Peritoneal sarcomatosis: site of origin for the establishment of an in vitro and in vivo cell line model to study therapeutic resistance in dedifferentiated liposarcoma.

Approximately 50-70 % of patients with retroperitoneal or intraabdominal sarcoma develop a relapse after surgical therapy, including peritoneal sarcomatosis, an extremely rare site of metastatic disease which is associated with an extremely poor prognosis. Accordingly, the establishment of a permanent cell line derived from peritoneal sarcomatosis might provide a helpful tool to understand the biological behavior and to develop new therapeutic strategies. Thus, we established and characterized a liposarcoma cell line (Lipo-DUE1) from a peritoneal sarcomatosis that was permanently cultured without showing any morphological changes. Lipo-DUE1 cells exhibited a spindle-shaped morphology and positive staining for S100. Tumorigenicity was demonstrated in vitro by invasion and migration assays and in vivo by using a subcutaneous xenograft mouse model. In addition, aCGH analysis revealed concordant copy number variations on chromosome 12q in the primary tumor, peritoneal sarcomatosis, and Lipo-DUE1 cells that are commonly observed in liposarcoma. Chemotherapeutic sensitivity assays revealed a pronounced drug-resistant phenotype of Lipo-DUE1 cells to conventionally used chemotherapeutic agents. In conclusion, we describe for the first time the establishment and characterization of a liposarcoma cell line derived from a peritoneal sarcomatosis. Hence, in the future, the newly established cell line Lipo-DUE1 might serve as a useful in vitro and in vivo model to investigate the biological behavior of liposarcoma and to assess novel targeted therapies.

Inhibition of Class I Histone Deacetylases 1 and 2 Promotes Urothelial Carcinoma Cell Death by Various Mechanisms.

Class I histone deacetylases HDAC1 and HDAC2 contribute to cell proliferation and are commonly upregulated in urothelial carcinoma. To evaluate whether specific inhibition of these enzymes might serve as an appropriate therapy for urothelial carcinoma, siRNA-mediated knockdown and specific pharmacologic inhibition of HDAC1 and HDAC2 were applied in urothelial carcinoma cell lines (UCC) with distinct HDAC1 and HDAC2 expression profiles. HDACs and response marker proteins were followed by Western blotting and qRT-PCR. Effects of class I HDAC suppression on UCCs were analyzed by viability, colony forming, and caspase-3/7 assays; flow cytometry, senescence and lactate dehydrogenase cytotoxicity assays; and immunofluorescence staining. Whereas single knockdowns of HDAC1 or HDAC2 were impeded by compensatory upregulation of the other isoenzyme, efficient double knockdown of HDAC1 and HDAC2 reduced proliferation by up to 80% and induced apoptosis-like cell death in all UCCs. Clonogenic growth was cell line- and HDAC-dependently reduced, with double knockdown of HDAC1 and HDAC2 being usually most efficient. Class I HDAC-specific inhibitors, especially the more specific HDAC1/2 inhibitors romidepsin and givinostat, significantly reduced proliferation of all UCCs (IC50, 3.36 nmol/L-4.59 µmol/L). Romidepsin and givinostat also significantly inhibited clonogenic growth of UCCs, with minor effects on nontumorigenic controls. Intriguingly, these compounds induced primarily S-phase disturbances and nonapoptotic cell death in UCCs. Thus, although both ways of inhibiting HDAC1/2 share mechanisms and efficaciously inhibit cell proliferation, their modes of action differ substantially. Regardless, combined inhibition of HDAC1/2 appears to represent a promising strategy for urothelial carcinoma therapy. Mol Cancer Ther; 15(2); 299-312. ©2016 AACR.

Epigenetic age predictions based on buccal swabs are more precise in combination with cell type-specific DNA methylation signatures.

Aging is reflected by highly reproducible DNA methylation (DNAm) changes that open new perspectives for estimation of chronological age in legal medicine. DNA can be harvested non-invasively from cells at the inside of a person's cheek using buccal swabs - but these specimens resemble heterogeneous mixtures of buccal epithelial cells and leukocytes with different epigenetic makeup. In this study, we have trained an age predictor based on three age-associated CpG sites (associated with the genesPDE4C, ASPA, and ITGA2B) for swab samples to reach a mean absolute deviation (MAD) between predicted and chronological age of 4.3 years in a training set and of 7.03 years in a validation set. Subsequently, the composition of buccal epithelial cells versus leukocytes was estimated by two additional CpGs (associated with the genes CD6 and SERPINB5). Results of this "Buccal-Cell-Signature" correlated with cell counts in cytological stains (R2 = 0.94). Combination of cell type-specific and age-associated CpGs into one multivariate model enabled age predictions with MADs of 5.09 years and 5.12 years in two independent validation sets. Our results demonstrate that the cellular composition in buccal swab samples can be determined by DNAm at two cell type-specific CpGs to improve epigenetic age predictions.

Phenotype plasticity rather than repopulation from CD90/CK14+ cancer stem cells leads to cisplatin resistance of urothelial carcinoma cell lines.

BACKGROUND: Tumour heterogeneity and resistance to systemic treatment in urothelial carcinoma (UC) may arise from cancer stem cells (CSC). A recent model describes cellular differentiation states within UC based on corresponding expression of surface markers (CD) and cytokeratins (CK) with CD90 and CK14 positive cells representing the least differentiated and most tumourigenic population. Based on the fact that this population is postulated to constitute CSCs and the origin of cisplatin resistance, we enriched urothelial carcinoma cell lines (UCCs) for CD90 and studied the tumour-initiating potential of these separated cells in vitro. METHODS: Magnetic- and fluorescence-activated- cell sorting were used for separation of CD90(+) and CD90(-) UCCs. Distribution of cell surface markers CD90, CD44, and CD49f and cytokeratins CK14, CK5, and CK20 as well as the effects of short- and long-term treatment with cisplatin were assessed in vitro and measured by qRT-PCR, immunocytochemistry, reporter assay and flow cytometry in 11 UCCs. RESULTS: We observed cell populations with surface markers according to those reported in tumour xenografts. However, expression of cytokeratins did not concord regularly with that of the surface markers. In particular, expression of CD90 and CK14 diverged during enrichment of CD90(+) cells by immunomagnetic sorting or following cisplatin treatment. Enriched CD90(+) cells did not exhibit CSC-like characteristics like enhanced clonogenicity and cisplatin resistance. Moreover, selection of cisplatin-resistant sublines by long-term drug treatment did not result in enrichment of CD90(+) cells. Rather, these sublines displayed significant phenotypic plasticity expressing EMT markers, an altered pattern of CKs, and WNT-pathway target genes. CONCLUSIONS: Our findings indicate that the correspondence between CD surface markers and cytokeratins reported in xenografts is not maintained in commonly used UCCs and that CD90 may not be a stable marker of CSC in UC. Moreover, UCCs cells are capable of substantial phenotypic plasticity that may significantly contribute to the emergence of cisplatin resistance.