Oncolytic Adenovirus-Induced Autophagy: Tumor-Suppressive Effect and Molecular Basis

Autophagy is a catabolic process that produces energy through lysosomal degradation of intracellular organelles. Autophagy functions as a cytoprotective factor under physiological conditions such as nutrient deprivation, hypoxia, and interruption of growth factors. On the other hand, infection with pathogenic viruses and bacteria also induces autophagy in infected cells. Oncolytic virotherapy with replication-competent viruses is thus a promising strategy to induce tumor-specific cell death. Oncolytic adenoviruses induce autophagy and subsequently contribute to cell death rather than cell survival in tumor cells. We previously developed a telomerase-specific replication-competent oncolytic adenovirus, OBP-301, which induces cell lysis in tumor cells with telomerase activities. OBP-301-mediated cytopathic activity is significantly associated with induction of autophagy biomarkers. In this review, we focus on the tumor-suppressive role and molecular basis of autophagic machinery induced by oncolytic adenoviruses. Addition of tumor-specific promoters and modification of the fiber knob of adenoviruses supports the oncolytic adenovirus-mediated autophagic cell death. Autophagy is cooperatively regulated by the E1-dependent activation pathway, E4-dependent inhibitory pathway, and microRNA-dependent fine-tuning. Thus, future exploration of the functional role and molecular mechanisms underlying oncolytic adenovirus-induced autophagy would provide novel insights and improve the therapeutic potential of oncolytic adenoviruses

Fluorescence-guided surgery of a highly-metastatic variant of human triple-negative breast cancer targeted with a cancer-specific GFP adenovirus prevents recurrence.

We have previously developed a genetically-engineered GFP-expressing telomerase-dependent adenovirus, OBP-401, which can selectively illuminate cancer cells. In the present report, we demonstrate that targeting a triple-negative high-invasive human breast cancer, orthotopically-growing in nude mice, with OBP-401 enables curative fluorescence-guided surgery (FGS). OBP-401 enabled complete resection and prevented local recurrence and greatly inhibited lymph-node metastasis due to the ability of the virus to selectively label and subsequently kill cancer cells. In contrast, residual breast cancer cells become more aggressive after bright (white)-light surgery (BLS). OBP-401-based FGS also improved the overall survival compared with conventional BLS. Thus, metastasis from a highly-aggressive triple-negative breast cancer can be prevented by FGS in a clinically-relevant mouse model

Tumor-targeting adenovirus OBP-401 inhibits primary and metastatic tumor growth of triple-negative breast cancer in orthotopic nude-mouse models.

Our laboratory previously developed a highly-invasive, triple-negative breast cancer (TNBC) variant using serial orthotopic implantation of the human MDA-MB-231 cell line in nude mice. The isolated variant was highly-invasive in the mammary gland and lymphatic channels and metastasized to lymph nodes in 10 of 12 mice compared to 2 of 12 of the parental cell line. In the present study, the tumor-selective telomerase dependent OBP-401 adenovirus was injected intratumorally (i.t.) (1 × 108 PFU) when the high-metastatic MDA-MB-231 primary tumor expressing red fluorescent protein (MDA-MB-231-RFP) reached approximately 500 mm3 (diameter; 10 mm). The mock-infected orthotopic primary tumor grew rapidly. After i.t. OBP-401 injection, the growth of the orthotopic tumors was arrested. Six weeks after implantation, the fluorescent area and fluorescence intensity showed no increase from the beginning of treatment. OBP-401 was then injected into high-metastatic MDA-MB-231-RFP primary orthotopic tumor growing in mice which already had developed metastasis within lymphatic ducts. All 7 of 7 control mice subsequently developed lymph node metastasis. In contrast, none of 7 mice which received OBP-401 had lymph node metastasis. Seven of 7 control mice also had gross lung metastasis. In contrast, none of the 7 mice which received OBP-401 had gross lung metastasis. Confocal laser microscopy imaging demonstrated that all control mice had diffuse lung metastases. In contrast, all 7 mice which received OBP-401 only had a few metastatic cells in the lung. OBP-401 treatment significantly extended survival of the treated mice

Massive Deposition and Accumulation of Hydroxyapatite Crystal after Total Hip Arthroplasty: A Case Report

We presented a case in which massive hydroxyapatite accumulation was observed around the artificial hip joint. A 66-year-old female showed a massive accumulation of fluid in and around the hip joint, and milk-like aspirate was obtained. Her aspirate culture was negative, and sediment analysis by X-ray diffraction showed that its component was hydroxyapatite. Since pain was mild, the patient was treated conservatively. To our knowledge, this is the first case in which liquid hydroxyapatite (milk of calcium) was accumulated around the artificial hip joint

Radiosensitization by telomerase-dependent oncolytic adenovirus

DNA修復機能阻害は放射線感受性を増強させるため，DNA修復に関与する因子の阻害剤は放射線増感剤となり得る．我々の開発したテロメラーゼ依存的腫瘍融解アデノウイルス製剤OBP-301（テロメライシン）は，アデノウイルスE1B55kDaタンパクを介して細胞のDNA修復に重要な役割を果たすMRN複合体（Mre11，Rad50，NBS1）を分解する機能を有する．このMRN複合体の分解によりATM（ataxia-telangiectasia mutated）の活性化が抑制され結果的にDNA修復機構が阻害される．我々はOBP-301と放射線との併用が強力な相乗効果を生み出すことをマウスの皮下腫瘍モデルおよび食道癌同所性モデルにおいて証明した．これらの結果はOBP-301が将来有望な放射線増感剤となり得ることだけでなく，E1B55kDaタンパクを産生する腫瘍融解アデノウイルス製剤と放射線との併用が悪性腫瘍に対する有力な治療戦略となり得ることを示す

A Genetically Engineered Oncolytic Adenovirus Decoys and Lethally Traps Quiescent Cancer Stem-like Cells in S/G(2)/M Phases

Purpose: Because chemoradiotherapy selectively targets proliferating cancer cells, quiescent cancer stem-like cells are resistant. Mobilization of the cell cycle in quiescent leukemia stem cells sensitizes them to cell death signals. However, it is unclear that mobilization of the cell cycle can eliminate quiescent cancer stem-like cells in solid cancers. Thus, we explored the use of a genetically-engineered telomerase-specific oncolytic adenovirus, OBP-301, to mobilize the cell cycle and kill quiescent cancer stem-like cells. Experimental Design: We established CD133(+) cancer stem-like cells from human gastric cancer MKN45 and MKN7 cells. We investigated the efficacy of OBP-301 against quiescent cancer stem-like cells. We visualized the treatment dynamics of OBP-301 killing of quiescent cancer stem-like cells in dormant tumor spheres and xenografts using a fluorescent ubiquitination cell-cycle indicator (FUCCI). Results: CD133(+) gastric cancer cells had stemness properties. OBP-301 efficiently killed CD133(+) cancer stem-like cells resistant to chemoradiotherapy. OBP-301 induced cell-cycle mobilization from G(0)-G(1) to S/G(2)/M phases and subsequent cell death in quiescent CD133(+) cancer stem-like cells by mobilizing cell-cycle-related proteins. FUCCI enabled visualization of quiescent CD133(+) cancer stem-like cells and proliferating CD133(-) non-cancer stem-like cells. Three-dimensional visualization of the cell-cycle behavior in tumor spheres showed that CD133(+) cancer stem-like cells maintained stemness by remaining in G(0)-G(1) phase. We showed that OBP-301 mobilized quiescent cancer stem-like cells in tumor spheres and xenografts into S/G(2)/M phases where they lost viability and cancer stem-like cell properties and became chemosensitive. Conclusion: Oncolytic adenoviralinfection is an effective mechanism of cancer cell killing in solid cancer and can be a new therapeutic paradigm to eliminate quiescent cancer stem-like cells

Biological Ablation of Sentinel Lymph Node Metastasis in Submucosally Invaded Early Gastrointestinal Cancer

Currently, early gastrointestinal cancers are treated endoscopically, as long as there are no lymph node metastases. However, once a gastrointestinal cancer invades the submucosal layer, the lymph node metastatic rate rises to higher than 10%. Therefore, surgery is still the gold standard to remove regional lymph nodes containing possible metastases. Here, to avoid prophylactic surgery, we propose a less-invasive biological ablation of lymph node metastasis in submucosally invaded gastrointestinal cancer patients. We have established an orthotopic early rectal cancer xenograft model with spontaneous lymph node metastasis by implantation of green fluorescent protein (GFP)-labeled human colon cancer cells into the submucosal layer of the murine rectum. A solution containing telomerase-specific oncolytic adenovirus was injected into the peritumoral submucosal space, followed by excision of the primary rectal tumors mimicking the endoscopic submucosal dissection (ESD) technique. Seven days after treatment, GFP signals had completely disappeared indicating that sentinel lymph node metastasis was selectively eradicated. Moreover, biologically treated mice were confirmed to be relapse-free even 4 weeks after treatment. These results indicate that virus-mediated biological ablation selectively targets lymph node metastasis and provides a potential alternative to surgery for submucosal invasive gastrointestinal cancer patients

Fluorescence virus-guided capturing system of human colorectal circulating tumour cells for non-invasive companion diagnostics

Background Molecular-based companion diagnostic tests are being used with increasing frequency to predict their clinical response to various drugs, particularly for molecularly targeted drugs. However, invasive procedures are typically required to obtain tissues for this analysis. Circulating tumour cells (CTCs) are novel biomarkers that can be used for the prediction of disease progression and are also important surrogate sources of cancer cells. Because current CTC detection strategies mainly depend on epithelial cell-surface markers, the presence of heterogeneous populations of CTCs with epithelial and/or mesenchymal characteristics may pose obstacles to the detection of CTCs. Methods We developed a new approach to capture live CTCs among millions of peripheral blood leukocytes using a green fluorescent protein (GFP)-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401, TelomeScan). Results Our biological capturing system can image epithelial and mesenchymal tumour cells with telomerase activities as GFP-positive cells. After sorting, direct sequencing or mutation-specific PCR can precisely detect different mutations in KRAS, BRAF and KIT genes in epithelial, mesenchymal or epithelial–mesenchymal transition-induced CTCs, and in clinical blood samples from patients with colorectal cancer. Conclusions This fluorescence virus-guided viable CTC capturing method provides a non-invasive alternative to tissue biopsy or surgical resection of primary tumours for companion diagnostics

成長する鳥類胚における心拍数解析 : 成長モードと卵質量の効果

Bird embryos may be regarded as developing in their thermo-neutral zone, at rest, and stay in the egg for a fixed period of time until hatching. It is therefore interesting to investigate if they follow the same "rule" set for adult homeotherms, which states that, within a taxonomically or functionally defined category such as mammals or birds, the number of heart beats throughout the life span (sL) is more or less constant. This rule stems from the allometric relationships between heart rate(HR) and body mass (mB) and between sL and mB. As a step towards understanding the general allometric nature of avian embryonic physiology we analyzed the HR values of avian embryos in relation to their incubation span (sI). Data from 30 species were selected from the scientific literature for the analyses. Values obtained from invasive methods which were judged to grossly alter natural incubation conditions, or from undefined or unmatched temperature conditions were not used. These include most values obtained below the first 30% of the incubation. Also, data obtained after internal pipping were discarded since hatching activity influences them. Values for sI and egg mass (mE) as representative of embryonic mass were also collected. Embryonic HR was normalized to 70.1-80% sI. At 20.1-30% sI it was only 85% of the value at 70.1-80% sI and increased to a plateau at about 50.1-60% sI. It was almost constant among species between 50.1- 60% sI and pre-internal pipping(PIP) time and thus, the mean HR value between 50.1-60% sI and 90.1-100% excluding pipped eggs (MHR) was taken as a representative value for each given species. The MHR(bpm) and the corresponding sI (d) values for the 30 species, scaled with mx (g) as follows: MHR=371.1 mE\u27-0.112\u27 and: sI=12.29 mE\u27+0.209\u27. Both powers were significantly different from 0. The product of MHR and sr(MHR ・ sr), representing the total number of heartbeats throughout the incubation, scaled with mE, for the entire data set as follows: MHR sr = 6.565 ・ 10+6 ・ mE+0.096, where the + 0.096 power is significantly different fram 0. Vaiues for MHR ・ sI, from embryos of altricial birds tended to concentrate at the low mE end of the plot while those of the precocial ones tended towards the high end. Separate analyses showed that the mE power for the combined altricial and semi-altricial species (ASA), and the combined precocial and semi precocial species (PSP), of log MHR ・ sr against log mE regressions, were both insignificantly different from 0. Thus, means of MHR ・ sI for ASA and PSP were calculated. The mean ASA value of 7.27 ・ 10+6 heartbeats for MHR ・ sr, was significantly different from the mean PSP value of 10.93 ・ 10+6. The difference of 3.66 ・ 10+6 (33.5%) heart beats can be attributed to either the more advanced stage of the PSP hatchlings at hatch, to the larger mE values of these hatchlings, to the difference on water fractien of the hatchlings or all. The result of a linear regression of MHR ・ sI (in d-1 units) against the rate of sI compietion (the inverse of incubation span, fI; d-1) was: MHR ・ 10-6 = 0.205 + 3.940 ・ fI. Thus, the faster is the average rate of development accomplished per day (shorter incubation) the higher is daily heart rate. Data tended to cluster such that large eggs, mostly of the PSP type with reiatively low MHR, complete 2 to 4% of their incubation per day, whiｌe small, ASA type eggs with relatively high MHR, complete 6 to 8% of their incubation time per day. We conclude that, at this stage of knowledge, the data is insufficient to resolve whether the different modes of hatch stage alone can explain differences in the total number of heartbeats throughout embryonic life among all bird species, or egg mass and water content differences contribute variability. This should be investigated on a larger sample of species in more depth.特集 : 「動物の心拍リズム」国際シンポジウム発表論文選

Current Femoral Stem Fixation Selection in Hip-Fracture Bipolar Hemiarthroplasties, and Factors Affecting Surgeons’ Confidence in Their Ability to Teach about Cemented Stems: A Questionnaire in a Region of Japan with Super-Aged Patients

Japan’s hip fracture management guidelines now recommend the use of cemented stems in cases of bone fragility. However, the current stem selection practices in bipolar hemiarthroplasty (BHA) in a super-aging area in Japan remain unclear. This study aimed to examine the stem selection policies, the surgeons’ concerns about cemented stems, and factors affecting their confidence in their ability to coach others on cemented stem procedures. Ninety-four orthopedic surgeons (27 facilities) responded to our web-based questionnaire conducted in January/February 2022. Cementless stem was the first choice of 97.8% of the surgeons; <15% of the respondents expected to increase their use of cemented stems in the future. The cement technique was the greatest concern; almost half of the surgeons described having insufficient experience with cemented stems. The factor that most affected the surgeons’ expertise in using cemented stems is the number of surgeries they had conducted with a cemented stem (multivariable analysis odds ratio 8.42, p=0.001). Greater experience was associated with increased expertise of the surgeons in using cemented stems, with a threshold of 11 cases showing sensitivity of 41.7% and specificity of 98.3% for their confidence to instruct cemented stems