AbstractIntroduction: Advanced radiotherapy techniques such as intensity modulated radiation therapy (IMRT) achieve highly conformal dose distributions within target tumour volumes. However, sequential delivery of multiple spatially and temporally modulated radiation fields have been shown in vitro and in vivo to impact radiobiological response in low dose (out-offield) regions. The efficacy of advanced radiotherapy is further limited by regions with low oxygen concentrations, commonly known as hypoxia. Previous studies have validated that hypoxic tumours result in genetic alterations which can allow a survival advantage and increase the tumorigenic properties of cancer cells. Additional understanding of the signalling responses in hypoxic microenvironments is therefore needed to optimise radiation treatment strategies in the future.
Aim: This study aimed to identify the signalling responses in low dose out-offield regions under oxic and hypoxic conditions at the cell survival, DNA damage, and gene expression level.
Method: Hypoxia was induced by incubating a range of cells with varying radiosensitivities at 95% N2; 5% CO2 prior to irradiation. A modulated beam was created by shielding 50% of the cells using a low melting point alloy. Out-of-field responses to modulated irradiation were determined by dose response survival assays and assessment of double strand breaks by 53BP1 fluorescent staining. Clariom-D microarrays were used to achieve extensive gene profiling analysis of localised high-risk PC3 prostate cancer cells following modulated beam exposures under hypoxic conditions. Gene expression levels were predicted by Transcriptome Analysis Console (TAC).
Results: We report a differential response of cells placed in- and out-of-field, impacted by oxygen status, time, and intercellular communication. Under both oxic and hypoxic conditions, significant increases in cell survival were observed in-field, while significant decreases in survival were observed out-of-field (p < 0.05). This was mirrored at the DNA damage level where decreases in foci counts were observed in-field while increases were observed out-of-field. The in-field response of MDA231 cells showed no significant time dependency up to 24 hours post-irradiation, however out-offield survival decreased significantly during the first 6 hours after irradiation. Whilst in-field responses were shown to be oxygen dependent, out-of-field effects were observed independent of oxygen with similar or greater cell killing under hypoxic conditions. At both the cell survival and DNA damage level, physical inhibition of intercellular communication was shown not only to abrogate out-of-field responses to that observed under their uniform counterparts in all oxygen concentrations investigated, but also restore the in-field response levels comparable to their uniform controls. Gene expression analysis confirmed that a hypoxic response was produced in PC3 cells as upregulation of common hypoxia signatures such as ADM, NDRG1, and BNIP3 were observed. Although trends in the microarray analysis mimicked that predicted by TAC, the magnitude of the genetic alterations in KCNMA1, KDM1A, PNISR, RBM6, and KMT2A out-of-field could not be replicated by RT-qPCR on this occasion.
Conclusion: Overall, the data presented here provides evidence for out-of-field cells relying on intercellular signalling generated by irradiated cells under hypoxic conditions, and highlights the need for further refinement of established radiobiological models for future applications in advanced radiotherapies. Determining gene expression signatures that can regulate out-of-field responses during modulated radiotherapy will also be of benefit to patients with hypoxic tumours.
|Date of Award||Jul 2020|
|Sponsors||Northern Ireland Department for the Economy|
|Supervisor||Kevin Prise (Supervisor), Karl Butterworth (Supervisor) & Alan Hounsell (Supervisor)|