The GENTIL Method for Isolation of Human Adult Cardiomyocytes from Cryopreserved Tissue for Proteomic Analyses.

Heart failure is a serious clinical and economic health care problem, and its clinical progression is linked to pathological cardiac remodeling. Due to the heterogeneity of heart failure, lack of animal models to accurately represent advanced heart failure, and limited access to fresh human cardiac tissue, little is known regarding cell-type-specific mechanisms and context-specific functions of cardiomyocytes during disease development processes. While mass spectrometry has been increasingly applied to unravel changes in the proteome associated with cardiovascular physiology and disease, most studies have used homogenized tissue. Therefore, new studies using isolated cardiomyocytes are necessary to gain a better understanding of the intricate cell-type-specific molecular mechanisms underlying the pathophysiology of heart failure. This chapter describes the GENTIL method, which incorporates recent technological developments in sample handling, for isolation of cardiomyocytes from cryopreserved human cardiac tissues for use in proteomic analyses.

Surfaceome mapping of primary human heart cells with CellSurfer uncovers cardiomyocyte surface protein LSMEM2 and proteome dynamics in failing hearts.

Cardiac cell surface proteins are drug targets and useful biomarkers for discriminating among cellular phenotypes and disease states. Here we developed an analytical platform, CellSurfer, that enables quantitative cell surface proteome (surfaceome) profiling of cells present in limited quantities, and we apply it to isolated primary human heart cells. We report experimental evidence of surface localization and extracellular domains for 1,144 N-glycoproteins, including cell-type-restricted and region-restricted glycoproteins. We identified a surface protein specific for healthy cardiomyocytes, LSMEM2, and validated an anti-LSMEM2 monoclonal antibody for flow cytometry and imaging. Surfaceome comparisons among pluripotent stem cell derivatives and their primary counterparts highlighted important differences with direct implications for drug screening and disease modeling. Finally, 20% of cell surface proteins, including LSMEM2, were differentially abundant between failing and non-failing cardiomyocytes. These results represent a rich resource to advance development of cell type and organ-specific targets for drug delivery, disease modeling, immunophenotyping and in vivo imaging.

Small cell variant of malignant melanoma masquerading as lymphoma on fine-needle aspiration cytology: a case report.

The morphologic and immunohistochemical diversity of malignant melanoma is well known to pathologists but despite this widespread awareness the diagnosis remains a constant challenge, particularly when a noncutaneous or metastatic tumor is evaluated by fine-needle aspiration cytology. We present a case of the small cell variant of malignant melanoma mimicking lymphoma in a 78-year-old previously healthy male who presented with multiple pulmonary masses and a right-sided pleural effusion. This particular case was also immunoreactive for the hematopoietic marker CD43, a feature not previously reported in malignant melanoma. Cytologic, histologic, and immunohistochemical features of this case are presented and discussed.

ETS gene fusions and prostate cancer.

Chromosomal rearrangements are common genetic alterations in solid tumors and hematologic neoplasias. Recently, gene fusions between erythroblastosis virus E26 transforming sequence (ETS) family of transcription factors and androgen-regulated, prostate-specific TMPRSS2 gene were detected in about 50% of prostate cancers. Further studies have shown a diversity of TMPRSS2:ETS hybrid transcripts and heterogeneity of the fusion genes in multifocal prostate cancer. The role of these gene fusions in prostate carcinogenesis, the protein products associated with the variant fusion transcripts and their association with tumor morphology, stage, and clinical outcomes have also been studied. Additional data have demonstrated ETS gene fusions as a potential biomarker for diagnosing and stratifying prostate cancer patients. The following summarizes these recent advances.

Identification of PDE4D as a proliferation promoting factor in prostate cancer using a Sleeping Beauty transposon-based somatic mutagenesis screen.

Retroviral and transposon-based mutagenesis screens in mice have been useful for identifying candidate cancer genes for some tumor types. However, many of the organs that exhibit the highest cancer rates in humans, including the prostate, have not previously been amenable to these approaches. This study shows for the first time that the Sleeping Beauty transposon system can be used to identify candidate prostate cancer genes in mice. Somatic mobilization of a mutagenic transposon resulted in focal epithelial proliferation and hyperplasia in the prostate. Efficient methods were established to identify transposon insertion sites in these lesions, and analysis of transposon insertions identified candidate prostate cancer genes at common insertion sites, including Pde4d. PDE4D was also overexpressed in human prostate cancer patient samples and cell lines, and changes in PDE4D mRNA isoform expression were observed in human prostate cancers. Furthermore, knockdown of PDE4D reduced the growth and migration of prostate cancer cells in vitro, and knockdown of PDE4D reduced the growth and proliferation rate of prostate cancer xenografts in vivo. These data indicate that PDE4D functions as a proliferation promoting factor in prostate cancer, and the Sleeping Beauty transposon system is a useful tool for identifying candidate prostate cancer genes.