In Vitro Radioenhancement using Ultrasound Stimulated Microbubbles
Creators: name: McCorkell, Giulia
Publisher: name: RMIT University
Year: 2025
Subjects: classificationCode: 510502
subject: Medical physics

description: Radiation Therapy (RT) is one of the core treatment options offered to patients with cancer. This treatment delivers therapeutic doses of ionising radiation to clinical targets in efforts to regain control over the hyperproliferative cells present in the disease, and/or, to irradicate all tumour cells entirely. However, as radiation is non-discriminatory in its action, a fine balance needs to be struck in leveraging radiation-induced cell killing of tumour cells whilst ensuring any unaffected normal/healthy cells in the vicinity do not suffer the same fate, giving rise to compromised organ and tissue functions and diminished quality of life for patients. There have been many attempts to enhance this balance using techniques that target the physical, chemical and biological phases of radiation action. One such agent that may have potential to enhance all three phases is the microbubble, which when stimulated by a suitably optimised ultrasound field, has been shown to enhance the uptake of other therapeutic agents commonly used in cancer treatment.

An extensive search of the literature showed that ultrasound stimulated microbubbles (USMB) could enhance the effects of radiation by acting as a biophysical disruptor of tumour vasculature, or by directly sensitising individual tumour cells to the effects of radiation. However, early in vitro research focussing on this second mechanism was plagued by inconsistencies in experimental conditions. Conflicting results were observed when cells were treated using low energy (kilovoltage, kV) compared to high energy (Megavoltage, MV) x-rays, and when cells were exposed in an adherent compared to a suspended cell culture state. Additionally, no research had been conducted in a lung cancer cell line, or a metastatic cell line, which is significant given the poor survival outcomes in primary lung cancer and the challenges of delivering radiotherapy to the lungs, a common site of metastatic disease. 

This thesis therefore aimed to address these gaps by investigating the radiosensitising potential of USMB using both a primary lung cancer cell line (NCI-H727) as well as a secondary thyroid metastasis in the lung (FTC-238) and comparing results across both kV and MV x-ray energies, and the suspension and adherent cell culture state.

Following a preliminary investigation to determine suitable microbubble concentrations and ultrasound sonication parameters, along with cell viability assay optimisation, both NCI-H727 and FTC-238 cells were treated using a combination of 1.6% v/v microbubbles, ~90 s of 2 MHz ultrasound (mechanical index=0.8); and 0-6 Gy of kV or MV x-rays. No difference in effect was observed using kV compared to MV radiation. Cells treated with the combination of USMB and radiation showed decreased normalised survival compared to those treated with radiation only. However, Analysis of Variance found this to be statistically significant only in the NCI-H727 cells, highlighting the influence of cell type on results. Even within the same cell type, varying results were achieved in NCI-H727 cells when in a suspended or adherent culture state. The effect of USMB-alone on these primary cell lines was more pronounced when cells were in a suspended cell culture state compared to the adherent culture state, with no such differences observed for the metastatic cell line. Given a feature of metastatic cell lines is their capacity to travel in the suspended cell culture state to a secondary site, this similarity in results is not unexpected. 

In light of these results observed with NCI-H727 cells, further investigation into the sonoporation effects elicited by USMB for these cells in a suspended versus adherent culture state was undertaken. Results from a Neutral Red Release (NRR) assay suggested that higher concentrations of USMB were required to compromise membrane integrity in adherent cells compared to suspended cells, highlighting another variable for consideration in this research area. However, the capacity to leverage these membrane effects for increased uptake of 1mM of gold nanoparticles appeared to be diminished. No discernible differences were observed in NCI-H727 cells in suspension treated with combinations of RT-alone, USMB-alone, gold nanoparticles-alone, or the presence of all three agents combined.

This research highlights the need to fully inform the experimental conditions used to investigate individual cell lines and cell culture states in preclinical in vitro studies. This is crucial to avoid misleading results that could significantly impact subsequent research and clinical applications. The insights gained from this thesis highlight the necessity of a systematic approach to parameter optimisation, particularly for USMB treatments alongside other radiosensitising agents, due to the number of individual treatment parameters requiring consideration.