Cell non-autonomous interactions during non-immune stromal progression in the breast tumor microenvironment

Summary The breast tumor microenvironment of primary and metastatic sites is a complex milieu of differing cell populations, consisting of tumor cells and the surrounding stroma. Despite recent progress in delineating the immune component of the stroma, the genomic expression landscape of the non-immune stroma (NIS) population and their role in mediating cancer progression and informing effective therapies are not well understood. Here we obtained 52 cell-sorted NIS and epithelial tissue samples across 37 patients from i) normal breast, ii) normal breast adjacent to primary tumor, iii) primary tumor, and iv) metastatic tumor sites. Deep RNA-seq revealed diverging gene expression profiles as the NIS evolves from normal to metastatic tumor tissue, with intra-patient normal-primary variation comparable to inter-patient variation. Significant expression changes between normal and adjacent normal tissue support the notion of a cancer field effect, but extended out to the NIS. Most differentially expressed protein-coding genes and lncRNAs were found to be associated with pattern formation, embryogenesis, and the epithelial-mesenchymal transition. We validated the protein expression changes of a novel candidate gene, C2orf88, by immunohistochemistry staining of representative tissues. Significant mutual information between epithelial ligand and NIS receptor gene expression, across primary and metastatic tissue, suggests a unidirectional model of molecular signaling between the two tissues. Furthermore, survival analyses of 827 luminal breast tumor samples demonstrated the predictive power of the NIS gene expression to inform clinical outcomes. Together, these results highlight the evolution of NIS gene expression in breast tumors and suggest novel therapeutic strategies targeting the microenvironment

A DNMT3B Alternatively Spliced Exon and Encoded Peptide Are Novel Biomarkers of Human Pluripotent Stem Cells

A major obstacle in human stem cell research is the limited number of reagents capable of distinguishing pluripotent stem cells from partially differentiated or incompletely reprogrammed derivatives. Although human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) express numerous alternatively spliced transcripts, little attention has been directed at developing splice variant-encoded protein isoforms as reagents for stem cell research. In this study, several genes encoding proteins involved in important signaling pathways were screened to detect alternatively spliced transcripts that exhibited differential expression in pluripotent stem cells (PSCs) relative to spontaneously differentiated cells (SDCs). Transcripts containing the alternatively spliced exon 10 of the de novo DNA methyltransferase gene, DNMT3B, were identified that are expressed in PSCs. To demonstrate the utility and superiority of splice variant specific reagents for stem cell research, a peptide encoded by DNMT3B exon 10 was used to generate an antibody, SG1. The SG1 antibody detects a single DNMT3B protein isoform that is expressed only in PSCs but not in SDCs. The SG1 antibody is also demonstrably superior to other antibodies at distinguishing PSCs from SDCs in mixed cultures containing both pluripotent stem cells and partially differentiated derivatives. The tightly controlled down regulation of DNMT3B exon 10 containing transcripts (and exon 10 encoded peptide) upon spontaneous differentiation of PSCs suggests that this DNMT3B splice isoform is characteristic of the pluripotent state. Alternatively spliced exons, and the proteins they encode, represent a vast untapped reservoir of novel biomarkers that can be used to develop superior reagents for stem cell research and to gain further insight into mechanisms controlling stem cell pluripotency

DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use

Astrocytes derived from trisomic human embryonic stem cells express markers of astrocytic cancer cells and premalignant stem-like progenitors

Abstract Background Trisomic variants of human embryonic stem cells (hESCs) arise spontaneously in culture. Although trisomic hESCs share many properties with diploid hESCs, they also exhibit features of cancer stem cells. Since most hESC-based therapies will utilize differentiated derivatives, it is imperative to investigate the potential of trisomic hESCs to undergo malignant transformation during differentiation prior to their use in the clinical setting. Methods Diploid and trisomic hESCs were differentiated into astrocytic progenitors cells (APCs), RNA extracted and hybridized to human exon-specific microarrays. Global gene expression profiles of diploid and trisomic APCs were compared to that of an astrocytoma cell line and glioblastoma samples, analyzed by others, using the same microarray platform. Results Bioinformatic analysis of microarray data indicates that differentiated trisomic APCs exhibit global expression profiles with similarities to the malignant astrocytoma cell line. An analogous trend is observed in comparison to glioblastoma samples indicating that trisomic APCs express markers of astrocytic cancer cells. The analysis also allowed identification of transcripts predicted to be differentially expressed in brain tumor stem cells. These data indicate that in vitro differentiation of trisomic hESCs along astrocytic pathways give rise to cells exhibiting properties of premalignant astrocytic stem/progenitor cells. Conclusions Given their occult nature, opportunities to study premalignant stem/progenitor cells in human have been few. The ability to propagate and direct the differentiation of aneuploid hESCs provides a powerful in vitro system for investigating biological properties of human cells exhibiting features of premalignant stem cells. This in vitro culture system can be used to elucidate changes in gene expression occurring enroute to malignant transformation and to identify molecular markers of cancer stem/progenitor cells. These markers are invaluable for diagnostic purposes and may be novel targets for therapeutic intervention.</p