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National Centre for Asbestos Related Diseases, Institute for Respiratory Health, Perth, AustraliaMedical School, University of Western Australia, Perth, Australia
National Centre for Asbestos Related Diseases, Institute for Respiratory Health, Perth, AustraliaSchool of Physics, Mathematics and Computing, University of Western Australia, Perth, Australia
National Centre for Asbestos Related Diseases, Institute for Respiratory Health, Perth, AustraliaSchool of Physics, Mathematics and Computing, University of Western Australia, Perth, Australia
School of Physics, Mathematics and Computing, University of Western Australia, Perth, AustraliaDepartment of Radiation Oncology, Sir Charles Gairdner Hospital, Perth, Australia
School of Physics, Mathematics and Computing, University of Western Australia, Perth, AustraliaDepartment of Radiation Oncology, Sir Charles Gairdner Hospital, Perth, Australia5D Clinics, Claremont, Australia
National Centre for Asbestos Related Diseases, Institute for Respiratory Health, Perth, AustraliaMedical School, University of Western Australia, Perth, AustraliaDepartment of Medical Oncology, Sir Charles Gairdner Hospital, Perth, Australia
National Centre for Asbestos Related Diseases, Institute for Respiratory Health, Perth, AustraliaSchool of Biomedical Sciences, University of Western Australia, Perth, Australia
To characterise the cellular responses of murine and human mesothelioma cell lines to different doses of photon radiation with a long-term aim of optimising a clinically relevant in vivo model in which to study the interaction of radiotherapy and immunotherapy combinations.
Materials and methods
Two murine mesothelioma cell lines (AB1 and AE17) and three human cell lines (BYE, MC, JU) were utilized in the study. Cells were treated with increasing doses of photon radiation. DNA damage, DNA repair, cell proliferation and apoptosis at different time-point post irradiation were quantified by flow cytometry and cell survival probability was examined using clonogenic survival assay.
Results
DNA damage increased with escalating dose in all cell lines. Evident G2/M arrest and reduced cell proliferation were observed following irradiation with 8 Gy. DNA repair was uniformly less efficient at higher compared with lower radiation-fraction doses. Apoptosis dose response varied between cell lines, with greater apoptosis observed at 16 Gy with human BYE and murine AB1 cell lines, but less for other studied cell lines regardless of dose and time. The α/β ratio from the cell survival fraction of human mesothelioma cell lines is smaller than from murine ones, suggesting human cell lines in our study are more sensitive to a change of dose per fraction than murine mesothelioma cell lines. However, in all studied cell lines, colony formation is completely inhibited at 8 Gy.
Conclusion
8 Gy appears to be an appropriate threshold dose for hypo-fractionated radiotherapy. However, the radiotherapy doses between 4 and 8 Gy remain to be systematically analysed. These observations provide an accurate picture of the in vitro response of mesothelioma cell lines to photon irradiation and characterise the heterogeneity between human and murine cell lines. This information will guide in vivo experiments and the strengths and limitations of extrapolation from murine experimentation to potential human translation.
Introduction
Malignant Pleural Mesothelioma (MPM) is an aggressive cancer of the pleura, predominantly arising from asbestos exposure1. Patients with MPM have an extremely poor prognosis, with a median survival of nine to twelve months2, 3. From 2003 until very recently, the standard first-line therapy for mesothelioma was cisplatin and pemetrexed chemotherapy; however, this combination only modestly improves patient survival4. Recently, immuno-oncology and anti-angiogenic agents have begun to challenge this standard of care. In 2021, results from the CheckMate 743 trial showed a benefit from treatment with the immune checkpoint inhibitors ipilimumab and nivolumab over combination chemotherapy, particularly in patients with non-epithelioid disease5. The vascular endothelial growth factor (VEGF) inhibitor bevacizumab showed increased survival of over two months when added to chemotherapy, but has not been adopted into standard of care treatment6. Localised radiotherapy is used for multiple purposes in treating mesothelioma - ranging from treatment of procedure tract metastases, adjuvant hemi-thoracic radiation as part of aggressive multimodality therapy, and as palliative therapy to individual lesions7. However, high dose radiotherapy for local control can have toxicities, and does not palliate symptoms in a wholly effective manner 8-10.
As demonstrated in preclinical models in other cancer types, radiotherapy can be combined with immune checkpoint blockade to enhance treatment efficacy - 11-13 due to the ability of radiotherapy to generate immunogenic cell death 14, activate the cGAS-Sting pathway (type I interferon) 15, increase intra-tumoral cytotoxic T lymphocytes (CD8+) 16, 17, decrease myeloid derived suppressor cells 18 and normalize tumour blood vessel 19-23. Synergy, increased survival and even abscopal effects from combinations of radiotherapy and immunotherapy (using agents such as anti-PD1/PD-L1, anti-CTLA-4) have been reported in several mouse studies including in breast cancer 12, intracranial glioma 13, colon adenocarcinoma 18, Kras-mutant lung cancer 24, and lymphoma plus Lewis lung carcinoma 25. With these encouraging outcomes from pre-clinical studies, many clinical trials are currently investigating radio-immunotherapy combinations in different cancer types - with promising preliminary outcomes.
In a recent phase II clinical trial in early-stage non-small cell lung cancer, patients (n=60) were randomly assigned to either durvalumab alone or durvalumab plus SBRT (8 Gy × 3); pathological responses were 53.3 (95% CI: 34.3-71.7) versus 6.7% (95% CI: 0.8-22.1) respectively, and several complete pathological responses were observed in the combination group 26. In a retrospective study examining the efficacy of hypo-fractionated radiotherapy plus ipilimumab (Ipi-RT) compared with ipilimumab alone in melanoma brain metastases, an increased median overall survival of 19 months was observed in the Ipi-RT arm compared with 10 months in the ipilimumab arm (p=0.001). Ipi-RT had a significantly higher complete response rate than ipilimumab alone (25 % vs. 6.5 %). Several other studies have reported an abscopal response in advanced melanoma patients following radiotherapy plus ipilimumab 27, 28 and increased survival following radiotherapy and immunotherapy 29-31.
For mesothelioma, however, the benefits of radiotherapy and immunotherapy are currently under-studied - in both the clinical and preclinical settings. To date, one mouse study using the AB12 mesothelioma model to examine the effects of radio-immunotherapy demonstrated an increased T cell influx into primary and secondary tumours following hypo-fractionated radiotherapy, with a highly activated CD8+ T cell phenotype observed following the addition of anti-CTLA-4. Delayed tumour growth in the secondary tumour following irradiation to the primary tumour compared with the sham-radiotherapy group was also observed. The authors also reported increased expression of genes such as interferon-γ, granzyme B, ICOS, CD80 & CD86 following radiotherapy plus anti-CTLA-4 32.
Due to the paucity of data from the clinic in combining radiotherapy and immunotherapy in mesothelioma, it is important to study this approach in a robust animal model33, 34. However, to understand the utility of the animal model, it is vital to first characterise the radiation-response of both murine and human mesothelioma cell lines because this work may enable us to assess the suitability of mouse cell lines and identify the approximate radiation dose to combine with immunotherapy for mesothelioma. As of 2020, according to the National Cancer Institute's clinical trial and evaluation program, a dose-fractionation schedule (8 Gy × 3) is considered a standard fractionation for combination with immunotherapy 35. However, each cancer type is different in their biology and microenvironment. Therefore, characterizing in-vitro radiation response may help guide us to optimally select doses to combine with immunotherapy in animal models.
The effects of radiotherapy are exerted primarily through damage to cellular DNA36. Ordinarily, DNA damage will activate DNA repair machinery and arrest the cell cycle to allow repairs to be carried out - therefore restoring genome integrity37. If cells are capable of repairing the DNA damage, they will resume normal cycling. However, if the damage is too extensive, cells may progress to cell death, or senescence38. Clinical reports suggest that mesothelioma is relatively resistant to radiotherapy39. However, in vitro studies revealed human mesothelioma cell lines were sensitive to conventional doses of radiation40, 41. Given the complex biological effects of radiotherapy delivered with variable dose and fractionation schedules, our present study investigated cellular damage in multiple human and murine mesothelioma cell lines following radiation by incorporating several outcome measures including: DNA damage, cell cycle, cell proliferation, survival, and apoptosis at varying doses of photon radiation. Here, we characterise the in vitro biological responses to different doses of radiation and identify the optimal radiation dose capable of inducing anti-tumour immune responses (particularly via DNA damage and clonogenic survival assay with characterization of the survival curve via the α/β ratio) for in vivo radio-immunotherapy studies. Finally, we compare the biological responses of murine and human mesothelioma cell lines to radiation thereby informing us of the suitability of the murine cell lines as preclinical models and selecting the optimal dose for future studies.
Materials and methods
Cell lines
Murine mesothelioma cell lines (AB1 and AE17) were generated as previously described42, 43. They were grown and maintained in complete RPMI medium containing 2mM glutamine, 20mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (Sigma Aldrich, Australia), 100U/mL Benzylpenicillin, 500 mg/mL Gentamicin, 0.05 mM 2-Mercapteothanol,10% neonatal calf serum (NCS). Human mesothelioma cell lines JU77 (JU), MC_P5001 (MC) and BYE10412 (BYE) were generated from patient pleural effusions and sourced from XXXXXXXX. The cell lines were maintained in RPMI medium containing 2mM glutamine, 20mM HEPES, 0.05 mM 2-Mercapteothanol, 5% fetal calf serum (FCS). The characteristics of the cell lines are summarized in supplementary table 1.
Irradiation
Exponentially-growing mesothelioma cell lines were irradiated in 50 mL tubes (0.5 × 106 cells/mL) at room temperature, using a Gammacell irradiator 3000 SN 211 (Nordion, Ottawa, Canada) at XXXXXX at various doses as specified. The dose rate from the 137Cs source was 3.87 Gy / min. The unit was calibrated according to National Institute of Standards and Technology (NIST) standards.
Cell proliferation
Assessment of cell proliferation after photon radiation exposure was done by pulse labelling the irradiated cells with 5-bromo-2′-deoxyuridine (BrdU), followed by quantification of the proportion of BrdU+ cells by flow cytometry. Briefly, cells were centrifuged (300 RCF, 3 minutes) to remove the irradiated media immediately after radiation. Supernatants were discarded and cell pellets were resuspended in fresh RPMI medium; cells (0.5 × 106) were transferred to a T175 flask and incubated at 37°C in a humidified 5% CO2 atmosphere. BrdU (20 µg) was added to the appropriate flask for the last hour of incubation, and cells were harvested at either 1 hr, 6 hrs, 24 hrs, 48 hrs, or 72 hrs following radiation exposure.
Cell fixation and storage
One hour after exposure, BrdU pulse labelling was completed at each time-point. Cell culture medium was transferred from flasks to 50 mL tubes. The flasks were washed with 10 mL 1X PBS and 2 ml warm trypsin was added to dislodge adherent cells, which were incubated for two minutes at 37°C in a humidified 5% CO2 atmosphere. The cells were washed by resuspension in 10 mL culture media to deactivate trypsin and centrifuged (300 RCF, three minutes). Supernatants were discarded, and cell pellets were resuspended in 1 ml culture media. Cell counts were performed using trypan blue exclusion; 0.5 × 106 cells were collected and washed twice with 2 mL 1X PBS and centrifuged (300 RCF, three minutes). Supernatants were discarded and cell pellets were fixed with 100 µl BD Cytofix/Cytoperm Fixation and Permeabilization solution for 20 minutes on ice. After cell fixation, cells were washed with staining buffer (1X PBS, 2% NCS), transferred into 1 mL freezing medium (10% DMSO, 90% NCS) and stored at -80°C until staining.
Flow cytometry
Flow cytometry staining was performed using ‘apoptosis, DNA damage and cell proliferation’ kits (BD Biosciences, NJ, USA) according to manufacturer's instructions. Briefly, frozen cells from -80°C were thawed at room temperature and transferred to corresponding labelled staining tubes. Cells were washed twice with 2 mL staining buffer and centrifuged (300 RCF, three minutes). Supernatants were discarded and cell pellets were fixed with 100 µL BD Cytofix/Cytoperm™Fixation and Permeabilization solution for 20 minutes on ice. After fixation, cells were washed with 500 µL 1 X PermWash from BD cytofix / cytoperm kit by centrifugation (300 RCF, three minutes). Supernatants were discarded and cell pellets were treated with 100 µL deoxyribonuclease (DNase) (300 µg/mL) in sterile 1X PBS to expose BrdU epitopes, and incubated for one hour at 37 ˚C in a humidified 5% CO2 atmosphere. After DNase treatment, the cells were washed with 500 µL 1X PermWash. Supernatants were discarded and cell pellets were stained using 20 µL antibody cocktail, mouse anti-BrdU-PerCP-C5.5 (Cat # 51-9007682, Clone: 34D, dilution: 1/40), mouse anti-γ-H2AX-AF467 (Cat # 560447, Clone, N1-431, dilution: 1/10), mouse anti-cleaved PARP-PE (Cat # 51-9007684, Clone:F21-852, dilution: 1/40) diluted in staining buffer, and incubated in the dark for 20 minutes at room temperature. After staining, cells were washed with 500 µL 1X PermWash by centrifugation (300 RCF, three minutes). Cell pellets were then stained with DAPI (Cat # 564907, dilution: 1/1000) for 15 minutes. After staining, cells were washed with 1 mL staining buffer and resuspended in 200 µL staining buffer. All staining tubes were acquired on BD LSRFortessa™ (BD Biosciences, USA), with 100,000 events were recorded per sample. Data were analysed using Flowjo_V10 (Treestar, OR, USA).
Clonogenic Survival Assay
Exponentially growing cells were irradiated (1 – 8 Gy) in 50 mL tubes, then seeded into T25 flasks in triplicate. Flasks were incubated at 37°C in a humidified 5% CO2 atmosphere and media changed every three days. Fifteen days after irradiation, cells were fixed with 100 % methanol for five minutes and stained with 0.01% crystal violet. Colonies comprising 50 cells, or more were counted using ColCountTM (Oxford Optronix, UK). According to the linear quadratic model, the survival fraction (SF) is mathematically related to dose, , using the relationship:
where and are constants for a particular tumour or normal tissue, that represent the linear and quadratic components of cell kill respectively. The / ratio is indicative of the sensitivity of that cell line to changes in dose-per-fraction 44. The conventional dose-per-fraction in cancer treatment is 2 Gy per day. Hypofractionation describes treatment delivered at a larger than 2 Gy per day schedule. Tumours with a small / ratio (for example an / ratio 2) are more sensitive to larger doses per fraction (hypofractionation) and are killed more effectively, and so total dose maybe lowered if hypofractionation is used. Tumours with a high / ratio (for example an / ratio of 10) indicates cells are relatively less sensitive to dose per fraction, and so need a higher total dose instead to achieve effective tumour kill45. Therefore, determining the / ratio of a particular mesothelioma cell line is vitally important for future studies where radiotherapy is being used to be able to compare results.
Statistical Analysis
Data were analysed and visualized using the R environment for statistical computing (version 4.0.5). Multiple group comparisons were performed using one-way analysis of variance with Tukey adjustment. The analysis of covariance was used to compare group mean in a multiple linear regression model for DNA repair with Tukey adjustment. A four-parameter log-logistic regression model for DNA damage dose response was fitted through the relationship:
where ,,, are fitting parameters46. Based on the exploration of the model, cell proliferation dose response was fitted using a generalized linear least square because it allowed us to examine the different variance structure for cell line strata. We also considered a log-transformation of response variable (BrdU), as this improved the model fit. The model formula is explained through the following relationship:
Where BrdUdi being the outcome at dose d (0, 2, 8, 16) on the cell ith (AB1, AE17, BYE, JU and MC) and that depends on the β parameters in linear fashion; α, β1, β2, and β3 represents the fixed intercept, fixed effects of Dose, Cell and Dose-/-Cell interaction respectively and εi is the overall error term in the model. Data are presented as mean ± one standard deviation unless otherwise stated and the experiment was independently repeated at least three times.
Results
Increased DNA damage responses following photon radiation exposure is proportional to doses
We first examined DNA damage response using the level of γ-H2AX at one-hour post-radiation. The flow cytometry gating strategy for γ-H2AX is demonstrated in supplementary Figure 1. The level of γ-H2AX increased as the radiation dose increased, reaching a maximum at approximately 8 Gy for AB1, AE17, BYE and JU cell lines (Figure 1A-D). Interestingly, the γ-H2AX level of the MC cell line did not differ between radiation doses of 0 Gy and 2 Gy, indicating a significant DNA damage response was not induced by 2 Gy. However, the level of γ-H2AX significantly increased at 8 Gy (Figure 1E). Comparison of the DNA damage responses of murine and human mesothelioma cell lines demonstrated a difference in ED50 (50 % DNA damage) between AB1 and MC cell lines (p=0.01, Figure 1F, supplementary table 2) and between BYE and MC cell lines (p=0.01, Figure 1F, supplementary table 2). There was no difference in slope or lower and upper limit parameters among all the studied cell lines.
Figure 1DNA damage increases as a function of dose. Murine and human mesothelioma cell lines were treated with increasing doses of photon radiation, whereupon DNA damage responses (γ-H2AX) were quantified by flow cytometry. (A-E) DNA damage responses of AB1, AE17, BYE, JU and MC cell lines respectively. (F) Differences in DNA damage responses specifically with ED50 parameter between BYE and MC cell lines and AB1 and MC cell line. One-way analysis of variance for multiple group comparison with Tukey adjustment for pair-wise comparison. A four-parameter log-logistic multiple regression model for DNA damage dose response analysis. * = p < 0.05, **= p < 0.01, *** = p < 0.001.
High-dose radiation arrests the cell cycle at G2/M phase in murine but not human mesothelioma cell line
DNA damage induces cell cycle arrest at specific checkpoints in order for cells to repair DNA47. Therefore, we next assessed the effects of photon radiation on the cell cycle of mesothelioma cell lines through the examination of DNA content (supplementary Figure 2). All pairwise comparisons of cell cycle for each dose at 24, 48 and 72 hours are summarised in supplementary table 3. Cell cycle arrest at G2/M was not observed at the earliest time-points (1 and 6 hours) post-irradiation, regardless of dose, in any cell line studied (Figure 2A-E). However, we observed a reduction in G0/G1 at 1 hour and 6 hours between 0 Gy and 8 Gy (Figure 2B, supplementary table 3). Doses of 1, 2 and 4 Gy did not significantly change the proportion of cells in G0/G1 and G2/M compared to 0 Gy at any time-point post irradiation in any cell line (Figure 2A-E). However, a decreasing proportion of cells in G0/G1 was observed in AB1 cells, reaching a nadir 24 hours following irradiation with 8 Gy when compared to untreated cells (p= 0.008, supplementary table 3A & Figure 2A). This was paralleled by an increase in G2/M arrest (Figure 2A). Moreover, the proportion of the G0/G1 and G2/M population of AB1 cells returned to normal levels at 48 and 72 hours, indicating that the cell cycle arrest at the G2/M phase following 8 Gy irradiation was temporary (Figure 2A). Conversely, doses of 16 and 32 Gy comprehensively arrested the cell cycle at G2/M, with a significant proportional increase in the G2/M population and decrease in G0/G1 cells at 24, 48, and 72 hours compared to untreated cells (supplementary table 3A & Figure 2A).
Figure 2High dose photon radiation arrests cell cycle of murine cell lines at the G2/M phase. (A-B) G2/M phase arrest of the AB1 and AE17 cell lines after 24 hours at 8, 16 and 32 Gy. (C-E) cell cycle pattern of BYE, JU and MC cell line. Data are generated from three independent repeats.
A temporary cell cycle arrest was also observed in the AE17 cell line following 8 Gy irradiation, as the level of G2/M was significantly higher than untreated cells at 24 hours (p=0.01, supplementary table 3B, Figure 2B). Additionally, a significant reduction in the G0/G1 population compared to the untreated group was also observed at 16 Gy from 24 hrs (supplementary table 3B & Figure 2B). However, G2/M arrest was only observed at 24 and 72 hours with 16 Gy (supplementary table 3B & Figure 2B) because the G2/M population did not differ from the untreated group at 48 hours (supplementary table 3B, Figure 2B).
We also observed a decreased proportion of the G0/G1 population in the BYE and JU cell lines at 72 hours at 8 and 16 Gy compared to 0 Gy (supplementary table 3C-D & Figure 2C-D). However, the G2/M arrest was not observed with these two cell lines (Figure 2C & D). For the MC cell line, the proportion of G0/G1 appeared lower at 16 Gy from 48 hours. However, it did not reach significant difference level compared with untreated group and the proportion of G2/M did not change regardless of dose and time (Figure 2E, Supplementary figure 3E).
DNA repair is different between low dose and higher doses, but similar between murine and human mesothelioma cell lines
Rapid reduction in γ-H2AX expression over time post-irradiation suggests efficient DNA repair48, 49. To examine DNA repair efficiency of murine and human mesothelioma cell lines, the decrease in γ-H2AX over time following irradiation was modelled for each dose and all studied cell lines. Pair-wise comparisons of DNA repair regression slopes between different doses of the AB1 cell line are summarised in supplementary table 4. We observed a rapid drop in the level of γ-H2AX in the AB1 cell line at 1 Gy, and the slope did not differ from those of 2 and 4 Gy (supplementary table 4 & Figure 3A). However, the level of γ-H2AX remained higher over a 72 hour period at 8 Gy and was different from 1 Gy (p=0.01, supplementary table 4 & Figure 3A). This pattern persisted at 16 and 32 Gy, with regression slopes significantly different from 1, 2, and 4 Gy (supplementary table 4 & Figure 3A), suggesting that the DNA repair machinery of the AB1 cell line was less efficient at doses higher than 8 Gy. Another murine cell line, AE17, had a similar DNA repair pattern to AB1 with a rapid decrease in γ-H2AX expression at 2 Gy, but not for higher doses of 8 and 16 Gy. A significant difference in the γ-H2AX slope was seen between 2 Gy and the higher doses of 8 and 16 Gy (supplementary table 4 & Figure 3B). This pattern was also observed for BYE, JU, and MC human cell lines (supplementary table 4 & Figure 3C-E). Moreover, there was no significant difference in global DNA repair in either murine and human mesothelioma cell lines following irradiation with 2, 8 and 16 Gy (Figure 3F), as the slopes between cell lines were not significantly different.
Figure 3Global DNA repair response following radiotherapy are similar for both murine and human mesothelioma cell lines. (A-E) DNA repair of AB1, AE17, BYE, MC and JU cell lines respectively after receiving different doses of radiation. (F) A comparison of global DNA repair of murine and human mesothelioma cell lines. Analysis of covariance for multiple linear regression model with interaction using Tukey adjustment: The linear model for global γ-H2AX with interaction was visualized as mean. Adjusted-R-squared is one of the indications of goodness-of-fit of the final model.
Inhibition of cell proliferation peaks at 8 Gy, and is similar between murine and human mesothelioma cell lines
We observed no change in the pattern of cell proliferation, when compared to un-irradiated cells, in any of our cell lines at any post-irradiation time-point following treatment with 1, 2 or 4 Gy (Supplementary Figure 3, Figure 4A-E). Interestingly, a decrease in cell proliferation was not observed at 1 or 6 hours regardless of radiation dose, suggesting a minimum 6-hour delay in the inhibitory effects of photon radiation (Figure 4A-E). However, a decrease in cell proliferation was observed 24 hrs following irradiation with 8 Gy for both murine and human cell lines (Figure 4A-E). The distinct reduction in cell proliferation was seen at higher doses of 16 and 32 Gy for AB1 (Figure 4A) and 16 Gy for AE17, BYE, JU and MC cell lines from 24 hrs (4B-E). Amongst all post irradiation time-points, inhibition of cell proliferation peaked after 72 hours; this time-point was therefore used to examine the dose-response relationship and compare cell proliferation patterns between murine and human mesothelioma cell lines. Overall, there was a significant association between radiation dose, and reduction in cell proliferation (Figure 4F). However, cell proliferation did not differ between cell lines at 72 hours because there slopes were similar (Figure 4F).
Figure 4Cell proliferation is greatly inhibited by 8 Gy radiation. (A-E) Cell proliferation of AB1, AE17, BYE, JU and MC cell lines respectively. (F) Cell proliferation of the murine and human cell line after exposing to 0 Gy, 2, 8 and 16 Gy at 72 hours fitted using multiple generalized linear least square regression.
Human mesothelioma cell lines are more sensitive than mouse cell lines to a change of dose per fraction
To examine the sensitivity of our cell lines to change of dose per fraction, we performed a colony formation assay to allow accurate calculation of α/β ratio (supplementary figure 4, supplementary table 5). Our data indicated the α/β ratio for AB1 and AE17 was similar with 3.34 and 4.79 respectively (Figure 5A, supplementary table 5). For human cell lines, BYE and JU, the α/β ratio was 1.76 and 0.97 respectively (supplementary table 5, Figure 5B). The α/β ratio of murine mesothelioma cell lines is higher than human cell lines, indicating that human cell lines exhibit a greater increase in response to increasing dose-per-fraction than murine cell lines. The MC cell line is extremely slow growing and did not form colonies. Therefore, this cell line was not used for the experiment.
Figure 5Decreasing cell survival fraction of murine and human mesothelioma cell lines. (A) Survival fraction of murine, AB1 and AE17 and (B) survival fraction of human mesothelioma cell lines, BYE and JU cell lines.
Radiation induces varying levels of apoptosis in a dose and cell-line dependent manner
Next we assessed apoptosis levels in response to radiation, through staining for the cleaved poly (ADP-ribose) polymerase (PARP-1) enzyme by flow cytometry (Supplementary Figure 5). Dose-responses were very variable between cell lines; human BYE and murine AB1 lines demonstrated approximately 10% of cells undergoing apoptosis 24h following a radiation dose of 16 Gy (increasing to 20% in the case of BYE after a further 24h) (Figure 6A, B, C). However, apoptotic responses appeared lower in other cell lines studied, only reaching 4 to 5 % of cells even 72 hrs following treatment with 16 Gy in AE17, JU and MC lines (Figure 6C & E). Comparison of apoptosis showed that the human cell line (BYE) had a higher proportion cell undergoing apoptosis than other cell lines (Figure 6F).
Figure 6Apoptosis levels following radiotherapy differ between murine and human mesothelioma cell lines. (A-E) Patterns of apoptosis of AB1, AE17, BYE, and MC cells respectively. (F) Comparison of the level of apoptosis among murine and human cell lines with 16 Gy at 72 hrs. Data is presented as mean ± standard error. One way analysis of variance for multiple group comparison with Tukey adjustment for pair-wise comparison
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.
Acknowledgements
We acknowledge the support from the Australian Government and the University of Western Australia through the Research Training Program and the University Postgraduate Awards respectively and the Douglas Peter Swift Scholarship, awarded to SK and Westpac Future Leaders Scholarship (Research Funds) awarded to KMM. We acknowledge the technical assistance from staff at the Centre for Microscopy, Characterization and Analysis, The University of Western Australia and thank to Dr. Tamara Abel and Dr. Blake Klyen at Telethon Kids Institute for assistance with Gamma irradiation. Our sincere thanks to Professor Jenette Creaney, head of Biomarker Discovery group and Ms Ebony Rouse at the National Centre for Asbestos Related Diseases for facilitating access to human cell lines in our study, and to Professor Natalka Suchowerska and Linda Rogers from Chris O'Brien Lifehouse for assistance with developing clonogenic survival assays and colony counting. We also thank Ms Tracy Hayward for help with manuscript preparation and submission.
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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Author Responsible for Statistical Analysis: Synat Keam, Email: [email protected]
Disclosures: Full disclosure details for Anna Nowak provided; no disclosures for any other authors.
Funding statement: This research was supported by the Cancer Australia Priority‐driven Collaborative Cancer Research Scheme (PdCCRS) (APP1163065) and the National Health and Medical Research Council (NHMRC) Centres of Research Excellence Scheme (APP1197652 and APP1107043). This material is the responsibility of the authors and does not reflect the views of these funding bodies.
Data Availability statement: Data is kept in our institutional repository and may be made available upon reasonable request to the corresponding author.
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