Tsuyoshi Chijiwa

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.

A new in vivo model to analyze hepatic metastasis of the human colon cancer cell line HCT116 in NOD/Shi-scid/IL-2Rγnull (NOG) mice by 18F-FDG PET/CT

Clinically, (18)F-fluorodeoxyglucose positron emission tomography/computed tomography ((18)F-FDG-PET/CT) is useful in the evaluation of various types of human cancers. While PET analysis has been established to evaluate subcutaneous lesions of human cancers in mice, its applications for internal lesions are still being developed. We are currently evaluating new PET approaches for the effective evaluation of in vivo metastatic lesions in the internal organs of small experimental animals. In this study, we analyzed in vivo hepatic metastases of human colonic cancer in immunodeficient mice (NOD/Shi-scid/IL-2Rγ(null), NOG) using PET imaging. This new PET approach has been proposed for the evaluation of in vivo metastatic lesions in internal organs. The human colon cancer line HCT116 (1.0x10(5) and 1.0x10(6) cells/mouse) was transplanted by intrasplenic injection. (18)F-FDG-PET/CT scans were performed 2 weeks after transplantation. After PET/CT scans, histopathological examinations were performed. PET/CT analysis disclosed multiple metastatic foci and increased standardized uptake values (SUV) of FDG in the livers of NOG mice (control, SUVmean 0.450±0.033, SUVmax 0.635±0.017; 1.0x10(5) cells, 0.853±0.087, 1.254±0.237; 1.0x10(6) cells, 1.211±0.108, 1.701±0.158). There were significant differences in FDG uptakes between the three groups (ANOVA, P=0.017 in SUVmean; P=0.044 in SUVmax, n=2). We clearly and quantitatively detected images of hepatic metastasis in the livers of NOG mice by (18)F-FDG-PET/CT in vivo. PET/CT analysis of internal organ lesions of human cancerous xenografts is a new reliable experimental system to simulate metastases. This model system is useful for analyzing metastatic mechanisms and for developing new novel drugs targeting hepatic metastases of cancer.

Efficient production of recombinant adeno-associated viral vector, serotype DJ/8, carrying the GFP gene

The purpose of this study was to establish an efficient method for the preparation of an adeno-associated viral (AAV), serotype DJ/8, carrying the GFP gene (AAV-DJ/8-GFP). We compared the yields of AAV-DJ/8 vector, which were produced by three different combination methods, consisting of two plasmid DNA transfection methods (lipofectamine and calcium phosphate co-precipitation; CaPi) and two virus DNA purification methods (iodixanol and cesium chloride; CsCl). The results showed that the highest yield of AAV-DJ/8-GFP vector was accomplished with the combination method of lipofectamine transfection and iodixanol purification. The viral protein expression levels and the transduction efficacy in HEK293 and CHO cells were not different among four different combination methods for AAV-DJ/8-GFP vectors. We confirmed that the AAV-DJ/8-GFP vector could transduce to human and murine hepatocyte-derived cell lines. These results show that AAV-DJ/8-GFP, purified by the combination of lipofectamine and iodixanol, produces an efficient yield without altering the characteristics of protein expression and AAV gene transduction.

Abstract 49: Zinc finger protein 185 is a key molecule of liver metastasis in colon cancer

Colorectal cancer is among the three leading causes of cancer-related deaths worldwide. Nearly 50% of patients with colon cancer develop liver metastases. Mortality associated with the advanced stage of colon cancer has principally been attributed to the development of liver metastases. Therefore, clarifying the biological mechanisms underlying liver metastasis from colon cancer and accelerating the development of new treatment strategies are needed. We isolated Zinc finger protein 185 (ZNF185) as a metastasis-related protein by global quantitative proteome analysis of the in vivo liver metastasis model of super immunodeficient NOG mice with the highly liver-metastatic subline SW48-LM2 (Matsuo E, et al. AACR, #3947, 2008). LIM domain proteins have many fundamental biological processes including cell lineage specification, cytoskeleton organization, and organ development. ZNF185 is one of the LIM domain proteins and is considered to be involved in regulating cellular differentiation and/or proliferation. However, the functions and properties of ZNF185 in the multi-step process of cancer biology have not yet been elucidated in detail. In the present study, we analyzed the relationships between ZNF185 and the clinicopathological features of colon cancer (age, gender, histological type, lymphatic involvement, venous involvement, T and N statuses, liver metastasis, and stage). The expression of the ZNF185 protein was immunohistochemically analyzed. The expression of ZNF185 was detected in the cancer cells of 78 of 87 colon cancer patients. A correlation was observed between ZNF185 and the histological type (p=0.010, G-test). The expression of ZNF185 was also correlated with liver metastasis (p=0.030, G-test). Multivariate analysis using the Cox proportional hazards model was performed among the cause-specific survival rate, ZNF185 expression, and clinicopathological features. The histological type, liver metastasis, and ZNF185 expression were found to be independent prognostic indicators (p= 0.028, p Citation Format: Daisuke Furukawa, Tsuyoshi Chijiwa, Masahiro Matsuyama, Masaya Mukai, Ei-ichi Matsuo, Osamu Nishimura, Kenji Kawai, Hiroshi Suemizu, Nobuyoshi Hiraoka, Toshio Nakagohri, Seiei Yasuda, Masato Nakamura. Zinc finger protein 185 is a key molecule of liver metastasis in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 49. doi:10.1158/1538-7445.AM2014-49

Plasma membrane expression of ZNF185 is a prognostic factor in pancreatic ductal carcinoma

Pancreatic ductal carcinoma (PDC) is one of the major causes of cancer-associated mortality globally due to its high potential for distant metastasis. To understand hematogenous metastasis, the molecular expression profiles of weak metastatic PDC cell subline BxPC-3 and highly liver-metastatic cell subline LM-BxPC-3 were compared, and zinc finger protein 185 (ZNF185) was identified as a molecule that is upregulated in LM-BxPC-3 cells. The aim of the present study was to evaluate the clinicopathological significance of ZNF185 in PDC. Using immunohistochemistry, ZNF185 expression was investigated in 182 patients with PDC, in association with numerous clinicopathological variables. The expression profile of ZNF185 was also characterized using xenograft models. In contrast to parent BxPC-3 cells in subcutaneous transplanted tumor foci, which only expressed ZNF185 on their plasma membrane (m)ZNF185, LM-BxPC-3 cells in liver-metastatic foci that were formed subsequent to transplantation all expressed cytoplasmic (c)ZNF185. Additionally, 51% of the cells at the periphery of the tumor foci expressed mZNF185. Expression of cZNF185, and of mZNF185 and cZNF185 combined was identified in 93 and 39% of clinical patients with PDC, respectively. Patients with mZNF185-negative and -positive PDC exhibited a median survival time of 30.2 months and 21.3 months, respectively. Multivariate analysis indicated that the expression of mZNF185 is closely associated with a shorter overall survival time. Increased marked venous invasion was more prevalent in patients who were mZNF185-positive, as compared with patients who were mZNF185-negative. These data suggest that the expression of mZNF185 is an independent and unfavorable prognosticator in patients with PDC. The results suggested that the amount and subcellular location of ZNF185 are correlated with the position of the cancer cells expressing it within the nests. Additionally, the subcellular location of ZNF185 may be important to its biological function.

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.

Abstract 1543: Clinical significance of ZNF185 intracellular localization in pancreatic ductal carcinoma

Pancreatic cancer is one of the most aggressive human tumors because of the challenges associated with detecting it at an early stage and its high potential for dissemination and distant metastasis. Therefore, clarifying the biological mechanisms underlying distant metastasis from pancreatic cancer and accelerating the development of new treatment strategies are needed. We previously isolated ZNF185, a metastasis-related protein, using a global quantitative proteome analysis. ZNF185 contains two zinc-finger motifs in the C-terminus that fit the consensus pattern of the LIM domains inducing tumor formation and cytoskeleton organization. In the present study, we investigated the expression of ZNF185 in human pancreatic cancer in vivo and in clinical cases, and also evaluated its prognostic significance. Immunohistochemical staining of resected specimens and a xenograft model of liver metastasis was used to examine the relationship between distant metastasis in and the prognosis of pancreatic cancer and the expression of ZNF185. Cells expressing ZNF185 in the plasma membrane were detected in 71 (39.0%) out of 182 patients with resected pancreatic cancer. Patients not expressing ZNF185 in the plasma membrane had a median survival of 30.2 months, whereas those expressing ZNF185 in the plasma membrane had a median survival of 21.3 months. A multivariate analysis indicated that the expression of ZNF185 in the plasma membrane was an independent poor prognostic factor (p = 0.016). A multivariate analysis indicated that patients with cells expressing ZNF185 in the plasma membrane were more likely to develop hematogenous metastasis (p = 0.081). NOD/Shi-scid/IL-2Rγnull(NOG) mice were used to compare the expression of ZNF185 in the subcutaneous tumors of the parent cell line BxPC-3 and liver metastatic tumors of the highly liver-metastatic subline LM-BxPC-3 established in our laboratory. Cells expressing ZNF185 in the plasma membrane were more prevalent in metastatic disease in the liver than in subcutaneous tumors, and these cells were often located at the tumor margins rather than in the center of the tumor. In conclusion, ZNF185 is a protein related to hematogenous metastasis, the expression of which in the plasma membrane was identified as an independent prognostic factor for pancreatic cancer. Citation Format: Daisuke Furukawa, Tsuyoshi Chijiwa, Masahiro Matsuyama, Masaya Mukai, Ei-ichi Matsuo, Osamu Nishimura, Kenji Kawai, Hiroshi Suemizu, Toshio Nakagohri, Seiei Yasuda, Kazuaki Shimada, Nobuyoshi Hiroka, Masato Nakamura. Clinical significance of ZNF185 intracellular localization in pancreatic ductal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1543.

Abstract 1449: The valuable experimental model system “Cancer Xenopatient” established in NOG mice

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Viable and stable human cancer materials combined with adequate clinical information are required for advance in cancer research and patient care. Conventional in vitro cancer cell lines are commonly available without enough information on the patient including disease phenotypes and drug-sensitivity. Mice bearing Patient-derived xenografts (PDXs) with clinical information (so-called “cancer xenopatients”) could be essential and useful systems to accelerate cancer medicine. In this study, we investigated the establishment efficiency of PDX using NOG mice with clinical factors of xeno-transplantaion. The NOG mouse, the NOD/Shi-scid/IL-2Rγnull (NOG) mice derived from the NOD/SCID mouse with a common gamma chain, has multifunctional defects in natural killer cell activity, macrophage function, complement activity, and dendritic cell function in addition to lacking functional T and B lymphocytes. NOG mice were reported to be the best appropriate immunodeficient host animal for direct xenografting of fresh tumor tissue. We also discuss herein its contribution for not only reliable preclinical studies of new anticancer drugs but also personalized anti-cancer therapies. Sixty-one PDX lines were established from 116 surgically removed tumor tissues inoculated into NOG mices subcutaneous (53%). PDX lines were established from various types of epithelial tumors and also from sarcomas including gastrointestinal stromal tumor and Ewing/PNET sarcoma. The metastatic tumors yielded PDX lines effectively (65%) than the primary tumors (27%, p<0.001). The group engrafted into NOG mice after 2 days or later from the surgical removal showed a higher establishment rate (61%) than that of the group engrafted at the day of surgery or the next day (51%), however non-significant (p = 0.49). Our PDX models were preserved well not only in morphological characteristics but also in gene expression and alteration patterns. In 8 cases (7%), the transplantable xenografts for several generations were composed of monotonous large non-epithelial cell growth of human origin, revealed to be the Epstein-Barr virus (EBV) infection associated lymphoprolipherative lesions prospected by chromogenic in situ hybridization for EBV-encoded RNA. We revealed efficient establishment rates both for primary tumors and metastatic tumors. PDXs linked with clinical information will contribute to reliable preclinical studies for new anticancer drugs. The fast and efficient establishment of individual PDXs may also contribute to future personalized anti-cancer therapies. Citation Format: Tsuyoshi Chijiwa, Kenji Kawai, Akira Noguchi, Hidemitsu Sato, Akimune Hayashi, Haruhiko Cho, Manabu Shiozawa, Takeshi Kishida, Soichiro Morinaga, Tomoyuki Yokose, Makoto Katayama, Nobuo Takenaka, Hiroshi Suemizu, Roppei Yamada, Yoshiyasu Nakamura, Yasuo Takano, Kohzoh Imai, Yohei Miyagi, Masato Nakamura. The valuable experimental model system “Cancer Xenopatient” established in NOG mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1449. doi:10.1158/1538-7445.AM2015-1449

Abstract 2675: 18F-FDG-PET/CT imaging analyses for liver metastases of human colon cancer xenografts in NOG mice.

Colorectal cancer is one of the most common malignant diseases in the world, and its prognosis is generally poor. Liver metastases of colorectal cancer significantly affect the fates of patients. To further understand the pathological aspects of distant metastases, we have established liver metastasis models of human colon cancer xenografts in immune-deficient NOD/Shi-scid/IL-2Rγ null (NOG) mice. 18 F-fluorodeoxyglucose positron emission tomography / x-ray computed tomography ( 18 F-FDG-PET/CT) is useful in the clinical evaluation of various human cancers. While PET analysis was established to evaluate subcutaneous lesions of human cancer xenografts in mice, its application to internal lesions including liver metastases is still being developed. We applied PET approaches for the evaluation of in vivo metastatic lesions in the internal organs of living small experimental animals. In this study, we analyzed in vivo liver metastases of human colon cancer cell lines in NOG mice using PET imaging. Experimental liver metastases were established by intrasplenic injection of human colon cancer cell line HCT116 (1.0 x 10 5 or 1.0 x 10 6 cells/mouse). 18 F-FDG-PET/CT scans were performed 2 weeks after xeno-transplantation. After PET/CT scans, histopathological examinations were also performed to precisely confirm lesions. 18 F-FDG-PET/CT imaging clearly demonstrated multiple hot spots on livers in spite of disadvantages in the contrast between tumor soft tissues and markedly swollen livers. PET/CT analysis also disclosed increased standardized uptake values (SUV) of FDG in metastatic foci of NOG mice (control, SUVmean 0.450 ± 0.033, SUVmax 0.635 ± 0.017; 1.0 x 10 5 cells, 0.853 ± 0.087, 1.254 ± 0.237; 1.0 x 10 6 cells, 1.211 ± 0.108, 1.701 ± 0.158). There were significant differences in FDG uptakes between the three groups (ANOVA, p=0.017 in SUVmean, p=0.044 in SUVmax, n=2). We clearly and quantitatively detected liver metastasis images in NOG mice by 18 F-FDG-PET/CT in vivo. Immunohistochemical examinations confirmed more metastatic foci and more severe hepatomegaly with 1.0 x 10 6 than 1.0 x 10 5 of HTC116 cell inoculations. PET/CT analysis of human cancer xenografts is a new and reliable system to evaluate metastatic lesions in internal organs. In conclusion, this model system is useful for analyzing metastatic mechanisms and developing new therapeutic approaches for the liver metastatic lesions of human cancer. Citation Format: Tsuyoshi Chijiwa, Kenji Kawai, Hideo Tsukada, Hiroshi Suemizu, Masato Nakamura. 18 F-FDG-PET/CT imaging analyses for liver metastases of human colon cancer xenografts in NOG mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2675. doi:10.1158/1538-7445.AM2013-2675