median survival from 20 to 23 days. In conclusion, this study provided preclinical evidence that FUS BBB-opening increased the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting its clinical potential for improving current brain tumor treatment.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Conceptual diagrams of focused ultrasound…
Figure 1. Conceptual diagrams of focused ultrasound induced blood-brain barrier opening to enhance chemotherapeutic agent delivery for brain tumor treatment.
(a) Focused ultrasound is used to transcranially steer the exposure to the targeted brain tumor region; (b) focused ultrasound induces a local and reversible increase in BBB permeability of tight junctions in cerebral vessels and capillaries in the tumor core/ peripheral region. EC = endothelial cell, N = neuron, P = pericytes, A = astrocyte, D = chemotherapeutic agent, MB = microbubble.
Figure 2. Time course for animal experiments…
Figure 2. Time course for animal experiments and longitudinal MRI follow-up in experimental group 3.
Figure 3. Representative Evans Blue dye stained…
Figure 3. Representative Evans Blue dye stained Brain sections and calibrations after inducing FUS-BBB opening.
(a, b) brain sections viewed from the top and in corresponding brain sections. Bar = 5 mm. (c) Calibration of Evans Blue dye concentration using its correlation with ELISA light absorption (r2 = 0.9992). (d) Evans Blue quantification of experimental group 1 animals. FUS-BBB opening reached a 3.8-fold increase in EB concentration in normal rats (p<0.001) and a 2.1-fold increase in tumor rats (p = 0.09).
Figure 4. Representative MR images before (upper…
Figure 4. Representative MR images before (upper panel) and after (lower panel) conducting FUS BBB-opening in rat brain tumors.
(a, e) T1-weighted images; (b, f) Gd-DTPA contrast-enhanced T1-weighted images; (c, g) subtracted after and before Gd-DTPA injection T1 images; (d, h) T2-weighted images.
Figure 5. In-vivo TMZ concentration measurement.
Figure 5. In-vivo TMZ concentration measurement.
(a) Measured TMZ concentrations (in µg/ µL) in cerebrospinal fluid (CSF) and blood plasma in animals treated with TMZ only (TMZ; n = 4) or combined TMZ with FUS-BBB opening (TMZ+FUS; n = 6). (b) Corresponding CSF/Plasma ratios (in %) determined from (a) (p = 0.06).
Figure 6. Representative T2-weighted MR images to…
Figure 6. Representative T2-weighted MR images to monitor brain tumor progression at day 10 and 17 in experimental group 3.
(a) sham control; (b) low dose TMZ oral delivery; (c) median dose TMZ oral delivery; (d) high median dose TMZ oral delivery; (e, f) median dose TMZ integrated with FUS-BBB opening procedures.
Figure 7. Tumor progression observation among groups.
Figure 7. Tumor progression observation among groups.
(a) Tumor volume (in mm3) between day 10 and 17 in experimental group 3; (b) Ratio of the tumor volume between day 17 and day 10 determined from (a).
Figure 8. Kaplan–Meier plot demonstrating animal survival…
Figure 8. Kaplan–Meier plot demonstrating animal survival in experimental group 3.
Figure 9. Hematoxylin and eosin (H&E) stained…
Figure 9. Hematoxylin and eosin (H&E) stained sections of rat brains.
Tissues were collected for analysis from the TMZ-delivery alone ((a) – (c); brain samples obtained immediately after animal died) or combined TMZ/FUS exposure animals ((d) – (f): brain samples obtained after day 90). (a) and (d): whole brain section; (b) and (e): 4 ×;(c) and (f): 10 ×.

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