Ultrasound microbubble potentiated enhancement of hyperthermia-effect in tumours

Autoři: Deepa Sharma aff001;  Anoja Giles aff001;  Amr Hashim aff001;  Jodi Yip aff001;  Yipeng Ji aff001;  Natalie Ngoc Anh Do aff001;  Juliana Sebastiani aff001;  William Tyler Tran aff001;  Golnaz Farhat aff001;  Michael Oelze aff005;  Gregory J. Czarnota aff001
Působiště autorů: Physical Sciences, Sunnybrook Research Institute, Toronto, ON, Canada aff001;  Department of Radiation Oncology, Sunnybrook Health Sciences Centre, Toronto, ON, Canada aff002;  Department of Radiation Oncology, University of Toronto, Toronto, ON, Canada aff003;  Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada aff004;  Department of Electrical and Computer Engineering, University of Illinois, Urbana-Champaign, IL, United States of America aff005
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226475


It is now well established that for tumour growth and survival, tumour vasculature is an important element. Studies have demonstrated that ultrasound-stimulated microbubble (USMB) treatment causes extensive endothelial cell death leading to tumour vascular disruption. The subsequent rapid vascular collapse translates to overall increases in tumour response to various therapies. In this study, we explored USMB involvement in the enhancement of hyperthermia (HT) treatment effects. Human prostate tumour (PC3) xenografts were grown in mice and were treated with USMB, HT, or with a combination of the two treatments. Treatment parameters consisted of ultrasound pressures of 0 to 740 kPa, the use of perfluorocarbon-filled microbubbles administered intravenously, and an HT temperature of 43°C delivered for various times (0–50 minutes). Single and multiple repeated treatments were evaluated. Tumour response was monitored 24 hours after treatments and tumour growth was monitored for up to over 30 days for a single treatment and 4 weeks for multiple treatments. Tumours exposed to USMB combined with HT exhibited enhanced cell death (p<0.05) and decreased vasculature (p<0.05) compared to untreated tumours or those treated with either USMB alone or HT alone within 24 hours. Deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and cluster of differentiation 31 (CD31) staining were used to assess cell death and vascular content, respectively. Further, tumours receiving a single combined USMB and HT treatment exhibited decreased tumour volumes (p<0.05) compared to those receiving either treatment alone when monitored over the duration of 30 days. Additionally, tumour response monitored weekly up to 4 weeks demonstrated a reduced vascular index and tumour volume, increased fibrosis and lesser number of proliferating cells with combined treatment of USMB and HT. Thus in this study, we characterize a novel therapeutic approach that combines USMB with HT to enhance treatment responses in a prostate cancer xenograft model in vivo.

Klíčová slova:

Apoptosis – Cancer treatment – Cell death – Cell staining – Collagens – Fibrosis – Hyperthermia – Radiation therapy


1. van der Zee J. Heating the patient: A promising approach? Annals of Oncology. 2002. doi: 10.1093/annonc/mdf280 12181239

2. Diederich CJ. Thermal ablation and high-temperature thermal therapy: Overview of technology and clinical implementation. International Journal of Hyperthermia. 2005. pp. 745–753. doi: 10.1080/02656730500271692 16338857

3. Hildebrandt B, Wust P, Ahlers O, Dieing A, Sreenivasa G, Kerner T, et al. The cellular and molecular basis of hyperthermia. Critical Reviews in Oncology/Hematology. 2002. doi: 10.1016/S1040-8428(01)00179-2

4. Diederich CJ, Hynynen K. Ultrasound technology for hyperthermia. Ultrasound in Medicine and Biology. 1999. doi: 10.1016/S0301-5629(99)00048-4

5. Wust P, Hildebrandt B, Sreenivasa G, Rau B, Gellermann J, Riess H, et al. Hyperthermia in combined treatment of cancer. Lancet Oncology. 2002. doi: 10.1016/S1470-2045(02)00818-5

6. Falk MH, Issels RD. Hyperthermia in oncology. Int J Hyperthermia. 2001; doi: 10.1080/02656730150201552 11212876

7. Pelz JOW, Vetterlein M, Grimmig T, Kerscher AG, Moll E, Lazariotou M, et al. Hyperthermic intraperitoneal chemotherapy in patients with peritoneal carcinomatosis: role of heat shock proteins and dissecting effects of hyperthermia. Ann Surg Oncol. 2013; doi: 10.1245/s10434-012-2784-6 23456378

8. Chicheł A, Skowronek J, Kubaszewska M, Kanikowski M. Hyperthermia–description of a method and a review of clinical applications. Reports Pract Oncol Radiother. 2007; doi: 10.1016/S1507-1367(10)60065-X

9. Marmottant P, Hilgenfeldt S. Controlled vesicle deformation and lysis by single oscillating bubbles. Nature. 2003; doi: 10.1038/nature01613 12736680

10. Sirsi S, Feshitan J, Kwan J, Homma S, Borden M. Effect of microbubble size on fundamental mode high frequency ultrasound imaging in mice. Ultrasound Med Biol. 2010; doi: 10.1016/j.ultrasmedbio.2010.03.015 20447755

11. McDannold N, Vykhodtseva N, Hynynen K. Blood-Brain Barrier Disruption Induced by Focused Ultrasound and Circulating Preformed Microbubbles Appears to Be Characterized by the Mechanical Index. Ultrasound Med Biol. 2008; doi: 10.1016/j.ultrasmedbio.2007.10.016 18207311

12. Karshafian R, Giles A, Burns PN, Czarnota GJ. Ultrasound-activated microbubbles as novel enhancers of radiotherapy in leukemia cells in vitro. Proceedings—IEEE Ultrasonics Symposium. 2009. doi: 10.1109/ULTSYM.2009.5441487

13. Ghoshal G, Oelze ML. Enhancing cell kill in vitro from hyperthermia through pre-sensitizing with ultrasound-stimulated microbubbles. J Acoust Soc Am. 2015; doi: 10.1121/1.4936644 26723356

14. Czarnota GJ, Karshafian R, Burns PN, Wong S, Al Mahrouki A, Lee JW, et al. Tumor radiation response enhancement by acoustical stimulation of the vasculature. Proc Natl Acad Sci. 2012; doi: 10.1073/pnas.1200053109 22778441

15. Tran WT, Iradji S, Sofroni E, Giles A, Eddy D, Czarnota GJ. Microbubble and ultrasound radioenhancement of bladder cancer. Br J Cancer. 2012; doi: 10.1038/bjc.2012.279\nbjc2012279 [pii]

16. Al-Mahrouki A a, Iradji S, Tran WT, Czarnota GJ. Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model. Dis Model Mech. 2014; doi: 10.1242/dmm.012922 24487407

17. Lai P, Tarapacki C, Tran WT, Kaffas A El, Hupple C, Iradji S, et al. Breast tumor response to ultrasound mediated excitation of microbubbles and radiation therapy in vivo. Oncosciencencoscience. 2016; 98–108.

18. Fajardo LF, Prionas SD, Kowalski J, Kwan HH. Hyperthermia Inhibits Angiogenesis. Radiat Res. 1988; doi: 10.2307/3577226

19. Norton KA, Popel AS. Effects of endothelial cell proliferation and migration rates in a computational model of sprouting angiogenesis. Sci Rep. 2016; doi: 10.1038/srep36992 27841344

20. Leung DW, Cachianes G, Kuang WJ, Goeddel D V., Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science (80-). 1989; doi: 10.1126/science.2479986 2479986

21. Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev. 1992; doi: 10.1210/edrv-13-1-18 1372863

22. Connolly DT, Olander J V., Heuvelman D, Nelson R, Monsell R, Siegel N, et al. Human vascular permeability factor. Isolation from U937 cells. J Biol Chem. 1989;

23. Sawaji Y, Sato T, Takeuchi A, Hirata M, Ito A. Anti-angiogenic action of hyperthermia by suppressing gene expression and production of tumour-derived vascular endothelial growth factor in vivo and in vitro. Br J Cancer. 2002; doi: 10.1038/sj.bjc.6600268 12085210

24. Bauman TM, Nicholson TM, Abler LL, Eliceiri KW, Huang W, Vezina CM, et al. Characterization of fibrillar collagens and extracellular matrix of glandular benign prostatic hyperplasia nodules. PLoS One. 2014; doi: 10.1371/journal.pone.0109102 25275645

25. Al-Mahrouki AA, Karshafian R, Giles A, Czarnota GJ. Bioeffects of Ultrasound-Stimulated Microbubbles on Endothelial Cells: Gene Expression Changes Associated with Radiation Enhancement In Vitro. Ultrasound Med Biol. 2012; doi: 10.1016/j.ultrasmedbio.2012.07.009 22980406

26. Kim HC, Al-Mahrouki A, Gorjizadeh A, Karshafian R, Czarnota GJ. Effects of biophysical parameters in enhancing radiation responses of prostate tumors with ultrasound-stimulated microbubbles. Ultrasound Med Biol. 2013; doi: 10.1016/j.ultrasmedbio.2013.01.012 23643061

27. El Kaffas A, Al-Mahrouki A, Hashim A, Law N, Giles A, Czarnota GJ. Role of acid sphingomyelinase and ceramide in mechano-acoustic enhancement of tumor radiation responses. J Natl Cancer Inst. 2018;110: 1009–1018. doi: 10.1093/jnci/djy011 29506145

28. Goertz DE, Todorova M, Mortazavi O, Agache V, Chen B, Karshafian R, et al. Antitumor Effects of Combining Docetaxel (Taxotere) with the Antivascular Action of Ultrasound Stimulated Microbubbles. PLoS One. 2012; doi: 10.1371/journal.pone.0052307 23284980

29. Todorova M, Agache V, Mortazavi O, Chen B, Karshafian R, Hynynen K, et al. Antitumor effects of combining metronomic chemotherapy with the antivascular action of ultrasound stimulated microbubbles. Int J Cancer. 2013;132: 2956–2966. doi: 10.1002/ijc.27977 23225339

30. Kim BM, Hong Y, Lee S, Liu P, Lim JH, Lee YH, et al. Therapeutic implications for overcoming radiation resistance in cancer therapy. International Journal of Molecular Sciences. 2015. doi: 10.3390/ijms161125991 26569225

31. Luqmani YA. Mechanisms of drug resistance in cancer chemotherapy. Medical Principles and Practice. 2005. doi: 10.1159/000086183 16103712

32. Martin T, Ye L, Sanders A, Lane J, Jiang W. Cancer Invasion and Metastasis: Molecular and Cellular Perspective. Cancer Invasion and Metastasis: Molecular and Cellular Perspective. 2014.

33. Weis SM, Cheresh DA. Tumor angiogenesis: Molecular pathways and therapeutic targets. Nature Medicine. 2011. doi: 10.1038/nm.2537 22064426

34. Ziyad S, Iruela-Arispe ML. Molecular Mechanisms of Tumor Angiogenesis. Genes and Cancer. 2011; doi: 10.1177/1947601911432334 22866200

35. Kim HC, Al-Mahrouki A, Gorjizadeh A, Karshafian R, Czarnota GJ. Effects of biophysical parameters in enhancing radiation responses of prostate tumors with ultrasound-stimulated microbubbles. Ultrasound Med Biol. 2013; doi: 10.1016/j.ultrasmedbio.2013.01.012 23643061

36. Lin FC, Hsu CH, Lin YY. Nano-therapeutic cancer immunotherapy using hyperthermia-induced heat shock proteins: Insights from mathematical modeling. Int J Nanomedicine. 2018; doi: 10.2147/IJN.S166000 29950833

37. Haghniaz R, Umrani RD, Paknikar KM. Temperature-dependent and time-dependent effects of hyperthermia mediated by dextran-coated La0.7Sr0.3MnO3: In vitro studies. Int J Nanomedicine. 2015; doi: 10.2147/IJN.S78167 25759583

38. Horsman MR, Overgaard J. Hyperthermia: a Potent Enhancer of Radiotherapy. Clin Oncol. 2007; doi: 10.1016/j.clon.2007.03.015 17493790

39. Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden D, Rafii S, Haimovitz-Friedman A, et al. Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science (80-). 2003;300: 1155–1159. doi: 10.1126/science.1082504 12750523

40. Peñ LA, Fuks Z, Kolesnick RN. Radiation-induced Apoptosis of Endothelial Cells in the Murine Central Nervous System: Protection by Fibroblast Growth Factor and Sphingomyelinase Deficiency. CANCER Res. 2000; doi: 10.1016/0360-3016(80)90175-3

41. Paris F, Fuks Z, Kang A, Capodieci P, Juan G, Ehleiter D, et al. Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science (80-). 2001;293: 293–297. doi: 10.1126/science.1060191 11452123

42. Roti JL. Cellular responses to hyperthermia (40–46°C): Cell killing and molecular events. International Journal of Hyperthermia. 2008. doi: 10.1080/02656730701769841 18214765

43. Li L, Tan H, Gu Z, Liu Z, Geng Y, Liu Y, et al. Heat stress induces apoptosis through a Ca2+-mediated mitochondrial apoptotic pathway in human umbilical vein endothelial cells. PLoS One. 2014; doi: 10.1371/journal.pone.0111083 25549352

44. Song CW. Effect of local hyperthermia on blood flow and microenvironment: A review. Cancer Res. 1984;

45. Song CW, Park HJ, Lee CK, Griffin R. Implications of increased tumor blood flow and oxygenation caused by mild temperature hyperthermia in tumor treatment. International Journal of Hyperthermia. 2005. doi: 10.1080/02656730500204487 16338859

46. Zhang Y, Zhan X, Xiong J, Peng S, Huang W, Joshi R, et al. Temperature-dependent cell death patterns induced by functionalized gold nanoparticle photothermal therapy in melanoma cells. Sci Rep. 2018; doi: 10.1038/s41598-018-26978-1 29880902

47. Vorotnikova E, Ivkov R, Foreman A, Tries M, Braunhut SJ. The magnitude and time-dependence of the apoptotic response of normal and malignant cells subjected to ionizing radiation versus hyperthermia. Int J Radiat Biol. 2006; doi: 10.1080/09553000600876678 16966182

48. Baronzio GF, Hager ED, Baronzio GF, Delia Seta R, D’Amico M, Baronzio A, et al. Effects of Local and Whole Body Hyperthermia on Immunity. Hyperthermia in Cancer Treatment: A Primer. 2006. doi: 10.1007/978-0-387-33441-7_20

49. Kondo T, Matsuda T, Tashima M, Umehara H, Domae N, Yokoyama K, et al. Suppression of heat shock protein-70 by ceramide in heat shock-induced HL-60 cell apoptosis. J Biol Chem. 2000; doi: 10.1074/jbc.275.12.8872 10722733

50. El Kaffas A, Nofiele J, Giles A, Cho S, Liu SK, Czarnota GJ. DLL4-notch signalling blockade synergizes combined ultrasound-stimulated microbubble and radiation therapy in human colon cancer xenografts. PLoS One. 2014; doi: 10.1371/journal.pone.0093888 24736631

51. Mantso T, Vasileiadis S, Lampri E, Botaitis S, Perente S, Simopoulos C, et al. Hyperthermia suppresses post—In vitro proliferation and tumor growth in murine malignant melanoma and colon carcinoma. Anticancer Res. 2019; doi: 10.21873/anticanres.13347 31092422

52. Zhang B, Zhou H, Cheng Q, Lei L, Hu B. Low-frequency low energy ultrasound combined with microbubbles induces distinct apoptosis of A7r5 cells. Mol Med Rep. 2014; doi: 10.3892/mmr.2014.2654 25324182

53. Li HX, Zheng JH, Ji L, Liu GY, Lv YK, Yang D, et al. Effects of low-intensity ultrasound combined with low-dose carboplatin in an orthotopic hamster model of tongue cancer: A preclinical study. Oncol Rep. 2018; doi: 10.3892/or.2018.6262 29436690

54. Nishimura Y, Urano M. Timing and sequence of hyperthermia in fractionated radiotherapy of a murine fibrosarcoma. Int J Radiat Oncol Biol Phys. 1993; doi: 10.1016/0360-3016(93)90386-A

55. Overgaard J. Influence of sequence and interval on the biological response to combined hyperthermia and radiation. Natl Cancer Inst Monogr. 1982;

56. Li GC, Kal HB. Effect of hyperthermia on the radiation response of two mammalian cell lines. Eur J Cancer. 1977; doi: 10.1016/0014-2964(77)90231-6

57. Dings RPM, Loren ML, Zhang Y, Mikkelson S, Mayo KH, Corry P, et al. Tumour thermotolerance, a physiological phenomenon involving vessel normalisation. Int J Hyperth. 2011; doi: 10.3109/02656736.2010.510495 21204622

58. Hegyi G, Szigeti GP, Szász A. Hyperthermia versus oncothermia: Cellular effects in complementary cancer therapy. Evidence-based Complementary and Alternative Medicine. 2013. doi: 10.1155/2013/672873 23662149

59. Andocs G, Rehman MU, Zhao Q-L, Tabuchi Y, Kanamori M, Kondo T. Comparison of biological effects of modulated electro-hyperthermia and conventional heat treatment in human lymphoma U937 cells. Cell Death Discov. 2016; doi: 10.1038/cddiscovery.2016.39 27551529

Článek vyšel v časopise


2019 Číslo 12