Zhiwei Qiao

Generation of novel patient-derived CIC- DUX4 sarcoma xenografts and cell lines

CIC-DUX4 sarcoma (CDS) is a group of rare, mesenchymal, small round cell tumours that harbour the unique CIC-DUX4 translocation, which causes aberrant gene expression. CDS exhibits an aggressive course and poor clinical outcome, thus novel therapeutic approaches are needed for CDS treatment. Although patient-derived cancer models are an essential modality to develop novel therapies, none currently exist for CDS. Thus, the present study successfully established CDS patient-derived xenografts and subsequently generated two CDS cell lines from the grafted tumours. Notably, xenografts were histologically similar to the original patient tumour, and the expression of typical biomarkers was confirmed in the xenografts and cell lines. Moreover, the xenograft tumours and cell lines displayed high Src kinase activities, as assessed by peptide-based tyrosine kinase array. Upon screening 119 FDA-approved anti-cancer drugs, we found that only actinomycine D and doxorubicin were effectively suppress the proliferation among the drugs for standard therapy for Ewing sarcoma. However, we identified molecular targeting reagents, such as bortezomib and crizotinib that markedly suppressed the growth of CDS cells. Our models will be useful modalities to develop novel therapeutic strategies against CDS.

Establishment and proteomic characterization of patient-derived clear cell sarcoma xenografts and cell lines

Clear cell sarcoma (CCS) is an aggressive mesenchymal malignancy characterized by the unique chimeric EWS-ATF1 fusion gene. Patient-derived cancer models are essential tools for the understanding of tumorigenesis and the development of anti-cancer drugs; however, only a limited number of CCS cell lines exist. The objective of this study was to establish patient-derived CCS models. We established patient-derived CCS models from a 43-yr-old female patient. We prepared the patient-derived xenografts (PDXs) from tumor tissues obtained through biopsy or surgery and isolated stable cell lines from PDXs and the original tumor tissue. The presence of gene fusions was examined by RT-PCR, and Sanger sequencing. The established cell lines were characterized by short tandem repeat, viability, colony and spheroid formation, and invasion analyses. Differences in gene enrichment between the primary tumor and cell lines were examined by mass spectrometry and KEGG pathway analysis. The cell lines were maintained for more than 80 passages, and had tumorigenic characteristics such as colony and spheroid formation and invasion. Mass spectrometric proteome analysis demonstrated that the cell lines were enriched for similar but distinct molecular pathways, compared to those in the xenografts and original tumor tissue. Next, tyrosine kinase inhibitors were screened for their suppressive effects on viability. We found that ponatinib, vandetanib, and doxorubicin suppressed the growth of cell lines, and had equivalent IC50 values. Further in-depth investigation and understanding of drug-sensitivity mechanisms will be important for the clinical applications of our cell lines.

Establishment and proteomic characterization of NCC-LMS1-C1, a novel cell line of primary leiomyosarcoma of the bone

Background Leiomyosarcoma (LMS) is one of most aggressive mesenchymal malignancies that differentiate towards smooth muscle. The clinical outcome of LMS patients is poor; as such, there is an urgent need for novel therapeutic approaches. Experimental models such as patient-derived cell lines are invaluable tools for pre-clinical studies. In the present study, we established a stable cell line from the tumor tissue of a patient with a primary LMS of the bone. Despite the urgent need for novel therapeutic strategies in LMS, there are only a few LMS cell lines available in public cell banks, none of which are primary to the bone. Methods Bone primary LMS tumor tissues were sampled to establish cell lines. Morphological and proteomic analyses were performed and sensitivity to pazopanib was evaluated. Results NCC-LMS1-C1 cells were maintained for over 100 passages. The cells exhibited a spindle shape and aggressive growth; they also expressed smooth muscle actin, reflecting the original LMS tissue (i.e. smooth muscle cells). The cells also showed tumor characteristics such as colony formation on soft agar and sensitivity to pazopanib, doxorubicin and cisplatin, with half-maximal inhibitory concentrations of 4.5, 0.11 and 20 μM, respectively. Proteomic analyses by mass spectrometry and antibody array revealed some differences in the protein expression profiles of these cells as compared to the original tumor tissue. Conclusions Our results indicate that the NCC-LMS1-C1 cell lines will be useful for LMS research.

Discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2) is a novel biomarker of myxofibrosarcoma invasion identified by global protein expression profiling

Myxofibrosarcoma (MFS) is a mesenchymal malignancy characterized by frequent recurrence even after radical wide resection. To optimize therapy for MFS patients, we aimed to identify candidate tissue biomarkers of MFS invasion potential. Invasion characteristics of MFS were evaluated by magnetic resonance imaging and protein expression profiling of primary tumor tissues performed using two-dimensional difference gel electrophoresis (2D-DIGE). Protein expression profiles were compared between invasive and non-invasive tumors surgically resected from 11 patients. Among the 3453 protein spots observed, 59 demonstrated statistically significant difference in intensity (≥2-fold) between invasive and non-invasive tumors (p<0.01 by Wilkoxon test), and were identified by mass spectrometry as 47 individual proteins. Among them, we further focused on discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2), a receptor tyrosine kinase with aberrant expression in malignant tumors. Immunohistochemistry analysis of 21 additional MFS cases revealed that higher DCBLD2 expression was significantly associated with invasive properties of tumor cells. DCBLD2 sensitivity and specificity, and positive and negative predictive values for MFS invasion were 69.2%, 87.5%, 90%, and 63.6%, respectively. The expression level of DCBLD2 was consistent in different portions of tumor tissues. Thus, DCBLD2 expression can be a useful biomarker to evaluate invasive properties of MFS. Further validation studies based on multi-institutional collaboration and comprehensive analysis of DCBLD2 biological functions in MFS are required to confirm its prognostic utility for clinical application.

Establishment and characterization of novel patient-derived osteosarcoma xenograft and cell line

Osteosarcoma is an aggressive mesenchymal malignancy of the bone. Patient-derived models are essential tools for elucidating the molecular mechanisms associated with poor prognosis and the development of novel anticancer drugs. This study described the establishment of a patient-derived cancer model of osteosarcoma. Primary osteosarcoma tumor tissues were obtained from an osteosarcoma patient and inoculated in the skin of immunodeficient mice, followed by transplantation to other mice upon growth. Cells were maintained in monolayer cultures, and the capability of spheroid formation was assessed by seeding the cells on culture dishes. The invasion ability of cells was monitored by Matrigel assay, and genomic and proteomic backgrounds were examined by mass spectrometry. A cell line was established from patient-derived tumors and showed similar histology to that of the primary tumor tissue. Additionally, these cells formed spheroids on low-attachment tissue-culture dishes and exhibited invasive capabilities, and we confirmed that the genomic backgrounds were similar between patient-derived xenograft tumors and the cell line. Furthermore, the proteome of the patient-derived tumors and the cells exhibited similar, but not identical, patterns to that of the original tumor tissue. Our results indicated that this patient-derived xenograft model and cell line would be useful resources for osteosarcoma research.

Establishment and characterization of the NCC–SS1–C1 synovial sarcoma cell line

Synovial sarcoma is an aggressive mesenchymal malignancy characterized by unique gene fusions. Tissue culture cells are essential tools for further understanding tumorigenesis and anti-cancer drug development; however, only a limited number of well-characterized synovial sarcoma cell lines exist. Thus, the objective of this study was to establish a patient-derived synovial sarcoma cell line. We established a synovial sarcoma cell line from tumor tissue isolated from a 72-year-old female patient. Prepared cells were analyzed for the presence of gene fusions by fluorescence in situ hybridization, RT-PCR, and karyotyping. In addition, the resulting cell line was characterized by viability, short tandem repeat, colony and spheroid formation, and invasion analyses. Differences in gene enrichment between the primary tumor and cell line were examined by mass spectrometric protein expression profiling and KEGG pathway analysis. Our analyses revealed that the primary tumor and NCC–SS1–C1 cell line harbored the SS18–SSX1 fusion gene typical of synovial sarcoma and similar proteomics profiles. In vitro analyses also confirmed that the established cell line harbored invasive, colony-forming, and spheroid-forming potentials. Moreover, drug screening with chemotherapeutic agents and tyrosine kinase inhibitors revealed that doxorubicin, a subset of tyrosine kinase inhibitors, and several molecular targeting drugs markedly decreased NCC–SS1–C1 cell viability. Results from the present study support that the NCC–SS1–C1 cell line will be an effective tool for sarcoma research.

Pazopanib-induced changes in protein expression signatures of extracellular vesicles in synovial sarcoma

Secreted proteins enclosed in extracellular vesicles can act as intercellular messengers. The objective of this study was to elucidate the role of proteins secreted from synovial sarcoma cells in the regulatory network underlying pazopanib response. We performed a comprehensive analysis of expression of proteins secreted from four synovial sarcoma cell lines (SYO-1, HS-SYII, 1273/99, and YaFuSS) using mass spectroscopy. Comparison of up-regulated proteins in cells, extracellular vesicles-free conditioned media, and extracellular vesicles revealed significantly up-regulated Wnt in synovial sarcoma vesicles. Furthermore, we compared protein signatures of cells, conditioned media, and extracellular vesicles before and after pazopanib treatment. Interestingly, protein signatures of extracellular vesicles showed robust changes in Wnt signaling pathways in response to pazopanib. Our findings provide insight into the potential role of Wnt, a protein secreted from the extracellular vesicles of synovial sarcoma cells, making it a potential candidate for use in sarcoma diagnosis.

Proteomic approach toward determining the molecular background of pazopanib resistance in synovial sarcoma

Pazopanib, a multitarget tyrosine kinase (TK) inhibitor, has been approved for treatment of soft tissue sarcoma. Elucidation of the molecular background of pazopanib resistance should lead to improved clinical outcomes in sarcomas; accordingly, we investigated this in synovial sarcoma using a proteomic approach. Pazopanib sensitivity was examined in four synovial sarcoma cell lines: SYO-1, HS-SYII, 1273/99, and YaFuSS. The 1273/99 cell line showed significantly higher IC50 values than the others for pazopanib. Expression levels of 90 TKs in the cell lines were examined by western blotting. Among these, the levels of PDGFRB, DDR1, AXL, MET, and PYK2 were higher, and those of FGFR1 and VEGFR3 were lower in the 1273/99 cell line than the other cell lines. Gene silencing analysis of the TKs upregulated in 1273/99 cells showed differing effects on cell growth: PDGFRB, MET, and PYK2 knockdown induced cell growth inhibition, whereas DDR1 and AXL knockdown did not influence cell growth. Using the PamChip peptide microarray, we found that 18 peptide substrates were highly phosphorylated in the 1273/99 cell line compared with other cell lines. Using the PhosphoNet database, we found that kinases FGFR3, RET, VEGFR1, EPHA2, EPHA4, TRKA, and SRC phosphorylated these 18 peptide substrates. Moreover, the results for overexpressed and aberrantly activated TKs in pazopanib-resistant cells showed no overlap. Taken together, our study indicates that identification of comprehensive TK profiles represents an essential approach to determining the molecular background of pazopanib resistance in synovial sarcoma.

miR-125b and miR-100 Are Predictive Biomarkers of Response to Induction Chemotherapy in Osteosarcoma.

Osteosarcoma is the most common primary malignancy in bone. Patients who respond poorly to induction chemotherapy are at higher risk of adverse prognosis. The molecular basis for such poor prognosis remains unclear. We investigated miRNA expression in eight open biopsy samples to identify miRNAs predictive of response to induction chemotherapy and thus maybe used for risk stratification therapy. The samples were obtained from four patients with inferior necrosis (Huvos I/II) and four patients with superior necrosis (Huvos III/IV) following induction chemotherapy. We found six miRNAs, including miR-125b and miR-100, that were differentially expressed > 2-fold (p < 0.05) in patients who respond poorly to treatment. The association between poor prognosis and the abundance of miR-125b and miR-100 was confirmed by quantitative reverse transcriptase-polymerase chain reaction in 20 additional osteosarcoma patients. Accordingly, overexpression of miR-125b and miR-100 in three osteosarcoma cell lines enhanced cell proliferation, invasiveness, and resistance to chemotherapeutic drugs such as methotrexate, doxorubicin, and cisplatin. In addition, overexpression of miR-125b blocked the ability of these chemotherapy agents to induce apoptosis. As open biopsy is routinely performed to diagnose osteosarcoma, levels of miR-125b and miR-100 in these samples may be used as basis for risk stratification therapy.