Establishment and characterization of a new human extragonadal germ cell line, SEM-1, and its comparison with TCam-2 and JKT-1.

OBJECTIVE: To describe the establishment and characterization of a human cell line, SEM-1, from a patient diagnosed with a mediastinal seminoma. METHODS: A small percentage of germ cell tumors develop as primary lesions in extragonadal sites, and the etiology of these tumors is poorly understood. Currently, only 2 cell lines from seminoma patients have been reported, JKT-1 and TCam-2, both derived from the testis. The cell line was characterized by heterotransplantation in Nude mice, cytogenetic studies, immunohistochemical and flow cytometry staining for germ cell tumor biomarkers, quantitative reverse-transcription polymerase chain reaction for cancer testis antigen expression, and BRAF mutation screening with quantitative polymerase chain reaction. RESULTS: Characterization studies confirmed the human extragonadal seminoma origin of SEM-1 and demonstrated that it had more features in common with TCam-2 than JKT-1. Specifically, SEM-1 was positive for Sal-like protein 4 (SALL-4), activator protein-2γ (AP-2γ), and cytokeratin CAM5.2, and demonstrated heterogeneous expression of stem cell markers octamer-binding transcription factor 3/4, NANOG, c-KIT, SOX17, and SOX2. Cytogenetic analysis revealed a hypotriploid chromosome number, with multiple copies of 12p, but isochromosome 12p and the BRAF mutation V600E were not identified. The cell lines also did not contain the BRD4/NUT gene rearrangement [t(15,19)] seen in midline carcinomas nor did they contain overexpressed nuclear protein in testis (NUT) genes. CONCLUSION: SEM-1 is the first cell line derived from an extragonadal germ cell tumor showing intermediate characteristics between seminoma and nonseminoma, and as such, is an important model to study the molecular pathogenesis of this malignancy.

Survival signals and targets for therapy in breast implant-associated ALK--anaplastic large cell lymphoma.

PURPOSE: Anaplastic lymphoma kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphoma (T-ALCL) in patients with textured saline and silicone breast implants is a recently recognized clinical entity for which the etiology and optimal treatment remain unknown. EXPERIMENTAL DESIGN: Using three newly established model cell lines from patient biopsy specimens, designated T-cell breast lymphoma (TLBR)-1 to -3, we characterized the phenotype and function of these tumors to identify mechanisms of cell survival and potential therapeutic targets. RESULTS: Cytogenetics revealed chromosomal atypia with partial or complete trisomy and absence of the NPM-ALK (2;5) translocation. Phenotypic characterization showed strong positivity for CD30, CD71, T-cell CD2/5/7, and antigen presentation (HLA-DR, CD80, CD86) markers, and interleukin (IL)-2 (CD25, CD122) and IL-6 receptors. Studies of these model cell lines showed strong activation of STAT3 signaling, likely related to autocrine production of IL-6 and decreased SHP-1. STAT3 inhibition, directly or by recovery of SHP-1, and cyclophosphamide-Adriamycin-vincristine-prednisone (CHOP) chemotherapy reagents, effectively kill cells of all three TLBR models in vitro and may be pursued as therapies for patients with breast implant-associated T-ALCLs. CONCLUSIONS: The TLBR cell lines closely resemble the primary breast implant-associated lymphomas from which they were derived and as such provide valuable preclinical models to study their unique biology.

USC-HN2, a new model cell line for recurrent oral cavity squamous cell carcinoma with immunosuppressive characteristics.

Head and neck squamous cell carcinomas (HNSCC) are common and aggressive tumors that have not seen an improvement in survival rates in decades. These tumors are believed to evade the immune system through a variety of mechanisms and are therefore highly immune modulatory. In order to elucidate their interaction with the immune system and develop new therapies targeting immune escape, new pre-clinical models are needed. A novel human cell line, USC-HN2, was established from a patient biopsy specimen of invasive, recurrent buccal HNSCC and characterized by morphology, heterotransplantation, cytogenetics, phenotype, gene expression, and immune modulation studies and compared to a similar HNSCC cell line; SCCL-MT1. Characterization studies confirmed the HNSCC origin of USC-HN2 and demonstrated a phenotype similar to the original tumor and typical of aggressive oral cavity HNSCC (EGFR(+)CD44v6(+)FABP5(+)Keratin(+) and HPV(-)). Gene and protein expression studies revealed USC-HN2 to have highly immune-modulatory cytokine production (IL-1ß, IL-6, IL-8, GM-CSF, and VEGF) and strong regulatory T and myeloid derived suppressor cell (MDSC) induction capacity in vitro. Of note, both USC-HN2 and SCCL-MT1 were found to have a more robust cytokine profile and MDSC induction capacity when compared to seven previously established HNSCC cell lines. Additionally, microarray gene expression profiling of both cell lines demonstrate up-regulation of antigen presenting genes. Because USC-HN2 is therefore highly immunogenic, it also induces strong immune suppression to evade immunologic destruction. Based upon these results, both cell lines provide an excellent model for the development of new suppressor cell-targeted immunotherapies.

Functional characterization of human Cd33+ and Cd11b+ myeloid-derived suppressor cell subsets induced from peripheral blood mononuclear cells co-cultured with a diverse set of human tumor cell lines.

BACKGROUND: Tumor immune tolerance can derive from the recruitment of suppressor cell populations, including myeloid-derived suppressor cells (MDSC). In cancer patients, MDSC accumulation correlates with increased tumor burden, but the mechanisms of MDSC induction remain poorly understood. METHODS: This study examined the ability of human tumor cell lines to induce MDSC from healthy donor PBMC using in vitro co-culture methods. These human MDSC were then characterized for morphology, phenotype, gene expression, and function. RESULTS: Of over 100 tumor cell lines examined, 45 generated canonical CD33+HLA-DR(low)Lineage- MDSC, with high frequency of induction by cervical, ovarian, colorectal, renal cell, and head and neck carcinoma cell lines. CD33+ MDSC could be induced by cancer cell lines from all tumor types with the notable exception of those derived from breast cancer (0/9, regardless of hormone and HER2 status). Upon further examination, these and others with infrequent CD33+ MDSC generation were found to induce a second subset characterized as CD11b+CD33(low)HLA-DR(low)Lineage-. Gene and protein expression, antibody neutralization, and cytokine-induction studies determined that the induction of CD33+ MDSC depended upon over-expression of IL-1ß, IL-6, TNFα, VEGF, and GM-CSF, while CD11b+ MDSC induction correlated with over-expression of FLT3L and TGFß. Morphologically, both CD33+ and CD11b+ MDSC subsets appeared as immature myeloid cells and had significantly up-regulated expression of iNOS, NADPH oxidase, and arginase-1 genes. Furthermore, increased expression of transcription factors HIF1α, STAT3, and C/EBPß distinguished MDSC from normal counterparts. CONCLUSIONS: These studies demonstrate the universal nature of MDSC induction by human solid tumors and characterize two distinct MDSC subsets: CD33+HLA-DR(low)HIF1α+/STAT3+ and CD11b+HLA-DR(low)C/EBPß+, which should enable the development of novel diagnostic and therapeutic reagents for cancer immunotherapy.