p53-Armed Oncolytic Virotherapy Improves Radiosensitivity in Soft-Tissue Sarcoma by Suppressing BCL-xL Expression.

Soft-tissue sarcoma (STS) is a heterogeneous group of rare tumors originating predominantly from the embryonic mesoderm. Despite the development of combined modalities including radiotherapy, STSs are often refractory to antitumor modalities, and novel strategies that improve the prognosis of STS patients are needed. We previously demonstrated the therapeutic potential of two telomerase-specific replication-competent oncolytic adenoviruses, OBP-301 and tumor suppressor p53-armed OBP-702, in human STS cells. Here, we demonstrate in vitro and in vivo antitumor effects of OBP-702 in combination with ionizing radiation against human STS cells (HT1080, NMS-2, SYO-1). OBP-702 synergistically promoted the antitumor effect of ionizing radiation in the STS cells by suppressing the expression of B-cell lymphoma-X large (BCL-xL) and enhancing ionizing radiation-induced apoptosis. The in vivo experiments demonstrated that this combination therapy significantly suppressed STS tumors' growth. Our results suggest that OBP-702 is a promising antitumor reagent for promoting the radiosensitivity of STS tumors.

Fluorescence-guided assessment of bone and soft-tissue sarcomas for predicting the efficacy of telomerase-specific oncolytic adenovirus.

Bone and soft-tissue sarcomas are rare malignancies with histological diversity and tumor heterogeneity, leading to the lack of a common molecular target. Telomerase is a key enzyme for keeping the telomere length and human telomerase reverse transcriptase (hTERT) expression is often activated in most human cancers, including bone and soft-tissue sarcomas. For targeting of telomerase-positive tumor cells, we developed OBP-301, a telomerase-specific replication-competent oncolytic adenovirus, in which the hTERT promoter regulates adenoviral E1 gene for tumor-specific viral replication. In this study, we present the diagnostic potential of green fluorescent protein (GFP)-expressing oncolytic adenovirus OBP-401 for assessing virotherapy sensitivity using bone and soft-tissue sarcomas. OBP-401-mediated GFP expression was significantly associated with the therapeutic efficacy of OBP-401 in human bone and soft-tissue sarcomas. In the tumor specimens from 68 patients, malignant and intermediate tumors demonstrated significantly higher expression levels of coxsackie and adenovirus receptor (CAR) and hTERT than benign tumors. OBP-401-mediated GFP expression was significantly increased in malignant and intermediate tumors with high expression levels of CAR and hTERT between 24 and 48 h after infection. Our results suggest that the OBP-401-based GFP expression system is a useful tool for predicting the therapeutic efficacy of oncolytic virotherapy on bone and soft-tissue sarcomas.

Oncolytic virotherapy reverses chemoresistance in osteosarcoma by suppressing MDR1 expression.

BACKGROUND: Osteosarcoma (OS) is a malignant bone tumor primarily affecting children and adolescents. The prognosis of chemotherapy-refractory OS patients is poor. We developed a tumor suppressor p53-expressing oncolytic adenovirus (OBP-702) that exhibits antitumor effects against human OS cells. Here, we demonstrate the chemosensitizing effect of OBP-702 in human OS cells. MATERIALS AND METHODS: The in vitro and in vivo antitumor activities of doxorubicin (DOX) and OBP-702 were assessed using parental and DOX-resistant OS cells (U2OS, MNNG/HOS) and a DOX-resistant MNNG/HOS xenograft tumor model. RESULTS: DOX-resistant OS cells exhibited high multidrug resistant 1 (MDR1) expression, which was suppressed by OBP-702 or MDR1 siRNA, resulting in enhanced DOX-induced apoptosis. Compared to monotherapy, OBP-702 and DOX combination therapy significantly suppressed tumor growth in the DOX-resistant MNNG/HOS xenograft tumor model. CONCLUSION: Our results suggest that MDR1 is an attractive therapeutic target for chemoresistant OS. Tumor-specific virotherapy is thus a promising strategy for reversing chemoresistance in OS patients via suppression of MDR1 expression.

Oncolytic virotherapy promotes radiosensitivity in soft tissue sarcoma by suppressing anti-apoptotic MCL1 expression.

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 previously 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. The in vitro antitumor effect of OBP-301 and ionizing radiation in monotherapy or combination therapy was assessed using highly radiosensitive (RD-ES and SK-ES-1) and moderately radiosensitive (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. The combination of OBP-301 and ionizing radiation showed a synergistic antitumor effect in all human STS cell lines tested, including those that show different radiosensitivity. 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 moderately radiosensitive cell lines. The combination of OBP-301 and ionizing radiation showed a more profound antitumor effect compared to monotherapy in SK-ES-1 (highly radiosensitive) and HT1080 (moderately radiosensitive) subcutaneous xenograft tumors. OBP-301 is a promising antitumor reagent to improve the therapeutic potential of radiotherapy by increasing radiation-induced apoptosis in STS.

Telomerase-specific oncolytic immunotherapy for promoting efficacy of PD-1 blockade in osteosarcoma.

Immune checkpoint inhibitors including anti-programmed cell death 1 (PD-1) antibody have recently improved clinical outcome in certain cancer patients; however, osteosarcoma (OS) patients are refractory to PD-1 blockade. Oncolytic virotherapy has emerged as novel immunogenic therapy to augment antitumor immune response. We developed a telomerase-specific replication-competent oncolytic adenovirus OBP-502 that induces lytic cell death via binding to integrins. In this study, we assessed the combined effect of PD-1 blockade and OBP-502 in OS cells. The expression of coxsackie and adenovirus receptor (CAR), integrins αvß3 and αvß5, and programmed cell death ligand 1 (PD-L1) was analyzed in two murine OS cells (K7M2, NHOS). The cytopathic activity of OBP-502 in both cells was analyzed using the XTT assay. OBP-502-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). Subcutaneous tumor models for K7M2 and NHOS cells were used to evaluate the antitumor effect and number of tumor-infiltrating CD8+ cells in combination therapy. K7M2 and NHOS cells showed high expression of integrins αvß3 and αvß5, but not CAR. OBP-502 significantly suppressed the viability of both cells, in which PD-L1 expression and the release of ATP and HMGB1 were significantly increased. Intratumoral injection of OBP-502 significantly augmented the efficacy of PD-1 blockade on subcutaneous K2M2 and NHOS tumor models via enhancement of tumor-infiltrating CD8+ T cells. Our results suggest that telomerase-specific oncolytic virotherapy is a promising antitumor strategy to promote the efficacy of PD-1 blockade in OS.

Early chondral damage following meniscus repairs with anterior cruciate ligament reconstruction.

BACKGROUND: Meniscal tears are commonly observed in patients with anterior cruciate ligament (ACL) injuries. Meniscal repair has become a common procedure for the injured meniscus, and good clinical outcomes have been reported in such cases when used concurrently with ACL reconstruction. However, it is unclear whether early chondral damage progression can be prevented following meniscal repair with ACL reconstruction, as meniscal damage is a potential risk factor for the development of osteoarthritis. The purpose of this study was to evaluate the zone-specific chondral damage that occurs after arthroscopic meniscal repair with concomitant ACL reconstruction. Our hypothesis was that meniscal repair with ACL reconstruction would not decrease the rate of progression of chondral damage compared to that observed in isolated ACL reconstruction with intact menisci. METHODS: This study included 40 patients who underwent anatomic double-bundle ACL reconstruction. We divided the patients into the following two groups: Group A with an intact meniscus (20 knees) and Group M requiring meniscal repair (20 knees). Chondral damage was evaluated arthroscopically in six compartments and 40 sub-compartments, and these features were graded using the International Cartilage Repair Society lesion classification. The cartilage damage in each sub-compartment and compartment was compared between the two groups both at reconstruction and at second-look arthroscopy (average 16 months postoperatively). At the latest follow-up examination (average 37 months postoperatively), the International Knee Documentation Committee (IKDC) score was compared between the two groups. RESULTS: Group M had a significantly worse cartilage status than Group A in five sub-compartments (mainly in the medial compartment) at reconstruction and in nine sub-compartments (mainly in the bilateral compartments) at second-look arthroscopy. The mean IKDC score was better in Group A than in Group M (Group A; 90 vs. Group M; 86). The overall success rate of meniscal repairs was 92% (23 of 25 menisci) at second-look arthroscopy. CONCLUSION: The progression of post-traumatic chondral damage may occur at a faster rate in patients who require ACL reconstruction and meniscal repair than in patients with intact menisci.

Posttraumatic cartilage degradation progresses following anterior cruciate ligament reconstruction: A second-look arthroscopic evaluation.

BACKGROUND: Several studies have demonstrated that posttraumatic knee osteoarthritis progresses even after anterior cruciate ligament reconstruction. Few reports described zone-specific cartilaginous damages after anterior cruciate ligament reconstruction. This study aimed to compare the status of articular cartilage at anterior cruciate ligament reconstruction with that at second-look arthroscopy. METHODS: This study included 20 patients (20 knees, 10 males and 10 females, mean age 22.4 years, Body mass index 24.4 kg/m2) that underwent arthroscopic anatomic double-bundle anterior cruciate ligament reconstruction and second-look arthroscopy. Mean periods from injury to reconstruction and from reconstruction to second-look arthroscopy were 3.4 and 15.3 months, respectively. Cartilage lesions were evaluated arthroscopically in the 6 articular surfaces and 40 articular subcompartments independently, and these features were graded with the International Cartilage Repair Society articular cartilage injury classification; comparisons were made between the grades at reconstruction and at second-look arthroscopy. Furthermore, clinical outcomes were assessed at reconstruction and at second-look arthroscopy, using the Lysholm knee score, Tegner activity scale, International Knee Documentation Committee score, Knee injury and Osteoarthritis Outcome Score, side-to-side difference of the KT-2000 arthrometer, and pivot shift test. RESULTS: Each compartment showed a deteriorated condition at second-look arthroscopy compared with the pre-reconstruction period. A significant worsening of the articular cartilage was noted in all compartments except the lateral tibial plateau and was also observed in the central region of the medial femoral condyle and trochlea after reconstruction. However, each clinical outcome was significantly improved postoperatively. CONCLUSIONS: Good cartilage conditions were restored in most subcompartments at second-look arthroscopy. Furthermore, posttraumatic osteoarthritic changes in the patellofemoral and medial compartments progressed even in the early postoperative period, although good knee stability and clinical outcomes were obtained. Care is necessary regarding the progression of osteoarthritis and the appearance of knee symptoms in patients undergoing anterior cruciate ligament reconstruction.

Post-traumatic Articular Cartilage Lesions Increase at Second-look Arthroscopy Following Primary Anterior Cruciate Ligament Reconstruction.

Anterior cruciate ligament (ACL) reconstruction (ACLR) after ACL rupture improves the instability of the knee joint and decreases mechanical stress to the meniscus and articular cartilage. However, there are reports that post-traumatic osteoarthritis (PTOA) is observed over time following ACLR. In this study, we assessed changes in cartilage lesions by arthroscopic findings following anatomical double-bundle ACLR and at post-operative second-look arthroscopy about 14 months later. We retrospectively evaluated 37 knees in cases with patients <40 years of age who had undergone an anatomical double-bundle ACL reconstruction <1 year after ACL rupture injury from March 2012 to December 2016. Clinical results and arthroscopic cartilage/meniscal lesion were evaluated and compared between a cartilage lesion-detected group and intact-cartilage group. Surgery improved anteroposterior laxity and other clinical measures; however, cartilage lesions were detected at 11 sites during ACLR and at 54 sites at second-look arthroscopy. The periods from injury to second-look arthroscopy and from ACLR to second-look arthroscopy were significantly longer in the cartilage-lesion group (n=23) than in the intact-cartilage group (n=14). Conversely, 96% of meniscal damage observed during ACLR was cured at the time of second-look arthroscopy. Knee articular cartilage lesions after ACL rupture cannot be completely suppressed, even using the anatomical ACL reconstruction technique. This study suggested that articular cartilage lesions can progress to a level that can be confirmed arthroscopically at approximately 17 months after ACL injury. Therefore, in ACLR patients, the possibility of developing knee articular cartilage lesions and PTOA should be considered.

Role of zoledronic acid in oncolytic virotherapy: Promotion of antitumor effect and prevention of bone destruction.

Osteosarcoma is an aggressive malignant bone tumor that causes bone destruction. Although tumor-specific replicating oncolytic adenovirus OBP-301 induces an antitumor effect in an osteosarcoma tumor, it cannot prevent bone destruction. Zoledronic acid (ZOL) is a clinically available agent that inhibits bone destruction. In this study, we investigated the potential of combination therapy with OBP-301 and ZOL against osteosarcomas with bone destruction. The antitumor activity of OBP-301 and ZOL in monotherapy or combination therapy was assessed using three human osteosarcoma cell lines (143B, MNNG/HOS, SaOS-2). The cytotoxic effect of OBP-301 and/or ZOL was measured by assay of cell apoptosis. The effect of OBP-301 and ZOL on osteoclast activation was investigated. The potential of combination therapy against tumor growth and bone destruction was analyzed using an orthotopic 143B osteosarcoma xenograft tumor model. OBP-301 and ZOL decreased the viability of human osteosarcoma cells. Combination therapy with OBP-301 and ZOL displayed a synergistic antitumor effect, in which OBP-301 promoted apoptosis through suppression of anti-apoptotic myeloid cell leukemia 1 (MCL1). Combination therapy significantly inhibited tumor-mediated osteoclast activation, tumor growth and bone destruction compared to monotherapy. These results suggest that combination therapy of OBP-301 and ZOL suppresses osteosarcoma progression via suppression of MCL1 and osteoclast activation.

Clinical significance of circulating miR-25-3p as a novel diagnostic and prognostic biomarker in osteosarcoma.

BACKGROUND: Emerging evidence has suggested that circulating microRNAs (miRNAs) in body fluids have novel diagnostic and prognostic significance for patients with malignant diseases. The lack of useful biomarkers is a crucial problem of bone and soft tissue sarcomas; therefore, we investigated the circulating miRNA signature and its clinical relevance in osteosarcoma. METHODS: Global miRNA profiling was performed using patient serum collected from a discovery cohort of osteosarcoma patients and controls and cell culture media. The secretion of the detected miRNAs from osteosarcoma cells and clinical relevance of serum miRNA levels were evaluated using in vitro and in vivo models and a validation patient cohort. RESULTS: Discovery screening identified 236 serum miRNAs that were highly expressed in osteosarcoma patients compared with controls, and eight among these were also identified in the cell culture media. Upregulated expression levels of miR-17-5p and miR-25-3p were identified in osteosarcoma cells, and these were abundantly secreted into the culture media in tumor-derived exosomes. Serum miR-25-3p levels were significantly higher in osteosarcoma patients than in control individuals in the validation cohort, with favorable sensitivity and specificity compared with serum alkaline phosphatase. Furthermore, serum miR-25-3p levels at diagnosis were correlated with patient prognosis and reflected tumor burden in both in vivo models and patients; these associations were more sensitive than those of serum alkaline phosphatase. CONCLUSIONS: Serum-based circulating miR-25-3p may serve as a non-invasive blood-based biomarker for tumor monitoring and prognostic prediction in osteosarcoma patients.

Ablation of MCL1 expression by virally induced microRNA-29 reverses chemoresistance in human osteosarcomas.

Osteosarcoma is a rare disease diagnosed as malignant bone tumor. It is generally refractory to chemotherapy, which contributes to its poor prognosis. The reversal of chemoresistance is a major clinical challenge to improve the prognostic outcome of osteosarcoma patients. We developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301 (telomelysin) and assessed its synergistic effects with chemotherapeutic agents (cisplatin and doxorubicin) using human osteosarcoma cell lines and a xenograft tumor model. The molecular mechanism underlying the chemosensitizing effect of OBP-301 was evaluated in aspects of apoptosis induction. OBP-301 inhibits anti-apoptotic myeloid cell leukemia 1 (MCL1) expression, which in turn leads to chemosensitization in human osteosarcoma cells. The siRNA-mediated knockdown of MCL1 expression sensitized human osteosarcoma cells to common chemotherapeutic agents. We also found that upregulation of microRNA-29 targeting MCL1 via virally induced transcriptional factor E2F-1 activation was critical for the enhancement of chemotherapy-induced apoptosis in osteosarcoma cells. Telomerase-specific oncolytic adenovirus synergistically suppressed the viability of human osteosarcoma cells in combination with chemotherapeutic agents. The combination treatment also significantly inhibited tumor growth, as compared to monotherapy, in an osteosarcoma xenograft tumor model. Our data suggest that replicative virus-mediated tumor-specific MCL1 ablation may be a promising strategy to attenuate chemoresistance in osteosarcoma patients.