Bacteriolytic therapy of experimental pancreatic carcinoma

AIM:To investigate the effectiveness of Clostridium novyi(C.novyi)-NT spores for the treatment of established subcutaneous pancreatic tumor in the syngeneic,immunocompetent Panc02/C57Bl/6 model. METHODS:C.novyi-NT spores were applied intravenously to animals carrying established pancreatic tumors of three different sizes.Systemic immune responses in peripheral blood and spleen were examined by flow cytometry.Supplementary,cytotoxic activity of lymphocytes against syngeneic tumor targets was analyzed. RESULTS:Application of spores identified,that(1) small tumors(<150 mm 3 )were completely unaffected (n=10);(2)very large tumors(>450 mm3)responded with substantial necrosis followed by shrinkage and significant lethality most likely due to tumor lysis syndrome (n=6);and(3)an optimal treatment window exists for tumors of approximately 250 mm 3 (n=21).In this latter group,all tumor-bearing animals had complete tu-mor regression and remained free of tumor recurrence. In subsequent tumor rechallenge experiments a significant delay in tumor growth compared to the initial tumor cell inoculation was observed(tumor volume at day 28:197.8±87.3 mm 3 vs 500.1±50.9 mm3,P<0.05). These effects were accompanied by systemic activation of immune response mechanisms predominantly mediated by the innate arm of the immune system. CONCLUSION:The observed complete tumor regression is encouraging and shows that immunotherapy with C.novyi-NT is an interesting strategy for the treatment of pancreatic carcinomas of defined sizes.

Establishment,functional and genetic characterization of a colon derived large cell neuroendocrine carcinoma cell line

AIM To establish cell line and patient-derived xenograft(PDX) models for neuroendocrine carcinomas(NEC) which is highly desirable for gaining insight into tumor development as well as preclinical research includingbiomarker testing and drug response prediction.METHODS Cell line establishment was conducted from direct in vitro culturing of colonic NEC tissue(HROC57). A PDX could also successfully be established from vitally frozen tumor samples. Morphological features, invasive and migratory behavior of the HROC57 cells as well as expression of neuroendocrine markers were vastly analyzed. Phenotypic analysis was done by microscopy and multicolor flow cytometry. The extensive molecular-pathological profiling included mutation analysis, assessment of chromosomal and microsatellite instability; and in addition, fingerprinting(i.e., STR analysis) was performed from the cell line in direct comparison to primary patient-derived tissues and the PDX model established. Drug responsiveness was examined for a panel of chemotherapeutics in clinical use for the treatment of solid cancers.RESULTS The established cell line HROC57 showed distinct morphological and molecular features of a poorly differentiated large-cell NEC with KI-67 > 50%. Molecular-pathological analysis revealed a Cp G island promoter methylation positive cell line with microsatellite instability being absent. The following mutation profile was observed: KRAS(wt), BRAF(mut). A high sensitivity to etoposide, cisplatin and 5-FU could be demonstrated while it was more resistant towards rapamycin. CONCLUSION We successfully established and characterized a novel patient-derived NEC cell line in parallel to a PDX model as a useful tool for further analysis of the biological characteristics and for development of novel diagnostic and therapeutic options for NEC.

Establishment,functional and genetic characterization of three novel patient-derived rectal cancer cell lines

AIM To establish patient-individual tumor models of rectal cancer for analyses of novel biomarkers, individual response prediction and individual therapy regimens.METHODS Establishment of cell lines was conducted by direct in vitro culturing and in vivo xenografting with subsequent in vitro culturing. Cell lines were in-depth characterized concerning morphological features, invasive and migratory behavior, phenotype, molecular profile including mutational analysis, protein expression, and confirmation of origin by DNA fingerprint. Assessment of chemosensitivity towards an extensive range of current chemotherapeutic drugs and of radiosensitivity was performed including analysis of a combined radioand chemotherapeutic treatment. In addition, glucose metabolism was assessed with 18 F-fluorodeoxyglucose(FDG) and proliferation with 18 F-fluorothymidine.RESULTS We describe the establishment of ultra-low passage rectal cancer cell lines of three patients suffering from rectal cancer. Two cell lines(HROC126, HROC284 Met) were established directly from tumor specimens while HROC239 T0 M1 was established subsequent to xenografting of the tumor. Molecular analysis classified all three cell lines as CIMP-0/non-MSI-H(sporadic standard) type. Mutational analysis revealed following mutational profiles: HROC126: APC^(wt), TP53^(wt), KRAS^(wt), BRAF^(wt), PTEN^(wt); HROC239 T0 M1: APC^(mut), P53^(wt), KRAS^(mut), BRAF^(wt), PTEN^(mut) and HROC284 Met: APC^(wt), P53^(mut), KRAS^(mut), BRAF^(wt), PTEN^(mut). All cell lines could be characterized as epithelial(EpCAM+) tumor cells with equivalent morphologic features and comparable growth kinetics. The cell lines displayed a heterogeneous response toward chemotherapy, radiotherapy and their combined application. HROC126 showed a highly radio-resistant phenotype and HROC284 Met was more susceptible to a combined radiochemotherapy than HROC126 and HROC239 T0 M1. Analysis of 18 F-FDG uptake displayed a markedly reduced FDG uptake of all three cell lines after combined radiochemotherapy. CONCLUSION These newly established and in-depth characterized ultra-low passage rectal cancer cell lines provide a useful instrument for analysis of biological characteristics of rectal cancer.

Establishment and characterization of cell lines from chromosomal instable colorectal cancer

AIM: To generate novel tumor models for preclinicalvalidation of biomarkers that allow drug response prediction and individual therapeutic decisions.METHODS: Cell line establishment was conducted by both direct in vitro culturing and in vivo xenografting followed by in vitro culturing procedure.A comprehensive characterization was subsequently performed.This included quality control,consisting of the confirmation of human and colorectal cancer(CRC) origin by DNA fingerprint and epithelial cell adhesion molecule(EpC AM) staining,as well as mycoplasma and human virus testing.Phenotypic analysis was done by light microscopy and multicolor flow cytometry.Histopathological examination(β-catenin and cytokeratin staining) was conducted in direct comparison to parental tumor tissues.Extensive molecular-pathological profiling included mutation analysis for CRC-associated driver mutations,assessment of chromosomal and microsatellite instability,and the grade of CpG island methylation.Additionally,an arraybased comparative genomic hybridization analysis was performed.Drug responsiveness was assessed for a panel of classical and novel substances in clinical use for the treatment of solid cancers.Finally,tumorigenicity of the cell lines was tested by xenografting into immunocompromised nude mice.RESULTS: Herein we describe the establishment of three ultra-low passage cell lines from two individual patients suffering from sporadic CRC.One cell line was derived directly from an early stage case(HROC18),whereas two cell lines could be established both direct from patient material and after xenografting from a late stage tumor(HROC32).All cell lines were free of contaminating mycoplasma and viruses.Molecularpathological analysis allowed all cell lines to be classified as chromosomal instable(CIN+).They were aneuploid,with CpG island promoter methylation and microsatellite instability being absent.The following mutational profile was observed both in the cell lines and the parental tumor tissue: HROC18: APCmut,p53 mut,K-raswt; HROC32:APCwt,p53 mut,K-rasmut.All cell lines were characterized as epithelial(EpC AM+) cells,showing distinct morphology and migration speed,but comparable growth kinetics.The cell lines showed different patterns of response towards clinically approved and novel drugs,with HROC18 being more resistant than HROC32 cells.Finally,in vivo tumorigenicity was demonstrated.CONCLUSION: We successfully established and characterized novel ultra-low passage patient-derived CRC models as useful instruments for analyzing biological characteristics associated with the CIN+ phenotype.