Susumu Miyazaki

Emerging Methods for Preparing iPS Cells

In 1998, human embryonic stem cells were first generated and were expected to contribute greatly to regenerative medicine. However, when medical treatments were performed using human embryonic stem cells, there were problems, such as transplant rejection, as well as bioethical issues. Induced pluripotent stem cells were generated from mouse and human fibroblasts in 2006 and 2007 by introducing four transcription factors (Oct3/4, Sox2, c-Myc and Klf4). This process was defined as direct reprogramming, and induced pluripotent stem cells were better tolerated. Although induced pluripotent stem cells have contributed greatly to biomedical research and regenerative medicine, high tumorigenic potential is still a critical problem due to the introduction of the oncogene c-Myc and reprogramming with a virus vector. To address this, we reprogrammed somatic cells by transfection with microribonucleic acids to avoid using virus vectors for genomic integration into the host genome. We found that it was possible to reprogram mouse and human cells to pluripotency by direct transfection of three mature microribonucleic acids (mir-200c, -302s and -369s) with increased expression levels in embryonic stem cells and induced pluripotent stem cells. The microribonucleic acid-induced pluripotent stem cells have a reduced risk of mutations and tumorigenesis. Our laboratory also introduced four transcription factors (Oct3/4, Sox2, c-Myc and Klf4) into cancer cells, generating induced pluripotent cancer cells that exhibited strikingly less malignant features, suggesting the possibility of a novel type of cancer therapy. However, the gene transduction method is not yet safe for clinical applications, due to a genomic integration that may cause tumor formation. We are currently investigating the reprogramming method using microribonucleic acids in cancer cells to develop a very safe, highly efficient and highly complete reprogramming for clinical applications.

A Cancer Reprogramming Method Using MicroRNAs as a Novel Therapeutic Approach against Colon Cancer

BackgroundWe previously generated induced pluripotent stem cells by reprograming adipose stem cells through the introduction of microRNAs targeting four transcription factors (Oct3/4, Sox2, c-Myc, and Klf4). In this study, we aimed to reprogram cancer cells using microRNAs to explore their therapeutic potential.MethodsMature microRNAs (mir-302a-d, 369-3p and 5p, and mir-200c, as needed) were introduced into colon cancer cells (DLD-1, RKO, and HCT116) using lipofection.ResultsThe transfected cells exhibited an embryonic stem cell-like morphology and expressed the undifferentiated marker genes Nanog, Oct3/4, SOX2, and Klf4, as well as tumor-related antigen-1-60. These cells expressed neurogenic or adipogenic markers, indicating that reprogramming of the cancer cells was partially successful. Moreover, we found that miRNA-expressing DLD-1 cells showed low proliferative activity in vitro and in vivo, accompanied by increased expression of the tumor suppressor genes p16ink4a and p21waf1. miRNA-expressing DLD-1 cells also exhibited enhanced sensitivity to 5-fluorouracil, possibly through the downregulation of multidrug-resistant protein 8. The reprogrammed cells from DLD-1, RKO, and HCT116 cells exhibited reduced c-Myc expression, in contrast to the high c-Myc expression in the induced pluripotent cancer cells that were generated using four transcription factors.ConclusionsOur cancer reprogramming method employing simple lipofection of mature microRNAs is safe and well suited for clinical application, because it avoids integration of exogenous genes into the host genome and allows escape from augmentation of c-Myc gene expression.

Reprogramming of gastrointestinal cancer cells

Cell reprogramming reverts cells to multipotent, preprogrammed states by re‐establishing epigenetic markers. It can also induce considerable malignant phenotype modification. Because key events in cancer relapse and metastasis, including epithelial–mesenchymal transition phenotypes, are regulated primarily by reversible and transient epigenetic modifications rather than the accumulation of irreversible and stable genetic abnormalities, studying dynamic mechanisms regulating these biological processes is important. Transcription factors for induced pluripotent stem cells and non‐coding microRNAs allow pluripotent phenotype induction. We present the current knowledge of the possible applications of cell reprogramming in reducing aggressive phenotype expression, which can induce tumor cell hibernation and maintain appropriate phenotypes, thereby minimizing relapse and metastasis after surgical resection of gastrointestinal cancer. (Cancer Sci 2012; 103: 393–399)

Biological and Clinical Availability of Adipose-Derived Stem Cells for Pelvic Dead Space Repair

Adipose‐derived stem cells (ADSCs) are a very attractive cell source for regenerative and reconstructive medicine. Although ADSCs have already been used in cardiovascular disease and cosmetic surgery, they have not yet been used in gastroenterological surgery. In this study, we clarified the utility of the combined application of ADSCs and resected intraperitoneal fatty tissues as a sealant for the pelvic dead space that sometimes causes severe and fatal complications in colorectal and gynecological surgeries. In pelvic dead space model mice, mouse ADSCs efficiently maintained transplanted intraperitoneal fatty tissues without any incidence of adhesion to surrounding organs. In vivo and in vitro analyses revealed that transplanted ADSCs differentiated into endothelial cells by expressing the angiogenic factors vascular endothelial growth factor and hepatocyte growth factor. Mouse and human ADSCs contained a CD45−CD34+ subset possessing high colony formation and sphere formation abilities. In addition, the CD45−CD34+ subset consisted of two characteristic subsets: the CD34+CD90+ angiogenic subset and the CD34+CD90− adipogenic subset. Grafts of human ADSCs with fat transplanted into mice were efficiently maintained for more than 12 months without volume reductions. A comparative study of graft maintenance efficacy between cultured human ADSCs and freshly isolated ADSCs indicated that the cultivation of ADSCs decreased their graft maintenance ability. These findings suggested that the angiogenic and adipogenic subsets act in coordination with each other and are essential for efficient graft maintenance.

Anxa9 gene expression in colorectal cancer: A novel marker for prognosis

Annexin A9 (ANXA9) is involved with the interaction with membrane phospholipids in a Ca2+-dependent manner. A previous study has shown that ANXA9 expression is associated with bone metastasis in breast cancer, whereas its significance in colorectal cancer (CRC) is unknown. The present study was comprised of 100 patients who underwent surgery for CRC. The correlation between gene expression and the clinical parameters of the patients was assessed. Patients with high ANXA9 expression were statistically susceptible to a relatively worse prognosis, and those with low ANXA9 expression showed improved overall survival compared with those with high expression. In conclusion, the present data suggests that ANXA9 expression is a prognostic factor in CRC patients.

Abstract 5319: Cancer reprogramming.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: The 2012 Nobel Prize success (Gurdon and Yamanaka) gives boost to medical science. While the nuclear transplantation but also the gene transfer of defined factors can elicit cellular reprogramming efficiently in terminally differentiated somatic cells, we have been studied the effects of coding genes (oncogenes [OG], tumor suppressor genes [TSG], and ES-like genes) and non-coding ones (microRNA) on the gastrointestinal cancer cells (reviewed in JAMA, 2001; Cancer Sci, 2008). Compared with known tumor-related genes [OG/TSG], the retroviral-mediated gene transfer of induced pluripotent stem cells (iPSCs) factors, such as Oct4, Sox2, Klf4 and c-Myc resulted in radical modifications of cell lineages, more sensitization to anti-cancer reagents, and significant suppression of tumorigenesis in immunodeficient NOD/SCID mice (PNAS, 2010; BBRC, 2010; IJO, 2012). Given that the viral vectors have potential risks for genome insertion causing carcinogenesis, which would offend clinical application, our extensive screening of ES cell-specific microRNAs allowed the identification of a set of microRNAs (microRNA302 s, 200c and 369 s), which could execute full reprogramming from differentiated cells to iPSCs (Cell Stem Cell, 2011; Cancer Sci, 2011). Here we studied the effect of those microRNAs on gastrointestinal cancer cells in vitro and in vivo. Methods: To inactivate cancer cells, we screened a small set of microRNAs by in vitro experiments, including cell growth, invasion, sphere formation, differentiation assay (three germs) and immunocytochemistry; and in vivo ones, such as chemo-sensitivity, teratoma assay and tumorigenesis. For in vivo assay, synthesized microRNAs were administered intravenously as conjugated forms with carbonate apatite, a novel microRNA delivery technology. The fluorescent labeled microRNAs monitored the efficiency in cancer tissue uptakes as well as the toxicity in normal tissues. Result: 1) Single microRNA302s were able to reprogram colorectal cancer HT29 cells in vitro, as shown by microscopic analysis and qRT-PCR: three germ line differentiation, reduced proliferation and suppressed invasion. 2) Cell fate analysis in vitro demonstrated that ∼60% of HT29 cells reprogrammed, while remaining cells were subjected to apoptosis induction. The reprogrammed cells were sensitized to chemotherapy (fluorouracil). 3) In vivo study showed the microRNA302s effect on tumors, but not on normal tissues, to suppress tumorigenesis significantly in immunodeficient NOD/SCID mice. Conclusion: In vitro and in vivo assay demonstrated clearly that cancer-reprogramming therapy using synthesized microRNAs could modify the cancer cell malignancy, and that the combination with efficient DDSs would facilitate the discovery of innovative cancer therapy. We study further the mechanism microRNA-mediated cancer reprogramming for the drug optimization. Citation Format: Hisataka Ogawa, Hirofumi Yamamoto, Masamitsu Konno, Shin Kure, Susumu Miyazaki, Shinpei Nishikawa, Shinichiro Hasegawa, Katsuya Ohta, Yoshihiro Kano, Takahito Fukusumi, Atsushi Hamabe, Takeshi Yamamoto, Satoshi Obika, Taroh Satoh, Hidetoshi Eguchi, Hiroaki Nagano, Hidenori Inohara, Yuichirou Doki, Masaki Mori, Hideshi Ishii. Cancer reprogramming. [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 5319. doi:10.1158/1538-7445.AM2013-5319

Efficacy of Negative-Pressure Wound Therapy for Preventing Surgical Site Infections after Surgery for Peritonitis Attributable to Lower-Gastrointestinal Perforation: A Single-Institution Experience

BACKGROUND AND PURPOSE For patients at high risk, such as those with lower-gastrointestinal perforations, it is important to establish a preventive method that reduces the incidence of surgical site infections (SSIs) significantly. We applied negative-pressure wound therapy (NPWT) as part of a delayed primary closure approach to prevent SSIs. This study evaluated the value of this technique. METHODS We included prospectively 28 patients undergoing abdominal surgery for peritonitis caused by a lower-gastrointestinal perforation between May 2014 and November 2015. Historical controls comprised retrospective data on 19 patients who had undergone primary suturing for managing peritonitis incisions for a lower-gastrointestinal perforation from January to December 2013. RESULTS We found a significant association between the SSI incidence and the type of incision management (10.7% with NPWT and delayed closure vs. 63.2% with primary suturing; p < 0.001). There was no significant difference between the groups in the length of the hospital stay (22 days for NPWT and delayed closure vs. 27 days for primary suturing; p = 0.45). No severe adverse events were observed related to NPWT. CONCLUSION The use of NPWT and delayed primary closure was an effective measure for preventing SSI in patients undergoing abdominal surgery for peritonitis caused by lower-gastrointestinal perforation.

Skeletal Muscle Loss during Neoadjuvant Chemotherapy Is an Independent Risk Factor for Postoperative Infectious Complications in Patients with Advanced Esophageal Cancer

Objective: Neoadjuvant therapy followed by surgery has been the standard treatment for advanced esophageal cancer. Severe toxicities may influence body composition, including skeletal muscle mass, and increase postoperative complications. The purpose of this study was to evaluate the influence of sarcopenia, changes in body composition, and adverse events during neoadjuvant chemotherapy (NACT) on postoperative complications in esophageal cancer patients. Methods: A total of 83 patients with esophageal cancer undergoing NACT followed by esophagectomy were included. Body composition was assessed before chemotherapy and before esophagectomy. The relationships between postoperative infectious complications and sarcopenia, changes in body composition, and adverse events during NACT were investigated. Results: Univariate analysis revealed that skeletal muscle loss during NACT, but not preoperative sarcopenia, was significantly higher in the complication (+) group. Febrile neutropenia tended to occur frequently in the complication (+) group. Multivariate analysis demonstrated that skeletal muscle loss was the only factor significantly associated with infectious complications (p = 0.029). Among adverse events, febrile neutropenia was significantly associated with a decrease in skeletal muscle mass. Conclusion: Loss of skeletal muscle mass during NACT was a significant risk factor for postoperative infectious complications in patients with esophageal cancer. Prevention of severe adverse events may reduce postoperative infectious complications.

Abstract 4460: Super carbonate apatite as simplified systemic nanoparticle carrier for therapy of solid tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: RNA interference technology is underway in clinical trials for limited diseases. RNAi technology for solid tumors, especially in systemic administration, still faces hurdles and has not emerged on the clinical stage. Here we introduce an in vivo pH sensitive delivery system of siRNA and microRNA using super carbonate apatite (sCA) nanoparticles (mean size: 10 nm), which simply consist of inorganic ions. In order to show the therapeutic superiority of the sCA system over currently available in vivo siRNA delivery systems, we perform further experiments comparing the sCA system with Invivofectamine 2.0 (IF) and Atelocollagen (Atelo). Finally we examine the toxicity of sCA nanoparticle system in mice and monkeys. Methods: To assess the bio-distribution of fluorescently labeled siRNA in normal organs and tumor tissues, we used the IVIS Spectrum CT system (PerkinElmer) and fluorescence microscopy. In addition, the multiphoton live imaging system (SP5; Leica) and light sheet fluorescence microscopy (Carl Zeiss) were employed for detailed analysis and clear visualization of extravasation and intracellular distribution of fluorescent siRNA in tumor cells. To examine therapeutic potential of the sCA system, we administered survivin siRNA in pre-established HCT116 tumor mice models. Results & Discussions: Ex vivo imaging analysis using IVIS Spectrum CT showed that the fluorescence of sCA-siRNA was highly accumulated into the tumors as early as 90 min. Light sheet fluorescence microscopy revealed that sCA-siRNA accumulated in the cytoplasm of HCT116 cells with considerable green fluorescence signals at 4 h after intravenous injection. On the other hand, IF-siRNA and Atelo-siRNA did not accumulate in tumor cells. sCA-survivin-siRNA exhibited anti-tumor effects on day 11 and the tumor volume of mice treated with sCA-survivin-siRNA was significantly smaller than in mice treated with sCA-control-siRNA (P = 0.0008), IF-survivin-siRNA (P = 0.0028), or Atelo-survivin-siRNA (P = 0.0046) on day 18. For in vivo safety, we carried out a toxicity study by intravenous injection of sCA to mice. Blood chemistry tests and HE stained sections of the liver, kidney and spleen showed no physiologically significant difference between the saline and sCA treated groups. To aim the final goal of therapy and diagnosis of human tumors, we made up approximately 70-fold sCA of that administered in mouse per injection and administered it to monkeys on 4 consecutive days (n = 3). Consequently intravenous administer of sCA did not cause any serious adverse events in monkeys. Conclusion: We have demonstrated a highly efficient in vivo siRNA delivery system targeting solid tumor models, which is characterized as 10 nm minimal particle size and quick accumulation in tumors, thus it can realize to deliver abundant intact siRNA into tumor cells. Citation Format: Hirofumi Yamamoto, Xin Wu, Susumu Miyazaki, Mamoru Uemura, Taishi Hata, Junichi Nishimura, Ichiro Takemasa, Tsunekazu Mizushima, Yuichiro Doki, Masaki Mori. Super carbonate apatite as simplified systemic nanoparticle carrier for therapy of solid tumors. [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 4460. doi:10.1158/1538-7445.AM2014-4460