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Background Gadolinium-enhanced magnetic resonance imaging and T2-weighted imaging/fluid-attenuated inversion recovery imaging are used to determine the efficacy of bevacizumab (Bev) against glioblastoma (GBM). Positron emission tomography (PET) using 18F-fluoromisonidazole (FMISO) reflects hypoxia in the tumor microenvironment (TME). This study compared FMISO-PET findings for alterations in tumor oxygenation in the TME of GBM during Bev treatment. Materials and Methods We retrospectively reviewed data from 7 patients with newly diagnosed IDH (isocitrate dehydrogenase)-wildtype GBM who underwent FMISO-PET during follow-up. Three patients received preoperative neoadjuvant Bev (neo-Bev) followed by radiation therapy and temozolomide, and subsequently underwent surgical resection. Re-operation was performed for recurrence. FMISO-PET was performed at each time point. Four patients who underwent tumor resection after FMISO-PET without any preoperative interventions were also included as a Control group. Surgically obtained tumor tissues were analyzed for expression of a marker of hypoxia (carbonic anhydrase; CA9), stem cell markers (nestin, FOXM1), and immunoregulatory molecules (CD163, FOXP3, PD-1, PD-L1) by immunohistochemistry (IHC). Results All 3 patients treated with preoperative chemoradiotherapy showed reduced FMISO accumulation (maximum tumor-to-blood ratio and hypoxic volume) and decreases in CA9 and FOXM1 compared with Controls. Two cases in the preoperative neo-Bev group showed recurrence with increasing FMISO accumulation. IHC showed increased CA9- and FOXM1-positive cells at recurrence in both cases. A trend toward fewer CA9-positive cells was seen in patients with low FMISO accumulation both with and without neo-Bev (r = 0.90, p = 0.006). Expressions of immunoregulatory molecules tended to be lower after neo-Bev compared with the Control group and increased at recurrence, but these differences were not significant. Conclusion FMISO-PET effectively visualized improvements in TME oxygenation after preoperative chemoradiotherapy including Bev. Increased FMISO accumulation at the time of recurrence, even under Bev treatment, suggests that FMISO-PET might be useful for monitoring the duration of Bev efficacy by reflecting tumor oxygenation.

Differentially expressed chemokine genes (fold change >2) in esophageal cancer tissues

Extracellular vesicles (EV) from cancer-associated fibroblasts (CAF) are composed of diverse payloads. Although CAFs impact the aggressive characteristics of gastric cancer cells, the contribution of CAF-EV to gastric cancer progression has not been elucidated. Here, we investigated the molecular mechanism of the changes in gastric cancer characteristics induced by CAF-EV. CAF abundance in gastric cancer tissues was associated with poor prognosis of patients with gastric cancer receiving chemotherapy. Moreover, CAF-EV induced tubular network formation and drug resistance of gastric cancer cells in the extracellular matrix (ECM). Comprehensive proteomic analysis of CAF-EV identified that Annexin A6 plays a pivotal role in network formation and drug resistance of gastric cancer cells in the ECM via activation of β1 integrin-focal adhesion kinase (FAK)-YAP. A peritoneal metastasis mouse model revealed that CAF-EV induced drug resistance in peritoneal tumors, and inhibition of FAK or YAP efficiently attenuated gastric cancer drug resistance in vitro and in vivo. These findings demonstrate that drug resistance is conferred by Annexin A6 in CAF-EV and provide a potential avenue for overcoming gastric cancer drug resistance through the inhibition of FAK-YAP signaling in combination with conventional chemotherapeutics.Significance:This study elucidates a novel molecular mechanism through which Annexin A6 in CAF-EV activates FAK-YAP by stabilizing β1 integrin at the cell surface of gastric cancer cells and subsequently induces drug resistance.

Purpose: Global DNA hypomethylation plays a crucial role in genomic instability and carcinogenesis. DNA methylation of the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of the global DNA methylation level, and is attracting interest as a useful marker for predicting cancer prognosis. Our previous study using more than 200 esophageal squamous cell carcinoma (ESCC) specimens demonstrated the significant relationship between LINE-1 hypomethylation and poor prognosis. However, the mechanism by which LINE-1 hypomethylation affects aggressive tumor behavior has yet to be revealed.Experimental Design: To examine the relationship between LINE-1 hypomethylation and DNA copy number variations, we investigated LINE-1–hypomethylated and LINE-1–hypermethylated ESCC tumors by comparative genomic hybridization array.Results: LINE-1–hypomethylated tumors showed highly frequent genomic gains at various loci containing candidate oncogenes such as CDK6. LINE-1 methylation levels were significantly associated with CDK6 mRNA and CDK6 protein expression levels in ESCC specimens. In our cohort of 129 patients with ESCC, cases with CDK6-positive expression experienced worse clinical outcome compared with those with CDK6-negative expression, supporting the oncogenic role of CDK6 in ESCC. In addition, we found that the prognostic impact of LINE-1 hypomethylation might be attenuated by CDK6 expression.Conclusion: LINE-1 hypomethylation (i.e., global DNA hypomethylation) in ESCC might contribute to the acquisition of aggressive tumor behavior through genomic gains of oncogenes such as CDK6. Clin Cancer Res; 20(5); 1114–24. ©2014 AACR.

PDF file - 55K, Signal counts of CDK6 and chromosome 7centromere in TE11 cells.

PDF file - 56K, LINE-1 methylation level in 129 esophageal cancers, and the association with the clinical and tumor features.

PDF file - 40K, Clinical and pathological characteristics of 6 cases selected for CGH array.

Previously reported Fusobacterium in cancer tissues and patient prognosis

Real-time imaging of AGS cells treated with CAF-CM in the ECM.

Real-time imaging of AGS cells treated with normal medium in the ECM.

PDF file - 195K, The result of CGH arrays in 6 cases.

Purpose: Fusobacterium nucleatum (F. nucleatum) is a component of the human microbiome that primarily inhabits the oral cavity. It causes periodontal disease and has also been implicated in the development of human cancers. Although there are several reports of the relationship between F. nucleatum and the clinical outcome in human cancers, its prognostic significance in esophageal cancer remains unclear.Experimental Design: We quantified F. nucleatum DNA in 325 resected esophageal cancer specimens by qPCR. Significant pathways in F. nucleatum–positive esophageal cancer tissues were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using microarray data.Results: Esophageal cancer tissues contained significantly more F. nucleatum DNA than matched normal esophageal mucosa (P = 0.021; n = 60). F. nucleatum DNA was detected in 74 of 325 cases (23%). F. nucleatum DNA positivity was significantly associated with tumor stage, but not with sex, age, performance status, tobacco use, alcohol use, histology, tumor location, or preoperative treatment. F. nucleatum DNA positivity was also significantly associated with cancer-specific survival [log-rank P = 0.0039; univariate HR = 2.01; 95% confidence interval (CI), 1.22–3.23; P = 0.0068; multivariate HR = 1.78; 95% CI, 1.06–2.94; P = 0.031]. The top-ranked KEGG pathway in F. nucleatum–positive tissues was “cytokine–cytokine receptor interaction.” A significant relationship between F. nucleatum and the chemokine CCL20 was validated by IHC.Conclusions: F. nucleatum in esophageal cancer tissues was associated with shorter survival, suggesting a potential role as a prognostic biomarker. F. nucleatum might also contribute to aggressive tumor behavior through activation of chemokines, such as CCL20. Clin Cancer Res; 22(22); 5574–81. ©2016 AACR.

Supplementary Figure 1: GC cells do not exhibit network formation or subsequent drug resistance after treatment with GC-CM in the ECM. Supplementary Figure 2: CAF-CM enhances the tubular network formation and anti-apoptotic signaling of GC cells. Supplementary Figure 3. Protocol for RNA sequencing and gene expression determination. Supplementary Figure 4. Identification of CAF-derived EVs. Supplementary Figure 5. Annexin A6 in CAF-EVs is correlated with tubular network formation. Supplementary Figure 6. CAF-EVs enhance the expression of integrins in GC cells. Supplementary Figure 7. Loss-of-function study using ITGB1 siRNA in CAFs and a GC cell line (AGS). Supplementary Figure 8. Loss-of-function study using ANXA6 siRNA in CAFs. Supplementary Figure 9. Increases in the YAP levels are correlated with increases in its nuclear localization. Supplementary Figure 10. Protocol used for evaluation of the chemoresistance of intraperitoneal mice after treatment with siCTRL- or siANXA6-transfected CAF-EVs.

PDF file - 188K, Cyclin D1 expression, LINE-1 methylation level, and patient prognosis in esophageal cancer.

The purpose of this study was to assess the diagnostic value of [18F]fluoromisonidazole (FMISO) and 2-deoxy-2-[18F]fluoro-D-glucose (FDG) PET to discriminate primary central nervous system lymphoma (PCNSL) from glioblastoma.FMISO and FDG PET/CT scans before therapy obtained in 13 patients with PCNSL and in 62 patients with glioblastoma were retrospectively reviewed. PET results were evaluated by visual and semiquantitative analysis. For semiquantitative analysis, the maximum standardized uptake value (SUV) for tumor (T) and the mean SUV for normal contralateral hemisphere (N) were calculated, and the tumor-to-normal (T/N) ratio was determined. The performance in discriminating PCNSL and glioblastoma was evaluated using a receiver-operating characteristics analysis. Area-under-the-curve (AUC) values for the discrimination were calculated.On visual analysis, 54% of PCNSL and 89% of glioblastoma showed positive on FMISO PET. All patients with PCNSL and glioblastoma were FDG positive. FMISO T/N ratio in PCNSL (mean ± SD = 1.80 ± 0.59) was significantly lower than that in glioblastoma (mean ± SD = 2.75 ± 0.84) (P 0.001). FDG T/N ratio in PCNSL (mean ± SD = 3.01 ± 1.11) was significantly higher than that in glioblastoma (mean ± SD = 1.77 ± 0.79) (P 0.001). For discrimination of patients with PCNSL from glioblastoma, the AUC values for the FMISO T/N ratio, FDG T/N ratio and combination of the two parameters were 0.833, 0.825 and 0.900, respectively.FMISO PET is as helpful for differentiating PCNSL from glioblastoma as FDG PET.

d-allulose is a low-calorie rare sugar. It has been reported that d-allulose supplementation significantly inhibits diet-induced hepatic fat accumulation. However, the underlying molecular mechanisms remain unclear. This study elucidates the mechanism underlying the suppressive effect of d-allulose on hepatic fat accumulation in terms of miRNA regulation.Male C57BL/6 mice are divided into three experimental groups-normal diet and distilled water (CC group), high-fat diet (HFD) and distilled water (HC group), and HFD and 5% d-allulose solution (HA group)-and fed the respective diets for 8 weeks. Weight gain is significantly lower in the HA group than that in the HC group, although the caloric intake is the same in both. Histological analysis of liver tissues reveals excessive lipid accumulation in the HC group; this is greatly attenuated in the HA group. Real-time PCR and western blot analyses demonstrate that, compared to the HC group, the HA group exhibits decreased hepatic PPARγ and CD36 expression. Hepatic miR-130 expression levels are higher in the HA group than those in the CC and HC groups.These results indicate that miRNA changes associated with PPARγ may underlie the suppression of hepatic lipid accumulation induced by d-allulose intake.

Although regenerative cell therapy is expected to be an alternative treatment for peripheral artery disease (PAD), many regenerative cell therapies have failed to show sufficient efficacy in clinical trials. Most preclinical studies have used acute ischemia models, despite PAD being a chronic disease. In addition, aging and atherosclerosis decrease the quality of a patient's stem cells. Therefore, using a non-acute ischemic preclinical model and stem cells with high regenerative potency are important for the development of effective regenerative therapy. In this study, we assessed the tissue regenerative potential of umbilical cord-derived mesenchymal stromal cells (UCMSCs), which could potentially be an ideal cell source, in a rat model of established ischemia.Methods and Results: The regenerative capacity of UCMSCs was analyzed in terms of angiogenesis and muscle regeneration. In vitro analysis showed that UCMSCs secrete high amounts of cytokines associated with angiogenesis and muscle regeneration. In vivo experiments in a rat non-acute ischemia model showed significant improvement in blood perfusion after intravenous injection of UCMSCs compared with injection of culture medium or saline. Histological analysis revealed UCMSCs injection enhanced angiogenesis, with an increased number of von Willebrand factor-positive microcapillaries, and improved muscle regeneration.These results suggest that intravenous administration of UCMSCs may be useful for treating patients with PAD.

Glioma is the most common type of central nervous system tumor reported worldwide. Current imaging technologies have limitations in the diagnosis and assessment of glioma. The present study aimed to confirm the diagnostic efficacy and safety of anti-1-amino-3-[18F]fluorocyclobutane carboxylic acid (18F-fluciclovine; anti-[18F]FACBC) as a radiotracer for patients undergoing combined positron emission tomography and computed tomography (PET/CT) for suspected glioma. Combined data from two multicenter, open-label phase III clinical trials were evaluated for this study. The two trials enrolled patients with suspected high- or low-grade glioma on the basis of clinical symptoms, clinical course, and magnetic resonance imaging findings, and who were scheduled for tumor resection surgery. Patients fasted for ≥ 4 h and received 2 mL of 18F-fluciclovine (radioactivity dose 78.3–297.0 MBq), followed by a 10-min PET scan 10–50 min after injection. The primary efficacy endpoint was the positive predictive value (PPV) of the gadolinium contrast-enhanced T1-weighted image negative [Gd (–)] and 18F-fluciclovine PET-positive [PET ( +)] area of the scans, using the histopathological diagnosis of the tissue sampled from that area as the standard of truth. All adverse events reported during the study were recorded for safety analysis. A total of 45 patients aged 23–89 years underwent 18F-fluciclovine PET; 31/45 patients (68.9%) were male, and 30/45 patients (66.7%) were suspected to have high-grade glioma. The PPV of 18F-fluciclovine PET in the Gd (–) PET ( +) area was 88.0% (22/25 areas, 95% confidence interval: 70.0–95.8). The extent of planned tumor resection was modified in 47.2% (17/36 cases) after 18F-fluciclovine PET scan, with an extension of area in 30.6% (11/36 cases) and reduction in 16.7% (6/36 cases). Furthermore, tissue samples collected from PET ( +) areas tended to have a higher malignancy grade compared with those from PET (–) areas. Overall, 18F-fluciclovine was well tolerated. 18F-fluciclovine PET/CT is useful for determining the extent of tumor resection at surgical planning, and may serve as a safe and effective diagnostic tool for patients with suspected glioma. These trials were registered in the Japan Pharmaceutical Information Center Clinical Trials Information (JapicCTI-152986, JapicCTI-152985).

To date, few clinical observations have been reported for changes of alternative angiogenic factors (other than vascular endothelial growth factor [VEGF]) in human gliomas under bevacizumab (Bev). The elucidation of the mechanism of action and resistance is imperative to overcome resistance to Bev and develop a more effective therapy. This study aims to investigate the status and change of alternative angiogenic factors regarding Bev usage.The present study used 56 glioblastoma tissues obtained at 3 different settings: tumors of initial resection (naive Bev group), tumors resected following Bev therapy (effective Bev group), and recurrent tumors after Bev therapy (refractory/autopsied Bev group). The expression of alternative angiogenic factors including basic fibroblast growth factor (bFGF), placental growth factor (PlGF), Angiopoietin1 (Ang-1), Angiopoietin2(Ang-2) and EphrinA2 were investigated via immunohistochemistry. In large tumor vasculature (diameter >15μm), the expression of PlGF was significantly higher in the effective Bev group than naive Bev group. The expression of Ang-2 was persistently suppressed in the refractory/autopsied Bev group. In microtumor vasculature (diameter

Background In chronic limb-threatening ischemia, maintenance or recovery of ambulatory function is an important goal of treatment. This study aimed to develop a predictive model for ambulatory ability 1 year after bypass based on preoperative risk factors, including the Wound, Ischemia, and foot Infection (WIfI) classification. Methods We analyzed 146 patients with chronic limb-threatening ischemia (154 limbs) who underwent bypass to below the knee arteries. The patients were classified into 2 groups based on ambulatory status 1 year postoperatively: postoperative ambulation (99 patients, 104 limbs) and postoperative nonambulation (47 patients, 50 limbs). Various factors associated with postoperative ambulation were analyzed and a predictive model of postoperative ambulation was developed. Results Multivariate logistic regression analysis detected preoperative nonambulatory status, functional nonindependence in daily living, older age, WIfI wound grade 3, chronic obstructive pulmonary disease, and hemodialysis as independent risk factors for postoperative nonambulation. The predictive scoring model (scores ranging from −5.0 to 4.4) comprising these risk factors discriminated the postoperative ambulatory status well: the probabilities of postoperative ambulatory ability were ≥85% in those with a score ≤−2, 50% in those with a score of zero, and ≤15% in those with a score ≥2. The area under the receiver operating characteristic curve was 0.898, indicating good performance of the model. Conclusions Preoperative nonambulatory status, functional nonindependence, advanced age, high WIfI wound grade, chronic obstructive pulmonary disease, and hemodialysis were important predictors of postoperative nonambulatory status. The predictive model will help us identify patients who will benefit from bypass surgery.

Extracellular vesicles (EV) from cancer-associated fibroblasts (CAF) are composed of diverse payloads. Although CAFs impact the aggressive characteristics of gastric cancer cells, the contribution of CAF-EV to gastric cancer progression has not been elucidated. Here, we investigated the molecular mechanism of the changes in gastric cancer characteristics induced by CAF-EV. CAF abundance in gastric cancer tissues was associated with poor prognosis of patients with gastric cancer receiving chemotherapy. Moreover, CAF-EV induced tubular network formation and drug resistance of gastric cancer cells in the extracellular matrix (ECM). Comprehensive proteomic analysis of CAF-EV identified that Annexin A6 plays a pivotal role in network formation and drug resistance of gastric cancer cells in the ECM via activation of β1 integrin-focal adhesion kinase (FAK)-YAP. A peritoneal metastasis mouse model revealed that CAF-EV induced drug resistance in peritoneal tumors, and inhibition of FAK or YAP efficiently attenuated gastric cancer drug resistance in vitro and in vivo. These findings demonstrate that drug resistance is conferred by Annexin A6 in CAF-EV and provide a potential avenue for overcoming gastric cancer drug resistance through the inhibition of FAK-YAP signaling in combination with conventional chemotherapeutics. Significance: This study elucidates a novel molecular mechanism through which Annexin A6 in CAF-EV activates FAK-YAP by stabilizing β1 integrin at the cell surface of gastric cancer cells and subsequently induces drug resistance.

In the field of peripheral artery disease (PAD), regenerative cell therapy has been expected to be a new alternative therapy especially for severely diseased patients. Although various preclinical studies showed promising results, those therapies failed to show sufficient efficacy for actual PAD patients and current guideline do not recommend regenerative therapy for PAD. The disappointing result may be caused by low retention rate of administered substance. Recently we published an article regarding novel method to augment the effect of cell therapy. In this review article, we would like to focus on the importance of improvement in cell survival and prerequisite factors to develop effective regenerative therapy for PAD patients.

Background Preoperative neoadjuvant bevacizumab (neoBev) reduces enhancement and perifocal edema by inhibition of angiogenesis and vascular permeability for glioblastoma (GBM). The aim of this study was to investigate safety and efficacy of neoBev for newly diagnosed GBM through an exploratory prospective multi-center study. METHODS & PATIENTS 15 patients with newly diagnosed GBM were enrolled in the present study. Eligibility was a patient with a brain tumor representing with ring-shaped enhancement and perifocal edema on magnetic resonance imaging (MRI). Based on neuroimage of typical GBM, neoBev and temozolomide (TMZ) were administered prior to craniotomy. Two weeks after neoBev, the tumor volume on T1-weighted gadolinium enhancement (T1Gd) and fluid attenuated inversion recovery (FLAIR) were assessed. Three to four weeks after neoBev and TMZ administration, patients underwent surgical resection. The primary endpoint was feasibility and safety, and the secondary endpoint was efficacy. Adverse events including systemic toxicity and wound healing delay during radiation (RT), TMZ, and Bev combined therapy were carefully monitored throughout clinical course including extent of resection. RESULTS Average of tumor regression rate two weeks after neoBev on T1Gd and FLAIR were 37.0% and 54.0%, respectively. Cerebral blood flow and cerebral blood volume were reduced after neoBev. Patients underwent surgical resection safely without excess blood loss due to less vascular and less degree of brain swelling. Two patients underwent awake surgery without any complications. Postoperative MRI showed that the all tumors were totally removed except one with multiple invasive tumors. Histological diagnosis of all patients was GBM, IDH-1 wild-type. Postoperative course was uneventful without neurological deficit and adverse effects except one postoperative hematoma in the resection cavity and one wound infection due to wound dehiscence. CONCLUSION Preoperative neoBev for newly diagnosed GBM might contribute to safe surgery. Clinical outcome of this therapeutics is now currently under investigation.