Hidemitsu Sato

Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors

We have created a novel cellular vehicle for gene therapy of malignant gliomas by transfection of murine bone marrow stroma cells (MSCs) with a cDNA encoding epidermal growth factor receptor (EGFR). These cells (EGFR-MSCs) demonstrate enhanced migratory responses toward glioma-conditioned media in comparison to primary MSCs in vitro. Enhanced migration of EGFR-MSC was at least partially dependent on EGF-EGFR, PI3-, MAP kinase kinase, and MAP kinases, protein kinase C, and actin polymerization. Unlike primary MSCs, EGFR-MSCs were resistant to FasL-mediated cytotoxicity and were capable of stimulating allogeneic mixed lymphocyte reaction, suggesting EGFR-MSCs possess suitable characteristics as vehicles for brain tumor immuno-gene therapy. Following injection at various sites, including the contralateral hemisphere in the brain of syngeneic mice, EGFR-MSCs were able to migrate toward GL261 gliomas or B16 melanoma in vivo. Finally, intratumoral injection with EGFR-MSC adenovirally engineered to secrete interferon-α to intracranial GL261 resulted in significantly prolonged survival in comparison to controls. These data indicate that EGFR-MSCs may serve as attractive vehicles for infiltrating brain malignancies such as malignant gliomas.

Identification of Interleukin-13 Receptor α2 Peptide Analogues Capable of Inducing Improved Antiglioma CTL Responses

Restricted and high-level expression of interleukin-13 receptor alpha2 (IL-13Ralpha2) in a majority of human malignant gliomas makes this protein an attractive vaccine target. We have previously described the identification of the IL-13Ralpha2(345-353) peptide as a human leukocyte antigen-A2 (HLA-A2)-restricted CTL epitope. However, as it remains unclear how efficiently peptide-based vaccines can induce specific CTLs in patients with malignant gliomas, we have examined whether analogue epitopes could elicit heteroclitic antitumor T-cell responses versus wild-type peptides. We have created three IL-13Ralpha2 analogue peptides by substitutions of the COOH-terminal isoleucine (I) for valine (V) and the NH(2)-terminal tryptophan (W) for either alanine (A), glutamic acid (E), or nonsubstituted (W; designated as 1A9V, 1E9V, and 9V, respectively). In comparison with the native IL-13Ralpha2 epitope, the analogue peptides 9V and 1A9V displayed higher levels of binding affinity and stability in HLA-A2 complexes and yielded an improved stimulatory index for patient-derived, specific CTLs against the native epitope expressed by HLA-A2(+) glioma cells. In HLA-A2-transgenic HHD mice, immunization with the peptides 9V and 1A9V induced enhanced levels of CTL reactivity and protective immunity against an intracranial challenge with IL13Ralpha2-expressing syngeneic tumors when compared with vaccines containing the native IL-13Ralpha2 epitope. These findings indicate highly immunogenic IL-13Ralpha2 peptide analogues may be useful for the development of vaccines capable of effectively expanding IL-13Ralpha2-specific, tumor-reactive CTLs in glioma patients.

Delivery of Dendritic Cells Engineered to Secrete IFN-α into Central Nervous System Tumors Enhances the Efficacy of Peripheral Tumor Cell Vaccines: Dependence on Apoptotic Pathways

We tested whether modulation of the CNS-tumor microenvironment by delivery of IFN-α-transduced dendritic cells (DCs: DC-IFN-α) would enhance the therapeutic efficacy of peripheral vaccinations with cytokine-gene transduced tumor cells. Mice bearing intracranial GL261 glioma or MCA205 sarcoma received peripheral immunizations with corresponding irradiated tumor cells engineered to express IL-4 or GM-CSFs, respectively, as well as intratumoral delivery of DC-IFN-α. This regimen prolonged survival of the animals and induced tumor-specific CTLs that expressed TRAIL, which in concert with perforin and Fas ligand (FasL) was involved in the tumor-specific CTL activity of these cells. The in vivo antitumor activity associated with this approach was abrogated by administration of neutralizing mAbs against TRAIL or FasL and was not observed in perforin−/−, IFN-γ−/−, or FasL−/− mice. Transduction of the tumor cells with antiapoptotic protein cellular FLIP rendered the gene-modified cells resistant to TRAIL- or FasL-mediated apoptosis and to CTL killing activity in vitro. Furthermore, the combination therapeutic regimen was ineffective in an intracranial cellular FLIP-transduced MCA205 brain tumor model. These results suggest that the combination of intratumoral delivery of DC-IFN-α and peripheral immunization with cytokine-gene transduced tumor cells may be an effective therapy for brain tumors that are sensitive to apoptotic signaling pathways.

Delivery of Interferon-α Transfected Dendritic Cells into Central Nervous System Tumors Enhances the Antitumor Efficacy of Peripheral Peptide-Based Vaccines

We evaluated the effects, on immunity and survival, of injection of interferon (IFN)-α-transfected dendritic cells (DC-IFN-α) into intracranial tumors in mice immunized previously with syngeneic dendritic cells (DCs) pulsed either with ovalbumin-derived CTL or T helper epitopes. These immunizations protected animals from s.c. challenge with ovalbumin-expressing M05 melanoma (class I+ and class II-negative). Notably, antiovalbumin CTL responses were observed in animals vaccinated with an ovalbumin-derived T helper epitope but only after the mice were challenged with M05 cells. This cross-priming of CTL was dependent on both CD4+ and CD8+ T cells. Because we observed that s.c., but not intracranial, tumors were infiltrated with CD11c+ DCs, and because IFN-α promotes the activation and survival of both DCs and T cells, we evaluated the combinational antitumor effects of injecting adenoviral (Ad)-IFN-α-engineered DCs into intracranial M05 tumors in preimmunized mice. Delivery of DC-IFN-α prolonged survival. This was most notable for animals prevaccinated with both the CTL and T helper ovalbumin epitopes, with 60% (6 of 10) of mice (versus 0 of 10 of control animals) surviving for >80 days after tumor challenge. DC-IFN-α appeared to persist longer than mock-transfected DCs within the intracranial tumor microenvironment, and DC-IFN-α-treated mice exhibited enhanced levels of ovalbumin-specific CTL in draining cervical lymph nodes. On the basis of these results, we believe that local expression of IFN-α by DCs within the intracranial tumor site may enhance the clinical efficacy of peripheral vaccine approaches for brain tumors.

Experimental gene therapy against subcutaneously implanted glioma with a herpes simplex virus-defective vector expressing interferon-γ

We investigated the feasibility of local treatment or tumor vaccination with a herpes simplex virus (HSV) type 1-defective vector. The vector was engineered to express murine interferon-γ (IFN-γ) for experimental gene therapy against mouse glioma Rous sarcoma virus (RSV). The murine IFN-γ gene was driven by the cytomegalovirus promoter. The helper virus (tsk) was thermosensitive; consequently, this vector could only proliferate at 31°C. A high level of murine IFN-γ expression was confirmed in vitro and in vivo by immunohistochemistry using anti-mouse IFN-γ monoclonal antibody. This engineered vector (dvHSV/MuIFN-γ) inhibited the proliferation of mouse glioma RSV cells in vitro, and an intratumoral (i.t.) local injection of the vector caused i.t. necrosis in vivo. The immunological effect of dvHSV/MuIFN-γ was also examined in a mouse glioma RSV cell implantation model. A subcutaneous (s.c.) implant of 1 × 106 mouse glioma RSV cells after treatment with dvHSV/MuIFN-γ was rejected. However, the implant after treatment with an engineered HSV-defective vector containing an antisense nucleotide sequence of the murine IFN-γ gene was not rejected. In addition, in another group of mice in which RSV cells treated with dvHSV/MuIFN-γ were implanted into a femoral (s.c.) region and nontreated RSV cells were implanted into a contralateral femoral (s.c.) region, the implanted RSV cells were rejected. The rejection of the implanted mouse glioma RSV was blocked by anti-asialo GM1, which was known to inhibit natural killer cell activity. These results revealed that the HSV-defective vector could realize a high efficiency of transfection to glioma cells through short-time treatment, and that the IFN-γ gene transferred to the cells had the effect of tumor vaccination, which was suggested be related to natural killer cells. In conclusion, dvHSV/MuIFN-γ may be useful for the gene therapy of malignant glioma through either i.t. local injection or a practical tumor vaccination with ex vivo gene transfer.

Establishment of patient-derived cancer xenografts in immunodeficient NOG mice

Viable and stable human cancer cell lines and animal models combined with adequate clinical information are essential for future advances in cancer research and patient care. Conventional in vitro cancer cell lines are commonly available; however, they lack detailed information on the patient from which they originate, including disease phenotype and drug sensitivity. Patient-derived xenografts (PDX) with clinical information (so-called ‘cancer xenopatients’) are a promising advance that may accelerate the development of anticancer therapies. We established 61 PDX lines from 116 surgically removed tumor tissues inoculated subcutaneously into NOG mice (53% success rate). PDX lines were established from various types of epithelial tumors and also from sarcomas, including gastrointestinal stromal tumors and Ewing/PNET sarcomas. The metastatic tumors yielded PDX lines more effectively (65%) than the primary tumors (27%, P<0.001). In our PDX models, morphological characteristics, gene expression profiles, and genetic alteration patterns were all well preserved. In eight cases (7%), the transplantable xenografts for several generations were composed of large monotonous nonepithelial cells of human origin, revealed to be Epstein-Barr virus infection-associated lymphoproliferative lesions. Despite this, PDX linked with clinical information offer many advantages for preclinical studies investigating new anticancer drugs. The fast and efficient establishment of individual PDX may also contribute to future personalized anticancer therapies.

The VHL tumor suppressor protein regulates tumorigenicity of U87-derived glioma stem-like cells by inhibiting the JAK/STAT signaling pathway

The signal transducer and activator of transcription 3 (STAT3) factor plays an important role in the tumorigenicity of cancer stem cells. The purpose of this study was to investigate the inhibitory mechanism of this pathway acting through the tumor suppressor von Hippel-Lindau (VHL) protein in glioma cancer stem cells. We isolated floating neurosphere-forming CD133+ cells as glioma stem-like cells (GSLCs) by the MACS method. Furthermore, we examined these cells for their growth rate, ability to form colonies and neurospheres in soft agar, capacity for implantation into SCID mice and expression of CD133, STAT3, JAK2, Elongin A, PTEN and VHL. Furthermore, we transferred the VHL gene, an inhibitor of STAT3, into GSLCs using an adenovirus vector and compared these transfectants with control vector-transfected GSLCs. GSLCs proved to be implantable and formed a tumor in the subcutaneous tissue of SCID mice, the histology of which was similar to that of human glioblastomas. In addition, GSLCs exhibited a high capacity for soft agar colony and neurosphere formation, nearly all of which were CD133 positive. The majority of GSLCs were immunopositive for STAT3, JAK2 and Elongin A, but immunonegative for PTEN and VHL. When the VHL gene was transferred to GSLCs and these cells were transplanted into SCID mice, they did not result in tumor formation. Their capacity for soft agar colony and neurosphere formation was significantly inhibited, although their proliferation was only moderately inhibited. Regarding the expression of various factors, that of CD133 was decreased in the VHL transfectants and those of STAT3, JAK2 and Elongin A were eliminated. However, the expression of PTEN and of VHL was upregulated. These findings suggest that VHL regulated the tumorigenicity and self-renewal ability of glioma cancer stem cells by inhibiting the JAK/STAT signaling pathway.

Collagen gel matrix assay as an in vitro chemosensitivity test for malignant astrocytic tumors

BackgroundThe efficacy of individual chemotherapy based on chemosensitivity has scarcely been studied.MethodsWe examined the chemosensitivites for four anticancer agents – 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3 (2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), carboplatin, cisplatin, and etoposide – of 43 malignant astrocytic tumors (21 anaplastic astrocytomas and 22 glioblastomas) by using a collagen gel matrix assay, and we also determined the survival periods of the tumor-bearing patients. The chemosensitivity was evaluated in terms of the growth inhibition rate, using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) method.ResultsFor the anaplastic astrocytomas, the mean growth inhibitory rate was 33.2% with cisplatin, 37.2% with carboplatin, 28.0% with ACNU, and 24.8% with etoposide. For the glioblastomas, these rates were 36.9%, 42.3%, 23.2%, and 34.8%, respectively. The median overall and progression-free survivals of anaplastic astrocytoma-bearing patients who had undergone chemotherapy with two anticancer drugs, both of which showed significant anticancer activity (growth inhibitory rate >30%) were significantly longer than those of the patients who had been treated with two drugs, one or both of which did not show significant anticancer activity. On the other hand, there was no significant difference in the overall or the progression-free survivals in the two corresponding groups of glioblastoma-bearing patients.ConclusionThe collagen gel matrix assay is clinically useful to determine in vitro chemosensitivity that reflects in vivo chemosensitivity. Individual chemotherapy for malignant astrocytic tumors, based on chemosensitivity data, could contribute to longer survival, particularly in anaplastic astrocytoma-bearing patients.

Abstract 2813: An interactome analysis for personalized chemotherapy using PDX/NOG models of non-small cell lung cancer

Lung cancer is one of the most common malignant diseases in the world, and its prognosis is generally poor. It is crucial to elucidate the biological mechanisms underlying lung cancer and accelerate the development of new treatment strategies. Xenografts derived from engrafting fresh surgical specimens directly into immunodeficient mice have recently enabled the development of more relevant in vivo models of human cancers. These patient-derived xenograft (PDX) models, established by the direct transfer of tumor tissue, retain similar morphologies, heterogeneities, and molecular signatures as the original cancers, and, thus, may be used in promising personalized medicine for cancer. We previously reported the rapid and efficient establishment of PDXs using super immunodeficient NOG mice (PDX/NOG model). In the present study, we analyzed the gene expression and cancer-stroma interaction profiles of PDXs established from NSCLC patients. We also discussed the possibility of individual PDX/NOG model simulations for personalized cancer chemotherapy. Ten NSCLC lines of PDX/NOG (Age 43-78 years; 8 men and 2 women; 7 adenocarcinoma, 1 adenosquamous carcinoma, 1 squamous carcinoma, and 1 large cell carcinoma) were presented. In these cases, clinical information regarding chemotherapy for donor patients was retrieved where possible. Genome sequencing and comprehensive analyses of tumor-stroma interactions (CAncer-STromal INteractome analysis, CASTIN) were performed on mRNA. CASTIN showed tumor-stroma interactions in PDX/NOG comprehensively and quantitatively at the gene expression level by distinguishing gene arrangements in human tissue (Cancer) from mouse tissue (Stroma) as signal strengths (ligand dependency (%) / receptor dependency (%)). Interactions of EGF(cancer)-EGFR(stroma) were observed from 1.2 (15%/0%) to 11.5 (94%/80%) as widely-distributed. Interactions of VEGFA(cancer)-KDR(stroma) were observed from 49.1 (31%/51%) to 301.7 (83%/100%) as closely-distributed. These interactions of EGF-EGFR or VEGFA-KDR in PDX/NOG closely reflected the clinical effectiveness of an EGFR inhibitor (Cetuximab) or VEGF-A inhibitor (Bevacizumab). The CASTIN results of PDX/NOG appear to be reliable for clinical simulations of chemotherapy and will definitely assist in the selection of the most sensitive anti-cancer drug. The fast and efficient establishment of individual PDXs will contribute to personalized anti-cancer therapies. Citation Format: Tsuyoshi Chijiwa, Daisuke Komura, Mizuha Haraguchi, Akira Noguchi, Hidemitsu Sato, Hiroaki Ito, Haruhiko Nakayama, Makoto Katayama, Naoki Miyao, Naruaki Matsui, Yuichi Tateishi, Hiroshi Suemizu, Yoshiyasu Nakamura, Daisuke Furukawa, Takayuki Isagawa, Hiroto Katoh, Shumpei Ishikawa, Masato Nakamura, Yohei Miyagi. An interactome analysis for personalized chemotherapy using PDX/NOG models of non-small cell lung cancer [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 2813. doi:10.1158/1538-7445.AM2017-2813

Abstract B38: Clinical applications of PDX/NOG models for personalized chemotherapy – possible use in chemo-sensitivity testing and clinical sequencing

Personalized medicine represents an ideal medical approach for cancer therapy. In the field of clinical oncology, personalized medicine or therapy involves the evolutionary expansion of conventional clinical approaches that progress from patient evaluation, differential diagnosis, to the treatment of diseases. A number of the complex techniques employed in personalized medicine, such as clinical genome sequencing, biochemical marker analyses, chemo-sensitivity testing, and cancer immunotherapy, require fresh, viable, and sufficient amounts of specimens for reliable estimations. Mice bearing patient-derived xenografts (PDXs) with clinical information (so-called “Cancer Xenopatients”) are remarkable systems in personalized medicine for cancer. We previously reported the rapid and efficient establishment of PDXs using NOG mice (PDX/NOG models, AACR2015 #1940, IJO 47 61-70 2015). NOG mice, NOD/Shi-scid/IL2Rγ null (NOG) mice derived from NOD/SCID mice with a common gamma chain, have multifunctional defects in natural killer cell activity, macrophage function, complement activity, and dendritic cell function in addition to the absence of functional T and B lymphocytes. NOG mice have been identified as the most appropriate immunodeficient host animal for the direct xenografting of fresh tumor tissue due to the preservation of cancer stem cells (CSCs). Fresh and valuable xenograft samples, similar to surgical samples with the preservation of CSC, are stably provided using PDX/NOG models. Moreover, human tissue (tumor) and mouse tissue (stroma) are clearly distinguished by immunohistochemical analysis or gene arrangement sequencing. In the present study, we discussed the possibility of using PDX/NOG model simulations for personalized cancer chemotherapy. We previously established 47 lines of gastrointestinal cancer xenografts. In these cases, clinical information regarding chemotherapy for donor patients was retrieved where possible. Collagen gel droplet-embedded culture-drug sensitivity tests (CD-DST) were performed on 16 lines of PDX/NOG. In 4 of these lines, CD-DST were successfully conducted on original surgical specimens. The results of CD-DST between original and PDX/NOG specimens generally correlated (R 2 =0.01-0.89). The results of CD-DST using PDX/NOG specimens were compatible with the clinical effects of anti-cancer drugs. Genome sequencing and interactome analyses, a comprehensive analysis of tumor-stroma interactions innovated by Professor Ishikawa S. at Tokyo Medical and Dental University, were also performed on mRNA from 17 lines of PDX/NOG. Our interactome analyses showed tumor-stroma interactions in PDX/NOG comprehensively and quantitatively at the gene-expression level by distinguishing gene arrangements in human tissue (tumor) from mouse tissue (stroma). The EGF-EGFR or VEGFA-KDR interactions observed closely reflected the clinical effectiveness of an EGFR inhibitor (Cetuximab) or VEGF-A inhibitor (Bevacizumab) as well as the results of in vivo chemo-sensitivity tests using PDX/NOG. The results of CD-DST and sequencing in PDX/NOG appear to be reliable for clinical simulations of chemotherapy and will definitely assist in the selection of the most sensitive anti-cancer drug for each patient. The fast and efficient establishment of individual PDXs will contribute to personalized anti-cancer therapies. Citation Format: Tsuyoshi Chijiwa, Takayuki Isagawa, Akira Noguchi, Hidemitsu Sato, Akimune Hayashi, Haruhiko Cho, Manabu Shiozawa, Takeshi Kishida, Soichiro Morinaga, Tomoyuki Yokose, Makoto Katayama, Nobuo Takenaka, Mizuha Haraguchi, Naoki Miyao, Yuichi Tateishi, Kenji Kawai, Hiroshi Suemizu, Roppei Yamada, Yoshiyasu Nakamura, Kohzoh Imai, Daisuke Komura, Shumpei Ishikawa, Masato Nakamura, Yohei Miyagi. Clinical applications of PDX/NOG models for personalized chemotherapy – possible use in chemo-sensitivity testing and clinical sequencing. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B38.