Skip to main content

Investigation of the tumoricidal effects of sonodynamic therapy in malignant glioblastoma brain tumors



Glioblastoma is the most common primary brain tumor; survival is typically 12–18 months after diagnosis. We sought to study the effects of sonodynamic therapy (SDT) using 5-Aminolevulinic acid hydrochloride (5-ALA) and high frequency focused ultrasound (FUS) on 2 glioblastoma cell lines.


Rat C6 and human U87 glioblastoma cells were studied under the following conditions: 1 mM 5-ALA (5-ALA); focused ultrasound (FUS); 5-ALA and focused ultrasound (SDT); control. Studied responses included cell viability using an MTT assay, microscopic changes using phase contract microscopy, apoptotic induction through a caspase-3 assay, and apoptosis staining to quantify cell death.


SDT led to a marked decrease in cell extension and reduction in cell size. For C6, the MTT assay showed reductions in cell viability for 5-ALA, FUS, and SDT groups of 5%, 16%, and 47%, respectively compared to control (p < 0.05). Caspase 3 induction in C6 cells relative to control showed increases of 109%, 110%, and 278% for 5-ALA, FUS, and SDT groups, respectively (p < 0.05). For the C6 cells, caspase 3 staining positivity was 2.1%, 6.7%, 11.2%, and 39.8% for control, 5-ALA, FUS, and SDT groups, respectively. C6 Parp-1 staining positivity was 1.9%, 6.5%, 9.0%, and 37.8% for control, 5-ALA, FUS, and SDT groups, respectively. U87 cells showed similar responses to the treatments.


Sonodynamic therapy resulted in appreciable glioblastoma cell death as compared to 5-ALA or FUS alone. The approach couples two already FDA approved techniques in a novel way to treat the most aggressive and malignant of brain tumors. Further study of this promising technique is planned.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Omuro A, DeAngelis LM (2013) Glioblastoma and other malignant gliomas: a clinical review. JAMA 310(17):1842–1850

    Article  CAS  Google Scholar 

  2. McDannold N, Clement GT, Black P, Jolesz F, Hynynen K (2010) Transcranial magnetic resonance imaging—guided focused ultrasound surgery of brain tumors: initial findings in 3 patients. Neurosurgery 66(2):323–332

    Article  Google Scholar 

  3. Walters H, Shah BB (2019) Focused ultrasound and other lesioning therapies in movement disorders. Curr Neurol Neurosci Rep 19(9):66

    Article  Google Scholar 

  4. Fishman PS, Frenkel V (2017) Focused ultrasound: an emerging therapeutic modality for neurologic disease. Neurotherapeutics 14(2):393–404

    Article  Google Scholar 

  5. Zhu L, Altman MB, Laszlo A et al (2019) Ultrasound hyperthermia technology for radiosensitization. Ultrasound Med Biol 45(5):1025–1043

    Article  Google Scholar 

  6. McHale AP, Callan JF, Nomikou N, Fowley C, Callan B (2016) Sonodynamic therapy: concept, mechanism and application to cancer treatment. Adv Exp Med Biol 880:429–450

    Article  CAS  Google Scholar 

  7. Yang Y, Tu J, Yang D, Raymond JL, Roy RA, Zhang D (2019) Photo- and sono-dynamic therapy: a review of mechanisms and considerations for pharmacological agents used in therapy incorporating light and sound. Curr Pharm Des 25(4):401–412

    Article  CAS  Google Scholar 

  8. Endo S, Kudo N, Yamaguchi S, Sumiyoshi K, Motegi H, Kobayashi H, Terasaka S, Houkin K (2015) Porphyrin derivatives-mediated sonodynamic therapy for malignant gliomas in vitro. Ultrasound Med Biol 41(9):2458–2465

    Article  Google Scholar 

  9. Jeong EJ, Seo SJ, Ahn YJ, Choi KH, Kim KH, Kim JK (2012) Sonodynamically induced antitumor effects of 5-Aminolevulinic acid and fractionated ultrasound irradiation in an orthotopic rat glioma model. Ultrasound Med Biol 38(12):2143–2150

    Article  Google Scholar 

  10. Lafond M, Yoshizawa S, Umemura SI (2019) Sonodynamic therapy: advances and challenges in clinical translation. J Ultrasound Med 38(3):567–580

    Article  Google Scholar 

  11. Pan X, Wang H, Wang S, Sun X, Wang L, Wang W, Shen H, Liu H (2018) Sonodynamic therapy (SDT): a novel strategy for cancer nanotheranostics. Sci China Lif Sci 61(4):415–429

    Article  Google Scholar 

  12. Hersh DS, Kim AJ, Winkles JA, Eisenberg HM, Woodworth GF, Frenkel V (2016) Emerging applications of therapeutic ultrasound in neuro-oncology: moving beyond tumor ablation. Neurosurgery 79(5):643–654

    Article  Google Scholar 

  13. Hadjipanayis CG, Widhalm G, Stummer W (2015) What is the surgical benefit of utilizing 5-Aminolevulinic acid for fluorescence-guided surgery of malignant gliomas? Neurosurgery 77(5):663–673

    Article  Google Scholar 

  14. Li YJ, Huang P, Jiang CL, de Jia X, Du XX, Zhou JH, Han Y, Sui H, Wei XL, Liu L, Yuan HH, Zhang TT, Zhang WJ, Xie R, Lang XH, Wang LY, Liu T, Bai YX, Tian Y (2014) Sonodynamically induced anti-tumor effect of 5-Aminolevulinic acid on pancreatic cancer cells. Ultrasound Med Biol 40(11):2671–2679

    Article  Google Scholar 

  15. Li Y, Zhou Q, Hu Z, Yang B, Li Q, Wang J, Zheng J, Cao W (2015) 5-Aminolevulinic acid-based sonodynamic therapy induces the apoptosis of osteosarcoma in mice. PLoS ONE 10(7):e0132074

    Article  Google Scholar 

  16. Ji C, Yang B, Yang YL, He SH, Miao DS, He L, Bi ZG (2010) Exogenous cell-permeable C6 ceramide sensitizes multiple cancer cell lines to Doxorubicin-induced apoptosis by promoting AMPK activation and mTORC1 inhibition. Oncogene 29(50):6557–6568

    Article  CAS  Google Scholar 

  17. Saraste A, Pulkki K (2000) Morphologic and biochemical hallmark of Apoptosis. Cardiovasc Res 45(3):528–537

    Article  CAS  Google Scholar 

  18. D’Amours D, Sallmann FR, Dixit VM, Poirier GG (2001) Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis. J Cell Sci 114(20):3771–3778

    PubMed  Google Scholar 

  19. Grisham J (2015) The Tumor. eBook.

  20. Fomenko A, Lozano AM (2019) Neuromodulation and ablation with focused ultrasound—toward the future of noninvasive brain therapy. Neural Regen Res 14(9):1509–1510

    Article  Google Scholar 

  21. Yoshida M, Kobayashi H, Terasaka S, Endo S, Yamaguchi S, Motegi H, Itay R, Suzuki S, Brokman O, Shapira Y, Moriyama K, Kawase Y, Akahane T, Kato Y, Kamada H, Houkin K (2019) Sonodynamic therapy for malignant glioma using 220-kHz transcranial magnetic resonance imaging-guided focused ultrasound and 5-Aminolevulinic acid. Ultrasound Med Biol 45(2):526–538

    Article  Google Scholar 

  22. Lv Y, Zheng J, Zhou Q, Jia L, Wang C, Liu N, Zhao H, Ji H, Li B, Cao W (2017) Antiproliferative and apoptosis-inducing effect of exo-protoporphyrin IX based sonodynamic therapy on human oral squamous cell carcinoma. Sci Rep 7:40967

    Article  CAS  Google Scholar 

  23. Yue W, Chen L, Yu L, Zhou B, Yin H, Ren W, Liu C, Guo L, Zhang Y, Sun L, Zhang K, Xu H, Chen Y (2019) Checkpoint blockade and nanosonosensitizer-augmented noninvasive sonodynamic therapy combination reduces tumour growth and metastases in mice. Nat Commun 10(1):2025

    Article  Google Scholar 

  24. Wan GY, Liu Y, Chen BW, Liu YY, Wang YS, Zhang N (2016) Recent advances of sonodynamic therapy in cancer treatment. Cancer Biol Med 13(3):325–338

    Article  CAS  Google Scholar 

  25. Wang X, Jia Y, Wang P, Liu Q, Zheng H (2017) Current status and future perspectives of sonodynamic therapy in glioma treatment. Ultrason Sonochem 37:592–599

    Article  CAS  Google Scholar 

  26. Wang X, Jia Y, Su X, Wang X, Zhang K, Feng X et al (2015) Combination of protoporphyrin IX-mediated sonodynamic treatment with doxorubicin synergistically induced apoptotic cell death of a multidrug-resistant leukemia K562/DOX cell line. Ultrasound Med Biol 41:2731–2739

    Article  Google Scholar 

Download references


We are grateful for the support of Drs. Eames and Moore who allowed us to use their 3D CAD software and 3D printer at the Focused Ultrasound Foundation. Finally, we appreciate the assistance of Dr. Pramoonjago at the University of Virginia’s Biorepository and Tissue Research Facility who processed the cells using automated instrumentation for the cytospin techniques for immunohistochemical staining.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Kimball Sheehan.

Ethics declarations

Ethical approval

The current research does not involve human participants and/or animals and therefore informed consent was not required.


Dr. Padilla and Dr. Moore are employees of the Focused Ultrasound Foundation. Otherwise, the authors have no disclosures related to this particular study.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.


Supplemental Figure 1: One of the 3-D printed devices for treating the glioblastoma cells with SDT. The ultrasound probe was slid into the cylindrical column and the plate or dish was placed on the upper flat surface. The entire system was placed in degassed water at the time of treatment. (JPEG 1,907 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sheehan, K., Sheehan, D., Sulaiman, M. et al. Investigation of the tumoricidal effects of sonodynamic therapy in malignant glioblastoma brain tumors. J Neurooncol 148, 9–16 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: