Keisuke Tamari

Identification of chemoradiation-resistant osteosarcoma stem cells using an imaging system for proteasome activity

Osteosarcoma is the most common primary bone malignancy in pediatric and adolescent populations. Recurrence and metastatic potential can be due to a subpopulation of cells with stem cell-like characteristics, such as tumor-initiating cells (TICs), which maintain the capacity to regenerate entire tumors. Targeting the TICs in osteosarcoma is a promising avenue for the development of new therapies for this devastating disease. TICs are usually quiescent with a low protein turnover, decreased metabolism, and downregulation of proteasome activity. Recently, cancer cells with low proteasome activity have been identified as TICs in several types of cancer. We stably infected two osteosarcoma cell lines, MG-63 and U2-OS, with an expression vector for a fusion protein between the green fluorescent protein, ZsGreen, and the C-terminal degron of the murine ornithine decarboxylase to monitor the 26S proteasome activity in living cells. We separated the osteosarcoma cells with low proteasome activity using fluorescence-activated cell sorting (FACS) and verified whether these ZsGreen+ cells had TIC-like properties. The ZsGreen+ cells showed enhanced sphere formation capacity and underwent asymmetric divisions into ZsGreen+ and ZsGreen- cells, whereas ZsGreen- cells underwent only symmetric divisions into ZsGreen- cells. Moreover, the ZsGreen+ cells were more chemo- and radioresistant. Thus, the present study demonstrated that chemoradiation-resistant TICs can be visualized by this system and suggested the rationale for further study of osteosarcoma stem cells.

Treatment outcomes using CyberKnife for brain metastases from lung cancer

We investigated the clinical outcomes following treatment using stereotactic radiosurgery (SRS) and fractionated stereotactic radiotherapy (SRT) for brain metastases from lung cancer. A total of 67 patients with 109 brain metastases from lung cancer treated using CyberKnife between 1998 and 2011 were retrospectively analyzed. SRS (median dose, 24 Gy) was used to treat 79 lesions, and 3-fraction SRT (median dose, 30 Gy) was used to treat 30 lesions. The median follow-up time was 9.4 months (range, 0.4–125 months). The 1-year local control rate was 83.3%, and the 1-year distant brain failure rate was 30.1%. The median survival time was 13.1 months, and the 1- and 3-year overall survival (OS) rates were 54.8% and 25.9%, respectively. On multivariate analysis, three factors were found to be statistically significant predictors of OS: (i) presence of uncontrolled primary disease [hazard ratio (HR) = 3.04; P = 0.002]; (ii) Brinkman index (BI) ≥ 1000 (HR = 2.75; P = 0.007); and (iii) pulmonary metastases (HR = 3.54; P = 0.009). Radionecrosis and worsening of neurocognitive function after radiosurgery were observed in 5 (7%) and 3 (4%) patients, respectively. Our results indicated that SRS/SRT for brain metastases from lung cancer was effective. Uncontrolled primary disease, high BI, and pulmonary metastases at treatment were significant risk factors for OS.

Radiation enhanced the local and distant anti-tumor efficacy in dual immune checkpoint blockade therapy in osteosarcoma

Radiation therapy has been long utilized as localized cancer treatment. Recent studies have also demonstrated that it has a distant effect by the enhanced immunity, but it rarely occurs. The purpose of this study was to investigate whether X-ray irradiation combined with anti-PD-L1 and anti-CTLA-4 antibodies (P1C4) provides a higher probability of this distant effect as well as enhanced local antitumor efficacy for osteosarcoma. LM8 mouse osteosarcoma cells were inoculated into both legs of C3H mice assigned to one of four groups, namely no treatment (No Tx), P1C4, X-ray irradiation (RAD) to the leg of one side, and combination (COMB) groups. Survival and treatment-related immune molecular changes were analyzed. Administration of P1C4 produced a tumor growth delay on day 30 in 18% of the mice. In contrast, combination therapy produced the strongest tumor growth inhibition not only at the irradiated tumor but also at unirradiated tumor in 67% of the mice. Accordingly, lung metastasis in the COMB group was strongly reduced by 98%, with a significant survival benefit. Unirradiated tumor in mice in the COMB group significantly recruited CD8 + tumor-infiltrating lymphocytes with a moderate reduction of Treg, producing a significant increase in the CD8/Treg ratio. These results suggest that radiation enhances the efficacy of P1C4 treatment against distant metastasis as well as local control in osteosarcoma. Our data suggest that radiation therapy combined with dual checkpoint blockade may be a promising therapeutic option for osteosarcoma.

Cancer stem cells: The potential of carbon ion beam radiation and new radiosensitizers (Review)

Cancer stem cells (CSCs) are a small population of cells in cancer with stem-like properties such as cell proliferation, multiple differentiation and tumor initiation capacities. CSCs are therapy-resistant and cause cancer metastasis and recurrence. One key issue in cancer therapy is how to target and eliminate CSCs, in order to cure cancer completely without relapse and metastasis. To target CSCs, many cell surface markers, DNAs and microRNAs are considered as CSC markers. To date, the majority of the reported markers are not very specific to CSCs and are also present in non-CSCs. However, the combination of several markers is quite valuable for identifying and targeting CSCs, although more specific identification methods are needed. While CSCs are considered as critical therapeutic targets, useful treatment methods remain to be established. Epigenetic gene regulators, microRNAs, are associated with tumor initiation and progression. MicroRNAs have been recently considered as promising therapeutic targets, which can alter the therapeutic resistance of CSCs through epigenetic modification. Moreover, carbon ion beam radiotherapy is a promising treatment for CSCs. Evidence indicates that the carbon ion beam is more effective against CSCs than the conventional X-ray beam. Combination therapies of radiosensitizing microRNAs and carbon ion beam radiotherapy may be a promising cancer strategy. This review focuses on the identification and treatment resistance of CSCs and the potential of microRNAs as new radiosensitizers and carbon ion beam radiotherapy as a promising therapeutic strategy against CSCs.

Novel drug discovery system for cancer stem cells in human squamous cell carcinoma of the esophagus

Cancer stem cells (CSCs) have been identified in several tumor tissues. Since CSCs are resistant to cancer therapies, including chemotherapy and radiation therapy, and can even remain after therapies, tumor tissue often regrows and relapses. Thus, identification of CSCs and treatment targeting CSCs are required to treat tumor tissues. Reportedly, a fluorescent vector consisting of fluorescein ZsGreen fused to the carboxyl-terminal region of ornithine decarboxylase (cODC) was used to detect CSCs or therapy-resistant cancer cells in tumor tissues of the brain, pancreas and liver. Cells transfected with the fluorescent vector can express a fluorescein fused to cODC and become fluorescent only when the fusion protein is accumulated. In the present study, CSCs or therapy-resistant cancer cells were identified with the fluorescent vector in esophageal squamous cell carcinoma. The use of this fluorescent vector in drug screening enabled the detection of three drugs, AKT inhibitor XI, ERK inhibitor II and JAK inhibitor I, which target malignant CSCs.

Visualization and characterization of cancer stem-like cells in cervical cancer.

Cancer stem cells (CSCs), defined by their differentiation capacity, self-renewal capacity, and maintenance of proliferation, have been identified in many tumors, including cervical cancer. Current studies identify CSCs by several specific biomarkers; however, it is difficult to monitor cervical CSCs in real-time in vitro and in vivo. Recent research reported the visualization of CSCs in breast cancer and gliomas using green fluorescent protein, ZsGreen, fused to a degron motif ornithine decarboxylase (ODC), which is destroyed by proteasomes. Accordingly, CSCs have low 26S proteasome activity, whereas non-CSCs have high 26S proteasome activity. Therefore, it is possible to observe CSCs by their accumulation of the fluorescent ZsGreen protein. In this study, we investigated optical imaging parameters to evaluate CSCs using two human cervical cancer cell lines: CaSki and HeLa. We defined populations as cell types having high- and low ZsGreen-cODC (high- and low-Zs, respectively) expression levels. The results of a sphere-forming assay revealed that the self-renewal ability of the high-Zs population was significantly higher than that of the low-Zs population. A tumorigenicity assay confirmed that the high-Zs population exhibited higher tumorigenic potential than the low-Zs population. The radioresistance of the high-Zs population of both HeLa and CaSki cells and the chemoresistance of the high-Zs population of CaSki cells were confirmed by a clonogenic survival assay and the tetrazolium dye assay, respectively. These results indicate that high-Zs populations of both the HeLa and CaSki cell lines possess CSC-like properties and therapeutic resistance. In conclusion, we successfully visualized CSC-like cells using a fluorescent protein system.

A robust measurement point for dose verification in delivery quality assurance for a robotic radiosurgery system

Abstract In this CyberKnife® dose verification study, we investigated the effectiveness of the novel potential error (PE) concept when applied to the determination of a robust measurement point for targeting errors. PE was calculated by dividing the differences between the maximum increases and decreases in dose distributions by the original distribution after obtaining the former by shifting the source-to-axis and off-axis distances of each beam by ±1.0 mm. Thus, PE values and measurement point dose heterogeneity were analyzed in 48 patients who underwent CyberKnife radiotherapy. Sixteen patients who received isocentric dose delivery were set as the control group, whereas 32 who received non-isocentric dose delivery were divided into two groups of smaller PE (SPE) and larger PE (LPE) by using their median PE value. The mean dose differences (± standard deviations) were 1.0 ± 0.9%, 0.5 ± 1.4% and 4.1 ± 2.8% in the control, SPE and LPE groups, respectively. We observed significant correlations of the dose difference with the PE value (r = 0.582, P < 0.001) and dose heterogeneity (r = 0.471, P < 0.001). We concluded that when determining a robust measurement point for CyberKnife point dose verification, PE evaluation was more effective than the conventional dose heterogeneity-based method that introduced optimal measurement point dose heterogeneity of <10% across the detector.

Robust plan optimization using edge-enhanced intensity for intrafraction organ deformation in prostate intensity-modulated radiation therapy

This study evaluated a method for prostate intensity-modulated radiation therapy (IMRT) based on edge-enhanced (EE) intensity in the presence of intrafraction organ deformation using the data of 37 patients treated with step-and-shoot IMRT. On the assumption that the patient setup error was already accounted for by image guidance, only organ deformation over the treatment course was considered. Once the clinical target volume (CTV), rectum, and bladder were delineated and assigned dose constraints for dose optimization, each voxel in the CTV derived from the DICOM RT-dose grid could have a stochastic dose from the different voxel location according to the probability density function as an organ deformation. The stochastic dose for the CTV was calculated as the mean dose at the location through changing the voxel location randomly 1000 times. In the EE approach, the underdose region in the CTV was delineated and optimized with higher dose constraints that resulted in an edge-enhanced intensity beam to the CTV. This was compared to a planning target volume (PTV) margin (PM) approach in which a CTV to PTV margin equivalent to the magnitude of organ deformation was added to obtain an optimized dose distribution. The total monitor units, number of segments, and conformity index were compared between the two approaches, and the dose based on the organ deformation of the CTV, rectum, and bladder was evaluated. The total monitor units, number of segments, and conformity index were significantly lower with the EE approach than with the PM approach, while maintaining the dose coverage to the CTV with organ deformation. The dose to the rectum and bladder were significantly reduced in the EE approach compared with the PM approach. We conclude that the EE approach is superior to the PM with regard to intrafraction organ deformation.

Abstract 4585: Radiation combined with checkpoint blockades enhanced antitumor efficacy for osteosarcoma

Osteosarcoama is one of the common malignancies at bone in children and adolescent. Recent study demonstrated that combination of anti-PD-L1 and anti-CTLA-4 antibodies provided grate control of metastatic osteosarcoma. However, a strong local treatment strategy including surgery or high precision radiation therapy is necessary to elucidate osteosarcoma. Radiation therapy plays an important role in local control for malignant tumors but previous studies demonstrated that radiation enhanced immune response in which not only local tumor regression at irradiated sites but also regression of metastatic tumor outside the radiation field were observed. Although this phenomenon, so called the abscopal effect, is rarely seen, recent studies demonstrated that combination of X-ray irradiation with checkpoint blockades provided higher probability of the abscopal effect for some kind of tumors. However, the effect of x-ray irradiation combined with checkpoint blockade on the abscopal effect for osteosarcoma has been totally unknown. We investigated whether local X-ray irradiation combined with the anti-PD-L1 and anti-CTLA-4 antibodies enhances local and distant antitumor efficacy for osteosarcoma. LM8 mouse osteosarcoma cells were inoculated into both legs of C3H mice. Mice were treated by 10 Gy X-ray irradiation alone to the tumor in the one side leg (RAD group) at day 12, 150 ug of anti-PD-L1 and anti-CTLA-4 antibodies (P1C4 group) at days 9, 12, and 15, or those combination (COMB group). Administration of anti-PD-L1 and anti-CTLA-4 antibodies provided tumor growth delay or complete response at day 37 for about 20% of the mice. X-ray irradiation strongly inhibited tumor growth at irradiated tumor but not in unirradiated tumor. On the other hand, the combination therapy provided the strongest tumor growth inhibition not only at irradiated tumor but also at unirradiated tumor for about 89% of the mice. Accordingly, lung metastasis in mice in COMB group was strongly reduced by 97% with the significant survival benefit compared with the mice in P1C4 group. Flow cytometric analysis revealed that mice in COMB group significantly recruited CD8 tumor infiltrating lymphocytes with moderate reduction of regularly T cells (Tregs), thereby increasing the CD8/Treg ratio. Furthermore, quantitative real time PCR showed significant induction of PD-L1 on irradiated LM8 cells in vitro. The radiation-induced upregulations of PD-L1, B7-1, and B7-2, the ligands of CTLA-4, were also confirmed by flow cytometry, indicating that the enhanced efficacy anti-PDL1 and CTLA-4 antibodies may associate with these upregulations by radiation. These results suggest that X-ray irradiation contributes to the enhancement of the efficacy for the distant metastasis as well as local control in the treatment of anti-PD-L1 and anti-CTLA-4 antibodies for osteosarcoma. Our data provide a rational to establish a new therapeutic strategy and start up a clinical trial for osteosarcoma. Citation Format: Yutaka Takahashi, Tomohiro Yasui, Keisuke Tamari, Kazumasa Minami, Masahiko Koizumi, Yuji Seo, Fumiaki Isohashi, Keisuke Ohtani, Ryosuke Kambe, Kazuhiko Ogawa. Radiation combined with checkpoint blockades enhanced antitumor efficacy for osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4585. doi:10.1158/1538-7445.AM2017-4585

Dose–volume analysis of predictors for chronic gastrointestinal complications in patients with cervical cancer treated with postoperative concurrent chemotherapy and whole-pelvic radiation therapy

The purpose of this study is to evaluate dose–volume histogram (DVH) predictors for the development of chronic gastrointestinal (GI) complications in patients with cervical cancer who have undergone postoperative concurrent chemotherapy and whole-pelvic radiation therapy (WPRT). The subjects were 135 patients who had undergone postoperative WPRT with concurrent nedaplatin-based chemotherapy between 2000 and 2014. Associations between selected DVH parameters and the incidence of chronic GI complications of G3 or higher were evaluated. Chronic GI complications of severity G3 occurred in 18 (13%) patients. Patients with GI complications had significantly greater V5–V45, mean dose and the generalized equivalent uniform dose (gEUD) of the small bowel loops, compared with those without GI complications. V30–V45, mean dose and gEUD of the bowel bag also showed significant differences between patients with and without GI complications. In contrast, no parameter for the large bowel loop was correlated with GI complications. Receiver operating characteristics curve analysis indicated that V30–V45 of the small bowel loops were better predictors than these respective parameters for the bowel bag. Next, patients were divided into four groups based on the median V15 and V40 of the small bowel loops. The group with both a high V15 and a high V40 showed a significantly higher probability of chronic GI complications. In conclusion, the small bowel loops are better predictors of chronic GI complications compared with the bowel bag, and a relatively high-dose volume (e.g. V40) of the small bowel loops is a useful predictor of chronic GI complications.