Hidetatsu Outani

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells.

Direct induction of chondrogenic cells from human dermal fibroblast culture by defined factors.

The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon) cells from human dermal fibroblast (HDF) culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.

Induction of chondrogenic cells from dermal fibroblast culture by defined factors does not involve a pluripotent state

There is a significant need for cell sources for cartilage regenerative medicine. It has been reported that the combined transduction of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) directly induces chondrogenic cells from mouse dermal fibroblast (MDF) culture. To gain insights into the process by which cellular characteristics are altered by transduction of c-Myc, Klf4 and SOX9, we examined marker gene expression in the MDF culture at various time points after transduction. The expression of fibroblast-markers was reduced first, followed by an increase in the expression of a chondrocyte-marker. We detected no expression of pluripotent markers at any time point examined. To determine whether or not induced chondrogenic cells go through a pluripotent state after transduction, we analyzed MDFs prepared from Nanog-GFP transgenic mice by monitoring expression of the GFP-labeled pluripotent marker Nanog-GFP in the MDF culture, using time-lapse microscopic observation. Whole-well time-lapse observation revealed that none of the induced chondrogenic cells displayed GFP fluorescence during induction. These results indicate that cells do not undergo a pluripotent state during direct induction of chondrogenic cells from fibroblast culture by transduction of c-Myc, Klf4 and SOX9.

Combined targeting of mTOR and c-MET signaling pathways for effective management of epithelioid sarcoma.

BackgroundEpithelioid sarcoma (EpS) is a high-grade malignant soft-tissue sarcoma characterized by local recurrences and distant metastases. Effective treatments for EpS have not been established and thus novel therapeutic approaches against EpS are urgently required. mTOR inhibitors exert antitumor effects on several malignancies but AKT reactivation by mTOR inhibition attenuates the antitumor effects of mTOR inhibitors. This reactivation is receptor tyrosine kinase (RTK)-dependent due to a release of negative feedback inhibition. We found that c-MET was the most highly activated RTK in two human EpS cell lines, Asra-EPS and VAESBJ. Here we investigated the functional and therapeutic relevance of mTOR and/or c-MET signaling pathways in EpS both in vitro and in vivo.MethodsWe first examined the effects of an mTOR inhibitor, RAD001 (everolimus), on cell proliferation, cell cycle, AKT/mTOR signaling, and xenograft tumor growth in EpS cell lines. Next, we determined whether RAD001-induced AKT reactivation was blocked by silencing of c-MET or treatment with a selective c-MET inhibitor, INC280. Finally, we evaluated the antitumor effects of RAD001 combined with INC280 on EpS cell lines compared with either single agent or control in vitro and in vivo.ResultsConstitutive AKT phosphorylation was observed in Asra-EPS and VAESBJ cells. RAD001 suppressed EpS cell growth by inducing cell cycle arrest but enhanced AKT phosphorylation, which resulted in intrinsic resistance to mTOR inhibitors. In both EpS cell lines, RAD001-induced AKT phosphorylation was dependent on c-MET signaling. INC280 inhibited phosphorylation of c-MET and its downstream molecules, and decreased RAD001-induced phosphorylation of both AKT and ERK in EpS. Compared with a single agent or control, the combination of RAD001 and INC280 exerted superior antitumor effects on the growth of EpS cell lines in vitro and in vivo.ConclusionsTargeting of mTOR and c-MET signaling pathways significantly abrogates the growth of EpS in preclinical models and may be a promising therapeutic approach for patients with EpS.

Tailored therapeutic strategies for synovial sarcoma : Receptor tyrosine kinase pathway analyses predict sensitivity to the mTOR inhibitor RAD001

We examined efficacy of the mTOR inhibitor RAD001 to seek novel therapies for synovial sarcoma (SS). Although RAD001 had significant anti-tumor effects, its sensitivity differed among cell lines. Phospho-receptor tyrosine kinase (RTK) array analyses revealed c-MET phosphorylation in highly mTOR inhibitor-sensitive cells and PDGFRα (which induces intrinsic resistance to mTOR inhibitor) activation in less sensitive cells. Combined treatment with RAD001 and the PDGFR inhibitor pazopanib showed anti-tumor effects in xenograft models with less sensitive cells. Thus, evaluating activated RTKs in clinical samples may predict sensitivity to mTOR inhibitors, raising the possibility of a tailored therapy for SS.

Establishment of a novel clear cell sarcoma cell line (Hewga-CCS), and investigation of the antitumor effects of pazopanib on Hewga-CCS

BackgroundClear cell sarcoma (CCS) is a therapeutically unresolved, aggressive, soft tissue sarcoma (STS) that predominantly affects young adults. This sarcoma is defined by t(12;22)(q13;q12) translocation, which leads to the fusion of Ewing sarcoma gene (EWS) to activating transcription factor 1 (ATF1) gene, producing a chimeric EWS-ATF1 fusion gene. We established a novel CCS cell line called Hewga-CCS and developed an orthotopic tumor xenograft model to enable comprehensive bench-side investigation for intensive basic and preclinical research in CCS with a paucity of experimental cell lines.MethodsHewga-CCS was derived from skin metastatic lesions of a CCS developed in a 34-year-old female. The karyotype and chimeric transcript were analyzed. Xenografts were established and characterized by morphology and immunohistochemical reactivity. Subsequently, the antitumor effects of pazopanib, a recently approved, novel, multitargeted, tyrosine kinase inhibitor (TKI) used for the treatment of advanced soft tissue sarcoma, on Hewga-CCS were assessed in vitro and in vivo.ResultsHewga-CCS harbored the type 2 EWS-ATF1 transcript. Xenografts morphologically mimicked the primary tumor and expressed S-100 protein and antigens associated with melanin synthesis (Melan-A, HMB45). Pazopanib suppressed the growth of Hewga-CCS both in vivo and in vitro. A phospho-receptor tyrosine kinase array revealed phosphorylation of c-MET, but not of VEGFR, in Hewga-CCS. Subsequent experiments showed that pazopanib exerted antitumor effects through the inhibition of HGF/c-MET signaling.ConclusionsCCS is a rare, devastating disease, and our established CCS cell line and xenograft model may be a useful tool for further in-depth investigation and understanding of the drug-sensitivity mechanism.

FDG-PET evaluation of chondromyxoid fibroma of left ilium.

Chondromyxoid fibroma is a rare benign tumor, which is composed of immature myxoid mesenchymal tissue with features of early primitive cartilaginous differentiation, accounting for less than 1% of all primary bone tumors. Chondromyxoid fibromas arising from the ilium are relatively rare. Reports on fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging for chondromyxoid fibroma are limited. We present a case of a chondromyxoid fibroma of the left ilium with fluorodeoxyglucose-positron emission tomography (FDG-PET) findings, which showed increased accumulation of fluorodeoxyglucose in spite of its characteristic benign pathology. The maximum standardized uptake value (SUV) of the mass was 3.20.

Radiofrequency ablation of osteoid osteoma using a three-dimensional navigation system

OBJECTIVE This study aimed to report the clinical outcomes for patients with osteoid osteoma (OO) treated by radiofrequency ablation (RFA) using a three-dimensional (3D) navigation system. METHODS We performed RFA using a 3D navigation system on 32 patients with clinically and radiologically diagnosed OO. This study included 25 males and 7 females with a median age of 20 years (range, 10-39 years). The median duration of follow-up was 18 months (range, 1-65 months). We investigated technical specifications, tumor localization, technical success, clinical success, biopsy success, complications, incomplete treatment, and recurrences. RESULTS Eighteen tumors were located in the femur, seven in the tibia, two in the humerus, and one each in the fibula, scapula, patella, lumbar vertebra, and acetabula. All procedures were technically successful, and pain relief was achieved in all patients. However, local recurrence developed in one patient, needing additional RFA. The clinical success rate was 96.8%. Biopsy showed OO in 12 patients (37%). Complications occurred in three patients (9%), two cases of fractures and one of osteomyelitis. CONCLUSIONS A 3D navigation provides real-time imaging and enables us to set the RFA needle in the correct position, particularly in case of OO-aroused complex anatomical structures. Our initial results indicated that radiofrequency ablation using a 3D navigation system is feasible and safe for patients with OO.

Atypical femoral fracture associated with bone-modifying agent for bone metastasis of breast cancer: A report of two cases:

Atypical femoral fractures (AFFs) are recently observed as a complication of long-term bone-modifying agent (BMA; bisphosphonate or denosumab) therapy for bone metastases. We describe the cases of two women diagnosed with breast cancer who developed incomplete AFF associated with BMAs prescribed for bone metastases. Radiographs of their femurs revealed thickening of the lateral subtrochanteric cortex, and tomosynthesis revealed a visible fracture line in the thickened cortex. They were initially treated with conservative management; however, the incomplete fracture resulted in a complete fracture. These cases highlight two major implications. First, symptomatic incomplete AFF associated with BMAs prescribed for bone metastases should be treated with surgical prophylaxis, given the fact that fracture healing is expected to require a longer duration and an incomplete fracture might potentially progress to a complete fracture during long-term conservative management. Second, tomosynthesis is useful in identifying radiolucent fracture lines that are reliable predictors of fracture propagation.

Functional and therapeutic relevance of hepatocyte growth factor/c-MET signaling in synovial sarcoma.

Synovial sarcoma (SS) is an aggressive soft tissue sarcoma with a poor prognosis and, thus, novel therapeutic strategies for SS are urgently required. In the present study, we investigated the functional and therapeutic relevance of hepatocyte growth factor (HGF)/c‐MET signaling in SS. Both HGF and c‐MET were highly expressed in Yamato‐SS cells, resulting in activation of c‐MET and its downstream AKT and extracellular signal‐regulated kinase signaling pathways, whereas c‐MET was expressed but not activated in SYO‐1 or HS‐SY‐II cells. c‐MET‐activated Yamato‐SS cells showed higher anchorage‐independent growth ability and less sensitivity to chemotherapeutic agents than did c‐MET‐inactivated SYO‐1 or HS‐SY‐II cells. INC280, a selective c‐MET inhibitor, inhibited growth of Yamato‐SS cells both in vitro and in vivo but not that of SYO‐1 or HS‐SY‐II cells. INC280 induced cell cycle arrest and apoptosis, and blocked phosphorylation of c‐MET and its downstream effectors in Yamato‐SS cells. Co‐expression of HGF and c‐MET in SS clinical samples correlated with a poor prognosis in patients with SS. Taken together, activation of HGF/c‐MET signaling in an autocrine fashion leads to an aggressive phenotype in SS and targeting of this signaling exerts superior antitumor effects on c‐MET‐activated SS. HGF/c‐MET expression status is a potential biomarker for identification of SS patients with a worse prognosis who can benefit from c‐MET inhibitors.