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Establishment and characterization of a highly immunogenic human renal carcinoma cell line.

ABSTRACT: Renal cell carcinoma (RCC) is the most common kidney cancer, and accounts for ~3% of all adult malignancies. RCC has proven refractory to conventional treatment modalities but appears to be the only histological form that shows any consistent response to immunotherapeutic approaches. The development of a clinically effective vaccine remains a major strategic target for devising active specific immunotherapy in RCC. We aimed to identify a highly immunogenic antigenic format for immunotherapeutic approaches, so as to boost immune responses in RCC patients. We established and cloned an immunogenic cell line, RCC85#21 named Elthem, which was derived from a non-aggressive and non-metastatic clear cell carcinoma. The cell line characterization was performed by genomics (real-time PCR, genome instability), proteomics (two dimensional electrophoresis, mass spectro-metry) and immunological analysis (mixed lymphocytes tumor cell cultures). Real-time PCR confirmed the RCC85#21 cell expression of tumor antigens and cytokine genes. No difference in microsatellite instability (MSI) in RCC85#21 cell line was found as compared to control, loss of heterozygosity was observed in the RCC85#21 clone, but not in the renal cancer cell lines from which it was generated. The image analysis of RCC85#21 by two-dimensional gels showed 700±26 spots and 119 spots were identified by mass spectrometry analysis. RCC85#21 promoted a significant RCC-specific T cells activation by exhibiting a cytotoxic phenotype after mixed lymphocyte and tumor cell cultures. CD8+ T cells isolated from RCC patients displayed an elevated reactivity against RCC85#21 and efficiently lysed the RCC85#21 clone. The RCC85#21 immunogenic cell line will be suitable for immune stimulation. The identification of novel tumor associated antigens will allow the evaluation of the immune response in vitro and, subsequently, in vivo paving the way for new immunotherapeutic strategies in the RCC setting.

SUBMITTER: Prattichizzo C

PROVIDER: S-EPMC4922831 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Establishment and characterization of a highly immunogenic human renal carcinoma cell line.

Prattichizzo Clelia C Gigante Margherita M Pontrelli Paola P Stella Alessandro A Rocchetti Maria Teresa MT Gigante Maddalena M Maiorano Eugenio E Herr Wolfgang W Battaglia Michele M Gesualdo Loreto L Ranieri Elena E

International journal of oncology 20160527 2

Renal cell carcinoma (RCC) is the most common kidney cancer, and accounts for ~3% of all adult malignancies. RCC has proven refractory to conventional treatment modalities but appears to be the only histological form that shows any consistent response to immunotherapeutic approaches. The development of a clinically effective vaccine remains a major strategic target for devising active specific immunotherapy in RCC. We aimed to identify a highly immunogenic antigenic format for immunotherapeutic ...[more]

PMID: 27278998

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Project description:PurposeEyelid sebaceous carcinoma (SeC) is the third most frequent eyelid malignancy worldwide and is relatively prevalent in Asian patients. An eyelid SeC cell line model is necessary for experimental research to explore the etiology and pathogenesis of eyelid SeC. This study established and characterized an eyelid SeC cell line with a TP53 mutation that might be useful for analyzing potential treatment options for eyelid SeC.MethodsThe eyelid SeC cell line SHNPH-SeC was obtained from a patient with eyelid SeC at Shanghai Ninth People's Hospital (SHNPH), Shanghai JiaoTong University School of Medicine. Immunofluorescence staining was employed to detect the origination and proliferation activity. Short tandem repeat (STR) profiling was performed for verification. Chromosome analysis was implemented to investigate chromosome aberrations. Whole exome sequencing (WES) was used to discover genomic mutations. Cell proliferation assays were performed to identify sensitivity to mitomycin-C (MMC) and 5-fluorouracil (5-FU).ResultsSHNPH-SeC cells were successively subcultured for more than 100 passages and demonstrated rapid proliferation and migration. Karyotype analysis revealed abundant chromosome aberrations, and WES revealed SeC-related mutations in TP53, KMT2C, and ERBB2. An in vivo tumor model was successfully established in NOD/SCID mice. Biomarkers of eyelid SeC, including cytokeratin 5 (CK5), epithelial membrane antigen (EMA), adipophilin, p53, and Ki-67, were detected in SHNPH-SeC cells, original tumors, and xenografts. MMC and 5-FU inhibited the proliferation and migration of SHNPH-SeC cells, and SHNPH-SeC cells presented a greater drug response than non-TP53-mutated SeC cells.ConclusionsThe newly established eyelid SeC cell line SHNPH-SeC demonstrates mutation in TP53, the most commonly mutated gene in SeC. It presents SeC properties and malignant characteristics that may facilitate the investigation of cellular behaviors and molecular mechanisms of SeC to explore promising therapeutic strategies.

Project description:Genuine racial differences in prostate cancer (PCa) biology have been considered among the potential reasons to explain PCa disparities. There is no animal model to represent all aspects of human PCa and, more specifically, to be used for PCa disparity research. The lack of a spontaneously transformed in vitro cell-based model system has been a significant impediment to investigating and understanding potential molecular mechanisms, and the hormonal, genetic, and epigenetic factors underlying the biological and clinical aggressiveness of PCa in African American (AA) men. In this study, we established and characterized the E006AA-hT cell line as a highly tumorigenic subline of the previously characterized primary AA-PCa cell line, E006AA. Extensive characterization of the E006AA-hT cell line was accomplished using cytodifferentiation and prostate-specific markers, spectral karyotyping, cell line authentication assays, cell proliferation and migration assays, and in vitro tumorigenesis assays. Spectral karyotyping of E006AA-hT showed a hypertriploid chromosome complement and shared cytogenetic changes similar to its parental cells such as diploid X, absence of Y-chromosomes, numerical gains in chromosomes 5,6,8,10,17,20,21, and marker chromosomes of unknown origin. In addition, E006AA-hT also presented numerous clonal and structural aberrations such as insertion, deletion, duplication, and translocations in chromosomes 1-5, 8, 9, 11, 13, 14, 17, and 18. The E006AA-hT cell line was shown to be highly tumorigenic and produced tumors at an accelerated growth rate in both athymic nude and triple-deficient SCID mice. Silencing the mutated androgen receptor (AR-599 Ser>Gly) did not affect proliferation (loss-of-function), but decreased migration (gain-of-function) in E006AA-hT and its parental cell type. These data support that AR-point mutations may lead simultaneously to different "loss-of-function" and "gain-of-function" phenotypes in PCa cells. E006AA-Par and its subline as the only available spontaneously transformed low- and highly-tumorigenic primary AA-PCa cell lines could be used for basic and translational research aimed in supporting prostate cancer disparity research.

Dataset Information

Establishment and characterization of a highly immunogenic human renal carcinoma cell line.

Publications

Establishment and characterization of a highly immunogenic human renal carcinoma cell line.

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