Focused ultrasound (FUS) is a less-invasive medical technique with the potential to improve the treatment outcome of many diseases by utilizing acoustic transducers to generate and concentrate the multiple intersecting ultrasonic waves on a targeted site in the body. The bio-effects induced by FUS are mostly classified into thermal and mechanical effects (mainly focus on cavitation effect). Cavitation is capable of disrupting tumor vasculature and cell membranes. Numerous studies reported that cavitation-induced sonoporation could provoke multiple anti-proliferative effects on cancer cells, including cell-cycle arrest, cell apoptosis, and clonogenicity suppression. Therefore, the combination of FUS-induced cavitation and other treatment modalities like radiation therapy is of great interest, but research in this field is inadequate.
A special high-throughput FUS system was used for cancer cell treatment with a customized 1.467 MHz single focused transducer. Characterization of acoustic behavior of gas-filled cavities was performed via a fiber-optic hydrophone (FOH) system and chemical terephthalic acid method helped to define the acoustic parameters, which could lead to occurrence of cavitation at the bottom of 96-well cell culture plates where cancer cells were located. Cavitation occurs at and above the acoustic intensity of 344 W/ cm2 for the 1.467 MHz transducer. The short- and long-term effects of FUS-induced cavitation and adjuvant effects to radiation therapy, standard hyperthermia and testosterone treatment (only for prostate cancer) were investigated comprehensively at the cellular and molecular levels in human prostate cancer (PC-3 and LNCap), glioblastoma (T98G) and head and neck (FaDu) cells in vitro.
The long-term additive effects of short FUS shots (with or without cavitation) to radiation therapy (RT) or hyperthermia (HT) were displayed by significantly reduced clonogenic survival in PC-3, T98G and FaDu cells compared to single treatments. The combination treatment of short FUS with cavitation (FUS-Cav) and RT led to a comparable radio-sensitization effect to HT at 45 °C for 30 min and showed a significant reduction in treatment duration, especially for PC-3 cells. The short-term additive effects of short FUS shots to RT or HT are manifested in reducing the potential of cells to invade and decreased metabolic activity. The induction of sonoporation by FUS-Cav was supposed to be the mechanism of cancer cell sensitization to other therapies at the cellular level. The dramatic decline of 5α-reductase type III (SRD5A3) level induced by combination treatment with FUS-Cav and HT is presumed as the underlying mechanism of additive effects of FUS-Cav to HT at the molecular level.
Besides, testosterone solutions with normal physiological levels were discovered to inhibit the long-term metabolic activity of androgen-dependent prostate cancer cells LNCap in vitro, while short FUS shots displayed a long-term additive effect to the testosterone treatment. The presented multilevel study demonstrates that short FUS shots using FUS-induced cavitation carry the potential to sensitize cancer cells to other cancer treatment modalities precisely and less-invasively, providing a promising adjuvant therapy to cancer treatments in the future.:1 Abbreviations
2 Summary
3 Introduction
4 Medical and technical background
4.1 The biological basis of prostate cancer treatment
4.1.1 Androgen receptor: an essential signaling pathway for progression of prostate cancer
4.1.2 5α-reductase: a promising therapeutic target for prostate cancer therapy
4.1.3 Testosterone: duality effects in prostate cancer development
4.2 Advantages and disadvantages of current clinical treatments of prostate cancer
4.3 Basics of focused ultrasound (FUS)
4.3.1 Medical application of FUS-induced thermal effects
4.3.1.1 High-intensity focused ultrasound (HIFU) induced thermal ablation
4.3.1.2 Hyperthermia: an alternative heating strategy to sensitize cancer cells for radiation therapy and chemotherapy
4.3.1.3 FUS-induced hyperthermia triggered drug delivery with thermo-sensitive drug carriers
4.3.2 Medical application of FUS-induced mechanical/cavitation effects
4.3.2.1 Cell sonoporation for drug delivery
4.3.2.2 Sonoporation induced anti-proliferative effects for cancer cells
4.3.2.3 Histotripsy
4.3.2.4 Anti-vascular and anti-metastatic effects
4.3.3 The state of art of cavitation detection in medical application
4.3.3.1 Sonoluminescence and sonochemistry
4.3.3.2 Passive cavitation detection
4.3.3.3 Active cavitation detection
4.3.3.4 High-speed sequential photography of cavitation dynamics
4.3.3.5 Laser scattering technique
4.3.3.6 Synchrotron X-ray imaging technique
4.3.3.7 MRI techniques
5 Aims of the thesis
6 Materials and methods
6.1 Materials
6.1.1 Devices
6.1.2 Chemicals and reagents
6.1.3 Consumables
6.1.4 Human cancer cell lines
6.2 Methods
6.2.1 Composition of the FUS system for in vitro treatment of cells
6.2.2 Physical characterization of the in vitro FUS system
6.2.2.1 Setup of fiber-optic hydrophone system to characterize the FUS apparatus
6.2.2.2 Data analysis in MATLAB
6.2.3 Cavitation measurement with FOH system
6.2.4 Chemical cavitation measurement with terephthalic acid (TA)
6.2.5 Culture of human cancer cell lines
6.2.6 FUS treatment of cancer cells
6.2.7 Water bath hyperthermia treatment
6.2.8 Radiation therapy in vitro
6.2.9 The protocol of FUS\FUS-Cav combined with RT or HT
6.2.10 Evaluation of cell ability to reproduce with clonogenic assay
6.2.11 Measurement of cellular metabolic activity with WST-1 assay
6.2.12 Evaluation of cell invasion ability with Transwell® assay
6.2.13 Detection of sonoporation by cell staining with propidium iodide (PI)
6.2.14 Detection of SRD5A as a therapeutic target for prostate cancer with immunofluorescence microscopy
6.2.15 Quantification for the reduction of SRD5A proteins with flow cytometry
6.2.16 Testosterone treatment
6.2.17 FUS/FUS-Cav combined with testosterone treatment
7 Results
7.1 Physical characterization of the in vitro FUS system
7.2 Cavitation occurs at a certain level of acoustic intensity
7.2.1 Characteristics of ultrasonic spectrograms
7.2.2 Cavitation dose depends on the acoustic intensity
7.3 FUS/FUS-Cav supports RT to reduce the long-term clonogenic survival of cancer cells
7.4 FUS/FUS-Cav increases the effects of HT by reducing the long-term clonogenic survival of cancer cells
7.5 FUS/FUS-Cav enhances the suppressive effects of RT in short-term cell potential to invade and metabolic activity of prostate cancer cells
7.6 FUS/FUS-Cav supports HT to diminish the short-term cell potential to invade and metabolic activity of prostate cancer cells
7.7 FUS-Cav treatment immediately induces sonoporation effects in PC-3 and FaDu cells
7.8 FUS/FUS-Cav enhances the effects of HT by inhibiting the SRD5A protein level in prostate cancer cell lines
7.9 FUS-Cav enhances the effects of the testosterone treatment by reducing the long-term cell metabolic activity of androgen-dependent prostate cancer cell line
8 Discussion
8.1 Cavitation measurement in a defined 96-well plate by PCD technique and sonochemistry method
8.2 Short-term and long-term additive effects of FUS-Cav to RT or HT
8.3 Inhibitory effects of FUS-cav in the potential of prostate cancer cells to invade
8.4 The reduction of the long-term metabolic activity of androgen-dependent prostate cancer cells by the combination treatment of FUS-Cav and testosterone
9 Conclusion
10 References
11 Appendix
11.1 Erklärung über die eigenständige Abfassung der Arbeit
11.2 List of figures
11.3 List of tables
11.4 Curriculum vitae
11.5 Acknowledgment
