Molecular radiosensitization of soft tissue sarcoma by oncolytic virus-mediated MCL1 ablation

Background: Soft tissue sarcoma (STS) is a rare cancer that develops from soft tissues in any part of the body. Despite major advances in the treatment of STS, patients are often refractory to conventional radiotherapy, leading to poor prognosis. Enhancement of sensitivity to radiotherapy would therefore improve the clinical outcome of STS patients. We recently revealed that the tumor-specific, replication-competent oncolytic adenovirus OBP-301 kills human sarcoma cells. In this study, we investigated the radiosensitizing effect of OBP-301 in human STS cells. Methods: The in vitro antitumor effect of OBP-301 and ionizing radiation in monotherapy or combination therapy was assessed using radiosensitive (RD-ES and SK-ES-1) and radioresistant (HT1080 and NMS-2) STS cell lines. The expression of markers for apoptosis and DNA damage were evaluated in STS cells after treatment. The therapeutic potential of combination therapy was further analyzed using SK-ES-1 and HT1080 cells in subcutaneous xenograft tumor models. Results: The combination of OBP-301 and ionizing radiation showed a synergistic antitumor effect in all human STS cell lines tested, including those that were radioresistant. OBP-301 was found to enhance irradiation-induced apoptosis and DNA damage via suppression of anti-apoptotic myeloid cell leukemia 1 (MCL1), which was expressed at higher levels in radioresistant cell lines. The combination of OBP-301 and ionizing radiation showed a more profound antitumor effect compared to monotherapy in SK-ES-1 (radiosensitive) and HT1080 (radioresistant) subcutaneous xenograft tumors. Conclusions: OBP-301 is a promising antitumor reagent to improve the therapeutic potential of radiotherapy by suppressing MCL1 expression in STS.

(Sigma-Aldrich, St. Louis, MO, USA). The secondary antibodies used were: horseradish peroxidaseconjugated antibodies against rabbit IgG or mouse IgG (GE Healthcare). Immunoreactive protein bands were visualized using enhanced chemiluminescence (ECL Plus; GE Healthcare).
Cells were irradiated at 1 Gy, and then 30 min, 1 h, or 3 h following irradiation, cells were fixed with chilled 1% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min on ice. The slides were subsequently incubated with primary mouse anti-γH2AX mAb (Millipore) for 24 h. After washing three times with PBS, slides were incubated with the secondary FITC-conjugated antibody against mouse IgG (Zymed Laboratories Inc., South San Francisco, CA, USA) for 30 min on ice. The slides were further stained with ProLong® Gold antifade reagent with DAPI (life technologies Co., Carlsbad, CA, USA) and then analyzed under a confocal laser microscope (FV10i; Olympus Co., Tokyo, Japan).

In vivo subcutaneous human STS xenograft tumor models
Animal experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of Okayama University School of Medicine. SK-ES-1 (5 × 10 6 cells/mouse) and HT1080 (3 × 10 6 cells/mouse) cells were subcutaneously inoculated into the flanks of 5-to 6-week-old female BALB/c nu/nu mice (CLEA Japan, Tokyo, Japan). When tumors reached 5 to 7 mm in diameter, the mice were irradiated at a dosage of 1 Gy/tumor (SK-ES-1) or 3 Gy/tumor (HT1080) every week for one or three cycles starting at day 0. During irradiation, mice were placed in a prone position, using custom-made holders that contain lead collimators to shield the upper half of the animal. OBP-301 at a dose of 1 × 10 8 PFU/tumor or PBS was injected into the tumors every week for one or three cycles.
The perpendicular diameter of each tumor was measured every 3 days, and tumor volume was calculated with the following formula: tumor volume (mm 3 ) = a × b 2 × 0.5, where a is the longest diameter, b is the shortest diameter, and 0.5 is a constant to calculate the volume of an ellipsoid. At the end of the experiment, euthanasia was performed by isoflurane inhalation.

Histopathologic analysis
Tumors were fixed in 10% neutralized formalin and embedded in paraffin blocks. Sections were stained with hematoxylin/eosin. Sections were also prepared for immunohistochemical examination using mouse anti-γH2AX mAb (Millipore) and rabbit anti-Ki67 mAb (Abcam, Cambridge, UK).

TUNEL staining
Sections were deparaffinized and put into 3% hydrogen peroxide for 10 min at room temperature.
After nonspecific binding sites were blocked, the sections were incubated for 60 min at 37°C with terminal deoxynucleotidyltransferase mediated dUTP nick end labeling (TUNEL; Roche Applied Science, Penzberg, Germany) and stained with DAB solution. Finally, all sections were counterstained with hematoxylin. Sections were rinsed with PBS after every step.

Statistical analysis
Data were expressed as mean ± SD. Differences between groups were examined for statistical significance with the Student's t test. P values < 0.05 were considered statistically significant.

Results
In vitro radiosensitizing effect of OBP-301 in human STS cells To evaluate the radiosensitizing effect of OBP-301 in STS cells, we first analyzed the baseline sensitivity of four human STS cell lines (RD-ES, SK-ES-1, HT1080, and NMS-2) to ionizing radiation.
The viability of RD-ES and SK-ES-1 (Ewing sarcoma) cells when irradiated at 2 Gy was decreased to approximately half that of non-irradiated cells, as measured by XTT assay (Fig. 1a). In contrast, the viability of HT1080 (fibrosarcoma) and NMS-2 (MPNST) was reduced by less than 50% compared to the viability of non-irradiated cells, even up to 10 Gy (Fig. 1a). These results indicate that RD-ES and SK-ES-1 cells are relatively radiosensitive, whereas HT1080 and NMS-2 cells are relatively radioresistant.
We next evaluated the combined effect of OBP-301 and ionizing radiation on viability. All four STS cell lines were irradiated 24 h after OBP-301 infection, and cell viability was assessed on day 4 after irradiation. The combination of OBP-301 and ionizing radiation decreased the viability of all STS cell lines more efficiently than single treatment (Fig. 1b). Calculation of the combination index indicated a synergistic antitumor effect of combination therapy in all STS cell lines, although in radioresistant NMS-2 cells there was an antagonistic effect found with low doses of ionizing radiation and OBP-301 ( Fig. 1c). These results suggest that OBP-301 may act as a potent radiosensitizer in human STS cells.
Suppression of anti-apoptotic MCL1 is critical for enhancing ionizing radiation-induced apoptosis by

OBP-301
To investigate the underlying molecular mechanism in the OBP-301-mediated enhancement of radiotherapy, we analyzed the level of anti-apoptotic MCL1 expression in all STS cells. Interestingly, radioresistant HT1080 and NMS-2 cells exhibited high expression of MCL1 protein, whereas radiosensitive RD-ES and SK-ES-1 cells showed low expression of MCL1 protein (Fig. 2a). These results suggest that the expression of MCL1 is associated with the radiosensitivity of STS cells.
We recently reported that OBP-301 enhances the cytotoxic effect of chemotherapeutic agents in human osteosarcoma cells by suppressing MCL1 expression through the activation of a transcription factor E2F1-microRNA pathway, resulting in the induction of apoptosis [12]. Therefore, we next assessed whether OBP-301 enhances ionizing radiation-induced apoptosis by suppressing MCL1 expression in radiosensitive SK-ES-1 and radioresistant HT1080 cells. OBP-301 infection at high doses increased the expression of adenoviral E1A and E2F1, whereas MCL1 expression was decreased in SK-ES-1 and HT1080 cells (Fig. 2b). The combination of OBP-301 and ionizing radiation induced apoptosis (PARP cleavage) in SK-ES-1 and HT1080 cells, which was associated with the upregulation of E2F1 and downregulation of MCL1 (Fig. 2c). To further confirm the role of MCL1 suppression in radiationinduced apoptosis, we assessed the effect of MCL1 knockdown by RNA interference. MCL1 siRNA suppressed MCL1 expression and resulted in the enhancement of ionizing radiation-induced apoptosis in SK-ES-1 and HT1080 cells compared to control siRNA (Fig. 2d). These results suggest that OBP-301 enhances ionizing radiation-mediated apoptosis induction via MCL1 suppression.

Enhancement of ionizing radiation-induced DNA damage by OBP-301
Exposure of cells to ionizing radiation leads to a number of types of DNA damage, including DNA double-stranded breaks, which can be detected by the accumulation of γH2AX protein [16]. We Subsequent immunohistochemical analysis demonstrated that the combination of OBP-301 and ionizing radiation significantly decreased the percentage of Ki67-positive proliferating cells compared to mock or single treatment in SK-ES-1 tumor tissues ( Fig. 4b and c). The number of TUNEL-positive cells was significantly increased in combination therapy-treated SK-ES-1 tumors compared with mock or monotherapy-treated tumors (Fig. 4c). Moreover, combination therapy significantly increased the number of γH2AX-positive cells within SK-ES-1 tumor tissues (Fig. 4c). Immunohistochemical analysis for radioresistant HT1080 tumors demonstrated similar findings, in that the percentage of Ki-67positive cells was significantly decreased, and the number of TUNEL-positive and γH2AX-positive cells was significantly increased, in combination therapy-treated tumor tissues (Fig. 5). These results suggest that the biological interaction between OBP-301 and ionizing radiation is induced in in vivo tumor tissues as well as in vitro.
Combination of OBP-301 and ionizing radiation inhibits the in vivo growth of STS tumors with different radiosensitivities To assess the in vivo therapeutic efficacy of OBP-301 in combination with ionizing radiation, we again used the subcutaneous xenograft models for radiosensitive SK-ES-1 (Fig. 6a) and radioresistant HT1080 cells (Fig. 6b). Radiosensitive SK-ES-1 tumors were injected with OBP-301 (1 × 10 8 PFU/tumor) or PBS, and subsequently irradiated at 1 Gy every week for three cycles, with tumor growth observed for 28 days after the first treatment. The combination of OBP-301 and ionizing radiation showed a more profound antitumor effect in radiosensitive SK-ES-1 tumors compared with OBP-301 or ionizing radiation alone (Fig. 6a). There was no significant difference in the mean body weight of mice between the treatment groups (Additional file 1a). Radioresistant HT1080 tumors were injected with OBP-301 (1 × 10 8 PFU/tumor) or PBS, and subsequently irradiated at 3 Gy every week for three cycles.
Consistent with the findings from radiosensitive SK-ES-1 tumors, the combination of OBP-301 and ionizing radiation resulted in significant suppression of tumor growth compared with monotherapy ( Fig. 6b). There was again no significant difference in the mean body weight of mice between the treatment groups (Additional file 1b). These results suggest that combination therapy with OBP-301 and ionizing radiation efficiently inhibits the growth of STS tumors in vivo.

Discussion
The multidisciplinary approach to STS treatment involves surgery, radiotherapy, and chemotherapy.
Although surgical resection and radiotherapy are the most frequent option for STS, resistance to radiotherapy contributes to tumor recurrence, metastasis, and poor prognosis. Therefore, the enhancement of radiosensitivity is a critical aspect of improving clinical outcomes for STS patients, including minimizing the risk of secondary cancer by reducing the required dose of ionizing radiation.
In this study, we demonstrated that combination therapy with OBP-301 and ionizing radiation has a synergistic antitumor effect in both radiosensitive and radioresistant STS cells. The profound antitumor effect of combination therapy was mainly due to OBP-301-mediated enhancement of ionizing radiation-induced apoptosis and DNA damage, via suppression of anti-apoptotic MCL1 expression. Thus, the combination of OBP-301 and ionizing radiation appears to be a promising antitumor strategy for improving the efficacy of radiotherapy in STS patients.
Although sensitivity to radiotherapy varies between the histological subtypes of STS, the underlying mechanism has not been clearly understood. An anti-apoptotic member of the BCL2 family, MCL1 is In various types of cancer, resistance to radiotherapy is thought to be associated with the existence of cancer stem-like cells [35]. The dormancy of such cells is a critical factor for the radioresistance of tumors, because radiotherapy targets proliferating cancer cells by inducing DNA damage-related cell cycle arrest. Recent reports have suggested that a CD133-positive subpopulation of radioresistant HT1080 cells exhibit cancer stem-like characteristics [36,37], such as activation of stemness-related markers, sphere-forming capacity, tumorigenicity, and resistance to chemotherapy [37]. CD133positive subpopulations have similarly been associated with the radioresistance of brain tumors [38], liver cancer [39], and gastric cancer [40]. We recently demonstrated that OBP-301 efficiently kills radioresistant CD133-positive cells, as well as radiosensitive CD133-negative cells, in human gastric cancer [40]. In that study, the dormancy of CD133-positive cancer stem-like cells, enriched by sphere HT, and TosF contributed to the writing, review, and/or revision of the manuscript; YU contributed to the administrative, technical and material support; HT, TKu, SK, TOz, and TosF contributed to the study supervision. All authors have read and approved the manuscript.      In vivo antitumor effect of combination therapy with OBP-301 and ionizing radiation in the radiosensitive SK-ES-1 and radioresistant HT1080 xenograft tumor models. a, b. SK-ES-1 cells (5 × 106 cells/mouse) or HT1080 cells (3 × 106 cells/mouse) were subcutaneously inoculated into the right flanks of mice. When tumors reached 5 to 7 mm in diameter, tumor-bearing mice were irradiated at 1 Gy (SK-ES-1) or 3 Gy (HT1080) after treatment with an intratumoral injection of OBP-301 (1 × 108 PFU/tumor) for three cycles every week (arrows indicate each treatment administration). The mock treatment group was sacrificed on days 21 and 24 in the SK-ES-1 and HT1080 tumor models, respectively, when tumor volumes reached approximately 4,000 mm3. Tumor growth is expressed as mean tumor volume ± SD (n = 4 to 7 in each group; *, P < 0.05).