Radiotherapy is widely used to treat patients with mesothelioma47. However, the in vitro effects of photon radiation on cellular damage of mesothelioma cell lines, and comparison between effects in mouse and human cell lines, have not previously been investigated intensively. Here, we employed a flow cytometry-based approach and clonogenic survival assays to examine the effects of photon radiation on the cellular damage of two well-characterized murine and three human mesothelioma cell lines. Our investigation assessed DNA damage, cell cycle, cell proliferation and survival, and apoptosis, which are the key indicators of cellular damage50.
As would be expected, we found that γ-H2AX increased with radiation dose in our studied cell lines, confirming a dose-response relationship. Maximum damage was induced by 8 Gy – above which, γ-H2AX generation saturated. This finding was in line with previous studies examining the phosphorylated histone H2AX by flow cytometry following exposure to ionizing radiation in human endothelial cells51 and several human cancer cell lines52. Gamma-H2AX was strongly induced in all murine and human cell lines at 2 Gy compared to 0 Gy at 1 hr post radiation, except for the MC cell line. MC cells are very slow-growing compared to the other murine and human cell lines in our study, which may explain the low level of γ-H2AX at 2 Gy - since slow-growing cells are less sensitive to radiation 53 than rapidly proliferating cells54, 55. This implies that MC cells are resistant to DNA damage at low radiation doses but radiosensitive at higher doses, as robust γ-H2AX levels are detected at 8 Gy.
Again, as expected, we showed that higher-dose radiation arrested cells at the G2/M phase, agreeing with previous studies demonstrating G2/M arrest by radiation in leukaemia and glioblastoma cell lines56-58. In these studies, U87 glioblastoma cells were treated with 0-8 Gy, and G2/M arrest was induced by 4 Gy and 8 Gy at 12 hrs and 24 hrs post-irradiation56, whilst doses of 1.5 Gy-6 Gy arrested leukaemia cells at G2/M at 8-12 hrs post-radiation. However, the arrest was reversible as cells re-entered the G0/G1 phase within 12 and 24 hrs58. Although we also observed G2/M arrest in our study, this did not occur at doses of 4 Gy or under, nor less than 24 hr post radiation, which differs from the aforementioned studies in U87 glioblastoma and leukaemia56, 58. Two possible reasons driving the differences may have resulted from the limited dose ranges in our study. Firstly, we treated the AB1 cell line with the full range of 1-32 Gy, whilst other cells lines were treated with only 2 Gy, 8 Gy and 16 Gy. Therefore, we could only assess the cell cycle phase distribution between larger dose increments, which allowed less accurate pinpointing of the exact dose at which G2/M phase arrest occurred. A second reason may have been the wider gap between each time-point. It is feasible that the G2/M phase may have been arrested at a time-point greater than 6 hrs and less than 24 hrs, not investigated in our study. However, a study led by Murad et al36 showed that 10 Gy radiation arrested T98G glioblastoma cells in G2/M from 24 hrs, similar to our finding in murine mesothelioma cell lines. Interestingly, G2/M phase arrest was not observed with human cell lines at 2, 8 or 16 Gy, although there was disappearance of the G0/G1 population (BYE, JU & MC) with 8 Gy and 16 Gy at 72 hrs. This was in line with a previous study in the U87-sph glioblastoma cell line demonstrating no cell cycle arrest regardless of dose 56. The possible mechanism may be largely related to the protein Cdc25 (a protein inducing radio-resistance) and the ATM/Chk1 signalling pathway. Previous studies have shown that the activation of the ATM/Chk1 pathway could inhibit Cdc25c activation, thereby preventing cell cycle arrest at the G2/M phase 56, 59-61. A recent study reported radiation-induced expression of an autophagy related protein, BECN1, a key mediator of the G2/M transition. Disruption of BECN1 using autophagy inhibitor 3-methyladenine (3-MA) sensitized cells to G2/M arrest post irradiation through the prevention of phosphorylation of Cdc25c 62. Therefore, γ-radiation may have activated ATM/Chk1 and upregulated BECN1 in our study, which interfered with Cdc25c in human cell lines, leading to unobservable G2/M arrest. Further experiments investigating ATM/Chk1, Cdc25 and BECN1 are required to validate this hypothesis. Converse to reports in the literature63, 64, we did not observe G0/G1 arrests at the proposed time-points in our study because the proportion of G0/G1 cells were not higher than untreated group. Cell cycle arrest following radiation, especially in G2/M, gives sufficient time for cells to repair DNA. In our present study, we found a dose of 8 Gy temporarily arrested the AB1 cells at G2/M at 24 hr post radiation. However, cells returned to normal level with increased G0/G1 peak at 48 hrs and 72 hrs. Doses greater than 8 Gy completely arrested cells at G2/M, suggesting the DNA damage induced by dose ≥ 8 Gy is too extensive and hence permanently arrested cells at G2/M.
We have also characterised DNA repair kinetics by modelling the decrease in γ-H2AX over time following photon irradiation. Gamma-H2AX is a DNA repair protein attracted to sites of DNA damage as the cells are arresting, thereby providing sufficient time for cellular repair50. Previous studies showed γ-H2AX is a reliable marker to measure DNA double-stranded break repair 65-68. We have demonstrated that murine and human mesothelioma cell lines repaired DNA effectively at lower radiation doses. However, the level of γ-H2AX remains higher after receiving 8 Gy compared to 1 Gy or 2 Gy. Temporal differences in γ-H2AX fluorescence intensity, between low and high radiation doses, may therefore infer different cellular responses of mesothelioma cell lines to low and high dose radiation in our study. One explanation may be development of radio-resistance by cells following low dose radiotherapy, because they can repair the DNA more effectively than at high doses and this was also described by Soto et al 69 in their review. Alternatively, differences could be accounted for by less efficient DNA repair at higher doses; prolonged elevated expression of γ-H2AX, when compared with lower doses, indicated excess acute and chronic toxicity induced by radiation, thereby impairing DNA repair machinery 68. Higher dose radiotherapy may severely affect non-homologous end joining (NHEJ) (a key pathway of double strand break repair) via the suppression of NHEJ proteins such as XRCC4, DNA Lig3 & Lig4, which are critical to forming a complex to ligate the broken ends during the NHEJ process. Decreases in XRCC4, DNA Lig3 & 4 are associated with the decline in NHEJ efficiency and fidelity 70 and impaired NEHJ has been reported following radiation in previous studies using myeloma cell lines 71. Therefore, the NEHJ pathway may have been badly disrupted in our human cells following higher doses of γ-irradiation. However, due to the technical difficulties with multi-fraction irradiation in-vitro, it is still difficult to conclude whether this phenomenon will occur in in-vivo - or indeed in the clinic, where fractionated radiotherapy is normally utilized; further research is therefore warranted. Moreover, by fitting multiple doses in a joint model of all cell lines, DNA repair was similar in all the studied cell lines - suggesting that this repair pattern occurs in both animal and human cells.
The most important measurable outcomes after radiation treatment in our study were cell proliferation and cell death (apoptosis), and cell survival as assessed by clonogenic survival assay. Our finding demonstrated that whilst lower dose radiation (1-4 Gy) did not inhibit cell proliferation, high doses (16-32 Gy) reduced proliferation almost completely from 24 hrs in murine cells, is similar to that from Yao et al72 demonstrating a significant decrease in cell proliferation with 20 Gy at 24 hrs. However, another study by Si Jie et al73 showed low dose radiation of 50, 75 and 100 mGy significantly inhibited cell proliferation of PC-3 cells compared to sham-controls. In that study, low dose radiation arrested cells at S and G2/M - differing from our findings since we did not observe G2/M arrest or inhibition of cell proliferation at low doses; this can be explained by different cell lines having differing sensitivities to radiation and this is also likely to mainly be a factor resulting from different proliferation rates between cell lines, although there are also likely to be other more nuanced factors at play such as the ability to repair DNA damage. Cell proliferation at 72 hrs post-irradiation showed the proliferation rate to be similar amongst all studied cell lines, as the slopes between cell and time was not significant. It is interesting to note that sham-irradiated MC human cells took up only 15 % BrdU in one hour pulse labelling compared to approximately 45 % BrdU uptake for murine cell lines. Therefore, if we examined cell proliferation at specific low doses i.e. 2 Gy at earlier time point post radiation (e.g. one or six hours), we would see the difference between MC to other cell lines. However, that might not suggest the inhibitory effect of radiation, but would be the slow growing nature of the MC cell line that may drive the difference. It is interesting to note that decreased cell proliferation was clearly seen in all human cell lines from 8 Gy at 24, 48 and 72 hours, even though our data did not show significant cell cycle arrest in G2/M; albeit reduction of the G0/G1 population in human cell lines 48-72 hours following doses ≥8 Gy were still observed. It may be that G2/M arrest peaks were not observed within the confines of the timepoints selected in our assay, for example occurring between 6-24 hours. One further explanation is that these human cell lines, (particularly BYE, which did not divide following higher dose radiation e.g., at 48 & 72 hours) may have undergone intrinsic apoptosis in a P53 dependent manner and did not require prior cell cycle arrest 74, 75 . P53 activated by radiotherapy can directly trigger the release of pro-apoptotic genes such as p53‐upregulated modulator of apoptosis (PUMA) and BCL2‐associated X protein (BAX), which subsequently activate caspase-7, 9 & 3 to induce intrinsic apoptosis 14. Alternatively, human cell lines that ceased proliferation may have died through other cell death pathways not investigated in this study, such as necrosis, ecroptosis and ferroptosis autophagy, independently of cell cycle arrest 76, 77. Further investigation is required to clarify the actual death mechanism. In addition to cell proliferation, cell survival probability decreased with increasing dose (1-8 Gy), with 8 Gy completely inhibiting colony formation in all studied cell lines. Interestingly, the α/β ratio of human cell lines was smaller than murine cell lines - indicating that human cells in our study are more sensitive to a change of dose per fraction than murine cell lines. Our findings showed a smaller α/β ratio relative to that reported in a previous study78, suggesting heterogeneity of response to radiation in human mesothelioma cell lines.
We found the AB1 and BYE human mesothelioma cell lines displayed more apoptosis between 8 Gy, 16 Gy and 32 Gy, with a time-dependence. Our finding is consistent with previous studies demonstrating the apoptotic sensitivity of colorectal cancer lines and human lung cancer79, 80. However, far lower levels of apoptosis were detected in other cell lines including AE17, JU and MC. Previously, several glioblastoma cell lines have shown no apoptosis after treatment with 20 Gy 72. Interestingly, a previous study in H661 and H520 non-small cell lung cancer lines observed greater apoptosis after radiation at 144 hrs81. The latest time-point in our study is 72 hrs, hence it is possible that we might have seen more apoptosis in AE17, JU, and MC if we had extended the observation time. To induce apoptosis, the phosphorylation of P53 and activation of caspase 3 and 7 after radiation are required14. Therefore, these signals might not yet have been fully activated in AE17, JU and MC cell lines in our study. Future studies may consider investigating other regulators of apoptosis such as cleaved caspase 3 and 7 or upregulation of Bax/Bcl-2 to validate our current finding14, 80. BRCA1 associated protein 1 gene (BAP1) mutations have been linked to early onset of MPM, and resistance to chemotherapy 82. In renal cell carcinoma, knockout of BAP-1 resulted in sensitization to apoptosis following γ-irradiation 83. The human cell lines used in our study were variably positive (MC line) or negative (JU and BYE) for BAP-1, but this did not seem to be indicative of responses to irradiation in our study.
In conclusion, in murine and human mesothelioma cell lines, radiation was shown to induce DNA damage and G2/M arrest, inhibit cell proliferation and cause apoptosis in a defined and quantifiable manner. Compared with lower doses (1-2 Gy), a dose of 8 Gy demonstrated significantly increased damaging effects. Considering DNA damage repair and the apoptotic mechanism of cells at the cellular level following different doses of irradiation, it can be concluded that an approximate dose of 8 Gy may be an optimal hypo-fractionated dose to induce cellular damages in our studied cell lines and may be able to generate immunogenic cell death to activate local and systemic immune response for future radio-immunotherapy study in a mouse model. However, since only epithelioid human mesothelioma cell lines were studied, our observation does not necessarily apply for other non-epithelioid mesothelioma, which are typically more resistant to radiation. Additionally, we have identified a 72 hour post radiation time point at which increased inhibition of cell proliferation was consistently observed across several cell lines; this may be an ideal time for combination with immunotherapies, e.g. anti-PD-L1, since response rates in mesothelioma are still generally low. Cytotoxic chemotherapy has been shown to improve response rates to anti-PD-L1 in mesothelioma84; radiotherapy may have the potential to perform a similar role, minus the systemic toxicities observed with chemotherapy. The similarity between mouse and human cell lines, particularly cell proliferation and cell survival following irradiation with 8 Gy and above, suggests mouse cell lines provide a good model that is broadly analogous to human cell lines, although human cell lines exhibit variable responsiveness to radiation, as seen in clinical practice. However, it is important to note that significant DNA damage induced by our prescribed radiotherapy dose (e.g., 8 Gy) may not be similarly observed with in-vivo mesothelioma tumours. There may be different effects when cell are irradiated in the context of intra-tumoral stromal cells such as cancer-associated fibroblasts, endothelial cells, suppressive immune cells, regulatory cytokines, the extracellular matrix, and regions of hypoxia. Stromal cells and hypoxia may drive the resistance of mesothelioma to radiotherapy compared with our current in-vitro study, where only single cell suspensions were irradiated.
Given that the recent approval of nivolumab plus ipilimumab as first line treatment for mesothelioma significantly improved progression free survival only in non-epithelioid patients5, improved therapies are still required. Radiotherapy is a potential partner for immune checkpoint blockade, through induction of immunogenic cell death - potentially converting tumours from being immunologically “cold” to “hot”, critical for immunotherapy responses85. The work we have described here represents an initial step towards developing an in vivo preclinical model system, in which robust studies into the optimal ways to combine radiation and immunotherapy can be run. We have shown our murine mesothelioma cell lines to be broadly similar to their human equivalents in regard to their responses to increasing photon radiation dose - mainly expressed as DNA damage, decreased cell proliferation and survival with a dose of 6 or 8 Gy. These clinically relevant hypo-fractionated doses may now be taken forward to preclinical radio-immunotherapy studies, with several further aspects now requiring investigation prior to generation of a fully optimised murine model; these will include scheduling of multiple radiotherapy fractions, the interaction of treatment with the full tumour microenvironment rather than just cancer cells (particularly in the context of an intact host immune system) and, ultimately, how best to combine with immune checkpoint inhibitors in an effort to unlock their full potential.
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