Development of a genotoxicity/carcinogenicity assessment method by DNA adductome analysis.

Safety evaluation is essential for the development of chemical substances. Since in vivo safety evaluation tests, such as carcinogenesis tests, require long-term observation using large numbers of experimental animals, it is necessary to develop alternative methods that can predict genotoxicity/carcinogenicity in the short term, taking into account the 3Rs (replacement, reduction, and refinement). We established a prediction model of the hepatotoxicity of chemicals using a DNA adductome, which is a comprehensive analysis of DNA adducts that may be used as an indicator of DNA damage in the liver. An adductome was generated with LC-high-resolution accurate mass spectrometer (HRAM) on liver of rats exposed to various chemicals for 24 h, based on two independent experimental protocols. The resulting adductome dataset obtained from each independent experiment (experiments 1 and 2) and integrated dataset were analyzed by linear discriminant analysis (LDA) and found to correctly classify the chemicals into the following four categories: non-genotoxic/non-hepatocarcinogens (-/-), genotoxic/non-hepatocarcinogens (+/-), non-genotoxic/hepatocarcinogens (-/+), and genotoxic/hepatocarcinogens (+/+), based on their genotoxicity/carcinogenicity properties. A prototype model for predicting the genotoxicity/carcinogenicity of the chemicals was established using machine learning methods (using random forest algorithm). When the prototype genotoxicity/carcinogenicity prediction model was used to make predictions for experiments 1 and 2 as well as the integrated dataset, the correct response rates were 89 % (genotoxicity), 94 % (carcinogenicity) and 87 % (genotoxicity/carcinogenicity) for experiment 1, 47 % (genotoxicity), 62 % (carcinogenicity) and 42 % (genotoxicity/carcinogenicity) for experiment 2, and 52 % (genotoxicity), 62 % (carcinogenicity), and 48 % (genotoxicity/carcinogenicity) for the integrated dataset. To improve the accuracy of the toxicity prediction model, the toxicity label was reconstructed as follows; Pattern 1: when +/+ and -/- chemicals were used from the toxicity labels +/+, +/-, -/+ and -/-; and Pattern 2: when +/+, +/-, and -/+ other than -/- were replaced with the label "Others". As a result, chemicals with only +/+ and -/- toxicity labels were used and the correct response rates were approximately 100 % for the measured data in experiment 1, 53 %-66 % for the data in experiment 2, and 59-73 % for the integrated data, all of which were 10 %-30 % higher compared with the data before the label change. In contrast, when the toxicity labels were replaced with -/- and "Others", they reached nearly 100 % in the measured data from experiment 1, 65 %-75 % in the data from experiment 2, and 70 %-78 % in the integrated data, all of which were 10 %-50 % higher compared with the data before the label change.

P53-negative status and gross total resection as predictive factors for autologous tumor vaccine treatment in newly diagnosed glioblastoma patients.

Background: Among primary brain tumors, glioblastoma (GBM) is the most common and aggressive in adults, with limited treatment options. Our previous study showed that autologous formalin-fixed tumor vaccine (AFTV) contributed to prognostic improvements in newly diagnosed GBM patients. However, some patients died early despite the treatment. The discovery of predictive factors in the treatment was warranted for efficient patient recruitment and studies to overcome resistance mechanisms. Identifying prognostic factors will establish AFTV guidelines for patients who may respond to the therapy. Methods: Data from 58 patients with newly diagnosed GBM, including 29 who received standard therapy plus AFTV (AFTV group) and 29 who received standard treatment (control group) were analyzed. Several data including patient age, sex, the extent of removal, and various cell immunohistochemistry (IHC) parameters were also included in the analysis. Results: Both univariate and multivariate analyses revealed that gross total resection (GTR) and negative p53 were associated with a better prognosis only in the AFTV group. In the IHC parameters, CD8 staining status was also one of the predictive factors in the univariate analysis. For blood cell-related data, lymphocyte counts of 1100 or more and monocyte counts of 280 or more before chemo-radiotherapy were significant factors for good prognosis in the univariate analysis. Conclusions: A p53-negative status in IHC and GTR were the predictive factors for AFTV treatment in newly diagnosed GBM patients. Microenvironment-targeted treatment and pretreatment blood cell status may be key factors to enhance therapy effects.

Quantum AI simulator using a hybrid CPU-FPGA approach.

The quantum kernel method has attracted considerable attention in the field of quantum machine learning. However, exploring the applicability of quantum kernels in more realistic settings has been hindered by the number of physical qubits current noisy quantum computers have, thereby limiting the number of features encoded for quantum kernels. Hence, there is a need for an efficient, application-specific simulator for quantum computing by using classical technology. Here we focus on quantum kernels empirically designed for image classification and demonstrate a field programmable gate arrays (FPGA) implementation. We show that the quantum kernel estimation by our heterogeneous CPU-FPGA computing is 470 times faster than that by a conventional CPU implementation. The co-design of our application-specific quantum kernel and its efficient FPGA implementation enabled us to perform one of the largest numerical simulations of a gate-based quantum kernel in terms of features, up to 780-dimensional features. We apply our quantum kernel to classification tasks using the Fashion-MNIST dataset and show that our quantum kernel is comparable to Gaussian kernels with the optimized hyperparameter.

[Benefits and Prospects of VEGF-targeted Anti-angiogenic Therapy and Immunotherapy for High-grade Glioma].

Elucidation of the ecological characteristics of malignant tumors has shown that angiogenesis and an immunosuppressive status in the tumor microenvironment are important for resolving treatment resistance and poor prognosis. Vascular endothelial growth factor(VEGF) and components of related signaling pathway can be targeted by anti-angiogenic therapy. Suppression of abundant angiogenesis using anti-angiogenic agents in high-grade gliomas inhibits rapid neurological deterioration in patients. Additionally, as VEGF promotes the formation of an immunosuppressive tumor microenvironment, anti-angiogenic therapy is expected to contribute to improving the immune status in the tumor microenvironment. In this review, we discuss the role of VEGF-targeted therapy and immunotherapy targeting immune checkpoint inhibitors and macrophages in high-grade gliomas. The authors also discuss the possibility of using these as combination therapies.

Anti-angiogenic and macrophage-based therapeutic strategies for glioma immunotherapy.

As a new concept of glioma therapy, immunotherapy combined with standard therapies is a promising modality to improve glioma patient survival. VEGF and its signaling pathway molecules not only inhibit angiogenesis but also may reinforce the immunosuppressive tumor microenvironment, including promotion of the accumulation of immunosuppressive tumor-associated macrophages (TAMs). In this review, we discuss VEGF-targeted therapy as a new treatment option of the TAM-targeted therapy for high-grade gliomas, as well as other TAM-targeted therapies. The authors also discuss the potential of these therapies combined with conventional immunotherapies.

Therapeutic Strategies for Overcoming Immunotherapy Resistance Mediated by Immunosuppressive Factors of the Glioblastoma Microenvironment.

Various mechanisms of treatment resistance have been reported for glioblastoma (GBM) and other tumors. Resistance to immunotherapy in GBM patients may be caused by acquisition of immunosuppressive ability by tumor cells and an altered tumor microenvironment. Although novel strategies using an immune-checkpoint inhibitor (ICI), such as anti-programmed cell death-1 antibody, have been clinically proven to be effective in many types of malignant tumors, such strategies may be insufficient to prevent regrowth in recurrent GBM. The main cause of GBM recurrence may be the existence of an immunosuppressive tumor microenvironment involving immunosuppressive cytokines, extracellular vesicles, chemokines produced by glioma and glioma-initiating cells, immunosuppressive cells, etc. Among these, recent research has paid attention to various immunosuppressive cells-including M2-type macrophages and myeloid-derived suppressor cells-that cause immunosuppression in GBM microenvironments. Here, we review the epidemiological features, tumor immune microenvironment, and associations between the expression of immune checkpoint molecules and the prognosis of GBM. We also reviewed various ongoing or future immunotherapies for GBM. Various strategies, such as a combination of ICI therapies, might overcome these immunosuppressive mechanisms in the GBM microenvironment.

Infiltration of CD163-positive macrophages in glioma tissues after treatment with anti-PD-L1 antibody and role of PI3Kγ inhibitor as a combination therapy with anti-PD-L1 antibody in in vivo model using temozolomide-resistant murine glioma-initiating cells.

Although chemoimmunotherapy often lengthens glioblastoma (GBM) survival, early relapses remain problematic as immunosuppressive M2 macrophages (Mϕ) that function via inhibitory cytokine and PD-L1 production cause immunotherapy resistance. Here, we detail anti-PD-L1 antibody effects on the tumor microenvironment, including Mϕ infiltration, using a temozolomide (TMZ)-treated glioma model. In addition, we tested combinations of anti-PD-L1 antibody and the M2Mϕ inhibitor IPI-549 on tumor growth. We simulated late TMZ treatment or relapse stage, persistent GBM cells by generating TMZ-resistant TS (TMZRTS) cells. M2Mϕ-associated cytokine production and PD-L1 expression in these cells were investigated. TMZRTS cells were then subcutaneously implanted into C57BL/6 mice to determine the effectiveness of an anti-PD-L1 antibody and/or IPI-549 treatment on infiltration of CD163-positive Mϕ, usually considered as an M2Mϕ marker into tumor tissues. CD163 expression in samples from human GBM patients were also evaluated. CD163-positive Mϕ heavily infiltrated TMZRS tumor tissues after in vivo anti-PD-L1 antibody treatment. Tumor growth was strongly inhibited by anti-PD-L1 antibody and IPI-549 combination therapy. Anti-PD-L1 antibody treatment significantly reduced infiltration of CD163-positive Mϕ into tumors, while combined PD-L1 antibody and IPI-549 therapy remarkably inhibited tumor growth. These therapies may be useful for recurrent or chronic GBM after TMZ treatment, but clinical safety and effectiveness studies are needed.

Rate of Clinical Complete Response for 1 Year or More in Bone-Metastatic Breast Cancer after Comprehensive Treatments including Autologous Formalin-Fixed Tumor Vaccine.

INTRODUCTION: No effective treatment has been developed for bone-metastatic breast cancer. We found 3 cases with clinical complete response (cCR) of the bone metastasis and longer overall survival of the retrospectively examined cohort treated comprehensively including autologous formalin-fixed tumor vaccine (AFTV). PATIENTS AND METHODS: AFTV was prepared individually for each patient from their own formalin-fixed and paraffin-embedded breast cancer tissues. RESULTS: Three patients maintained cCR status of the bone metastasis for 17 months or more. Rate of cCR for 1 year or more appeared to be 15% (3/20) after comprehensive treatments including AFTV. The median overall survival time (60.0 months) and the 3- to 8-year survival rates after diagnosis of bone metastasis were greater than those of historical control cohorts in Japan (1988-2002) and in the nationwide population-based cohort study of Denmark (1999-2007). CONCLUSION: Bone-metastatic breast cancer may be curable after comprehensive treatments including AFTV, although larger scale clinical trial is required.

Long-lasting complete response status of advanced stage IV gall bladder cancer and colon cancer after combined treatment including autologous formalin-fixed tumor vaccine: two case reports.

BACKGROUND: The prognosis of advanced (stage IV) cancer of the digestive organs is very poor. We have previously reported a case of advanced breast cancer with bone metastasis that was successfully treated with combined treatments including autologous formalin-fixed tumor vaccine (AFTV). Herein, we report the success of this approach in advanced stage IV (heavily metastasized) cases of gall bladder cancer and colon cancer. CASE PRESENTATION: Case 1: A 61-year-old woman with stage IV gall bladder cancer (liver metastasis and lymph node metastasis) underwent surgery in May 2011, including partial resection of the liver. She was treated with AFTV as the first-line adjuvant therapy, followed by conventional chemotherapy. This patient is still alive without any recurrence, as confirmed with computed tomography, for more than 5 years. Case 2: A 64-year-old man with stage IV colon cancer (multiple para-aortic lymph node metastases and direct abdominal wall invasion) underwent non-curative surgery in May 2006. Following conventional chemotherapy, two courses of AFTV and radiation therapy were administered sequentially. This patient has had no recurrence for more than 5 years. CONCLUSION: We report the success of combination therapy including AFTV in cases of liver-metastasized gall bladder cancer and abdominal wall-metastasized colon cancer. Both patients experienced long-lasting, complete remission. Therefore, combination therapies including AFTV should be considered in patients with advanced cancer of the digestive organs.

Assessment of PD-1 positive cells on initial and secondary resected tumor specimens of newly diagnosed glioblastoma and its implications on patient outcome.

Glioblastoma (GBM) is the most common type of malignant brain tumor and has a very poor prognosis. Most patients relapse within 12 months despite aggressive treatment and patient outcome after recurrent is extremely worse. This study was designed to clarify the change of the molecular expression, including programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), on the initial and secondary resected tumor specimens and to address the influence of these expressions for patient outcome after second surgery of glioblastoma. We investigated 16 patients, ranging in age from 14 to 65 years, with histologically verified WHO grade IV GBM, whose original tumor was resected between 2008 and 2014, and treated with fractionated radiotherapy and temozolomide. Four patients who were treated with immunotherapy using autologous formalin-fixed tumor vaccine were enrolled. All of the patients underwent secondary resection after tumor recurrence within 24 months. We carried out an immunohistochemical examination of the initial and secondary resected tumors from patients using a panel of immune system molecular markers, and assessed whether marker expression correlated with clinical outcomes. CD3, CD8 and PD-1 on tumor-infiltrating lymphocytes was significantly increased in secondary resected specimens compared with initially resected specimens (p ≤ 0.05). All patients expressed PD-L1 on tumor cells in initial and secondary resection specimens. Patients were divided into high or low expression group by median IHC score of PD-1 on initial or secondary resected specimens. No significant differences in patient outcomes were observed between high and low PD-1 or PD-L1 groups of initially resected specimens. In high expression group of secondary resected specimens, most patients score had increased which compared with initial resected tumor specimens. The PD-1 high expression score group of secondary resected specimens was associated with long progression-free survival and short survival after recurrence. PD-L1 expression was detected in almost all initial and secondary specimens. Patients with high PD-1 expression of secondary specimen had bad prognosis after secondary resection. PD-1/PD-L1 pathway may be associated with patient outcome after second surgery of glioblastoma.

Immunocytochemistry for vancomycin using a monoclonal antibody that reveals accumulation of the drug in rat kidney and liver.

We prepared monoclonal antibodies against N-(γ-maleimidobutyryloxy)succinimide-conjugated vancomycin (VM). The monoclonal antibody was specific for conjugated or free VM. The monoclonal antibody enabled us to develop an immunocytochemical method for detecting the uptake of VM in the rat kidney and liver. Three hours after a single intravenous (i.v.) injection of VM at the therapeutic dose, the immunocytochemistry revealed that VM accumulated in large amounts in both the S1 and S2 segments and in much smaller amounts in the S3 segment of the proximal tubules as well as in the distal tubules and collecting ducts. The drug was detected in the cytoplasm, cytoplasmic irregular granules, nuclei, and microvilli of the proximal tubule cells. The distal tubules and collecting ducts contained scattered swollen cells in which both the nuclei and cytoplasm were heavily immunostained. Twenty-four hours after injection, most of the swollen cells returned back to normal size and had somewhat decreased immunostaining. Also, significant amounts of VM remained accumulated for as long as 8 days postadministration. In the liver, similar drug accumulation was observed in the Kupffer cells and the endothelial cells of the hepatic sinusoids but not in the hepatocytes, suggesting that vancomycin cannot be eliminated via the liver. Immunoelectron microscopic studies demonstrated that in the collecting ducts, uptake of VM occurred exclusively in the lysosomes and cytoplasm of the principal cells and scarcely in the intercalated cells. Furthermore, double fluorescence staining using rats simultaneously administered with VM and gentamicin strongly suggests that both drugs colocalized in lysosomes in the proximal tubule cells of kidneys.

An in vivo role of Mrp2 in the rat hepatocytes by immunocytochemistry for amoxicillin using the transporter-deficient EHBR.

An in vivo role of the multidrug resistant-associated protein (Mrp2) in rat hepatocytes was examined by immunocytochemistry (ICC) for amoxicillin (AMPC) by the use of the transporter-deficient Eisai hyperbilirubinemic rats (EHBR). The ICC revealed that in the liver of EHBR at 3-h post-administration, amoxicillin accumulated in the cytoplasmic pools and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries. However, no amoxicillin was observed on the surface of the lumina ranging from the bile capillaries to the interlobular bile ducts. The drug persisted at least for 6-h after administration. In contrast, in the control rat liver at 3-h post-administration, AMPC-adsorption occurred on such luminal surface, while AMPC accumulated to a less level in both the cytoplasm and nuclei of the hepatocytes. The drug completely disappeared in the hepatocytes at 6-h post-administration. These results strongly suggest that AMPC taken up into the cytoplasm of the hepatocytes excretes via Mrp2 into the bile flow. Furthermore, electron microscopy demonstrated that the lower electron density areas in large sizes, corresponding to the cytoplasmic pools in ICC for AMPC, occurred in the cytoplasm peripheral to the nuclei of the hepatocytes in EHBR at 3-h post-administration, and then disappeared 24 h after administration.

Immunocytochemistry for bestatin and its application to drug accumulation studies in rat intestine and kidney.

The in vivo role of transporters in drug disposition, in the context of other transporters, and metabolism has not been established. We prepared an anti-bestatin serum against bestatin conjugated to albumin with glutaraldehyde (GA). The antiserum was specific for GA-conjugated bestatin and weakly reacted with free bestatin, but no reaction occurred with structurally unrelated compounds according to both the inhibition and binding ELISAs. The antiserum allowed us to develop an immunocytochemical (ICC) method for detecting the uptake of bestatin in the rat intestine and kidney. Three hours after a single oral administration of bestatin, the ICC method revealed that the drug distributed in the microvilli, cytoplasm and nuclei of the absorptive epithelial cells at much larger amounts than in all other cell types in the small intestine. However, no drug was detected in the mucin goblets in the epithelium. In the kidney, the drug distributed to a greater extent in the S3 segment than in the S1 and S2 segments of the proximal tubule, and also was detected in the microvilli of the proximal tubule cells (S1, S2 and S3). These findings that bestatin accumulated in large amounts, especially in the cells and/or at the sites where the transporters PEPT1 and PEPT2 occur, corresponded well to those observed with ß-lactam amoxicillin in the previous ICC studies. Thus, this may indicate a possibility that both the transporters might be involved, at least in part, in the distribution of bestatin in the small intestine and kidney under the conditions examined.

A probable relationship between characteristic accumulation of doxorubicin and P-glycoprotein transporter in rat liver.

Correlations between the distribution of anthracycline antibiotics doxorubicin (DX) and daunorubicin (DR) in the liver of rats injected with a single i.v. injection of each drug and the reported distribution of P-glycoprotein transporter for the drug was histochemically examined. Immunocytochemical studies for DX or DR using monoclonal antibody that equally detects both drugs, as well as conventional electron microscopy were employed. DX persisted for more than 5 days in the cytoplasm and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries. Meanwhile, DR distributed in almost the same pattern, but more rapidly decreased to a level that almost no granular morphology in the hepatocytes was visible 24 h after the injection. Also, unknown large cells that strongly reacted with the antibody appeared in the hepatic sinusoids near the interlobular triads rather than the central vein. The accumulation sites of DX or DR on the bile capillaries seems to correspond to specific sites where the ATP-binding cassette transport protein P-glycoprotein for the anthracycline occurs, suggesting a possibility that DX or DR is actually and actively excreted at these sites, possibly through P-glycoprotein. This is supported by conventional electron microscopic studies, since no specific subcellular organelles such as lysosomes assemble in the vicinity of the bile capillaries of the hepatocytes.

Immunocytochemistry for amoxicillin and its use for studying uptake of the drug in the intestine, liver, and kidney of rats.

Specific transport systems for penicillins have been recognized, but their in vivo role in the context of other transporters remains unclear. We produced a serum against amoxicillin (anti-AMPC) conjugated to albumin with glutaraldehyde. The antiserum was specific for AMPC and ampicillin (ABPC) but cross-reacted weakly with cephalexin. This enabled us to develop an immunocytochemical (ICC) method for detecting the uptake of AMPC in the rat intestine, liver, and kidney. Three hours after a single oral administration of AMPC, the ICC method revealed that AMPC distributed to a high degree in the microvilli, nuclei, and cytoplasm of the absorptive epithelial cells of the intestine. AMPC distributed in the cytoplasm and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries, and in addition, AMPC adsorption was observed on the luminal surface of the capillaries, intercalated portions, and interlobular bile ducts on the bile flow. Almost no AMPC could be detected 6 h postadministration in either the intestine or the liver. Meanwhile, in the kidney, AMPC persisted until 12 h postadministration to a high degree in the proximal tubules, especially in the S3 segment cells in the tubular lumen, in which numerous small bodies that strongly reacted with the antibody were observed. All these sites of AMPC accumulation correspond well to specific sites where certain transporter systems for penicillins occur, suggesting that AMPC is actually and actively absorbed, eliminated, or excreted at these sites, possibly through such certain penicillin transporters.