Immunogenetics of glioblastoma: the future of personalized patient management.

The prognosis of glioblastoma has changed little over the past two decades, with only minor improvements in length of overall survival through the addition of temozolomide (temodal) to standard of care and the recommended use of alternating electric field therapy (optune) to newly diagnosed patients. In an effort to define novel therapeutic targets across molecularly heterogeneous disease subgroups, researchers have begun to uncover the complex interplay between epigenetics, cell signaling, metabolism, and the immunosuppressive tumor microenvironment. Indeed, IDH mutations are now recognized as a defining differential factor not only influencing global hypermethylation and patient prognosis but also degree of immune infiltration within individual tumors. Likewise, next-generation sequencing has defined subgroup-specific transcriptional profiles that correlate with different mechanisms of immune evasion, including increased PD-L1 and CTLA-4 among mesenchymal tumors. Interestingly, sequencing of the T cell repertoire from numerous patient samples suggests that the correlation between mutational burden and enrichment of tumor-specific peptides may be less convincing than originally suspected. While this raises questions over the efficacy of dendritic cell or tumor-lysate vaccines and CAR-T therapies, these avenues continue to be explored. In addition to these active immunotherapies, inhibitors of molecular hubs with wide reaching effects, including STAT3, IDO, and TGF-ß, are now in early-phase clinical trials. With the potential to block intrinsic biological properties of tumor growth and invasion while bolstering the immunogenic profile of the tumor microenvironment, these new targets represent a new direction for GBM therapies. In this review, we show the advances in molecular profiling and immunophenotyping of GBM, which may lead to the development of new personalized therapeutic strategies.

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model's ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Systematic mapping of occluded genes by cell fusion reveals prevalence and stability of cis-mediated silencing in somatic cells.

Both diffusible factors acting in trans and chromatin components acting in cis are implicated in gene regulation, but the extent to which either process causally determines a cell's transcriptional identity is unclear. We recently used cell fusion to define a class of silent genes termed "cis-silenced" (or "occluded") genes, which remain silent even in the presence of trans-acting transcriptional activators. We further showed that occlusion of lineage-inappropriate genes plays a critical role in maintaining the transcriptional identities of somatic cells. Here, we present, for the first time, a comprehensive map of occluded genes in somatic cells. Specifically, we mapped occluded genes in mouse fibroblasts via fusion to a dozen different rat cell types followed by whole-transcriptome profiling. We found that occluded genes are highly prevalent and stable in somatic cells, representing a sizeable fraction of silent genes. Occluded genes are also highly enriched for important developmental regulators of alternative lineages, consistent with the role of occlusion in safeguarding cell identities. Alongside this map, we also present whole-genome maps of DNA methylation and eight other chromatin marks. These maps uncover a complex relationship between chromatin state and occlusion. Furthermore, we found that DNA methylation functions as the memory of occlusion in a subset of occluded genes, while histone deacetylation contributes to the implementation but not memory of occlusion. Our data suggest that the identities of individual cell types are defined largely by the occlusion status of their genomes. The comprehensive reference maps reported here provide the foundation for future studies aimed at understanding the role of occlusion in development and disease.

Embryonic stem cells induce pluripotency in somatic cell fusion through biphasic reprogramming.

It is a long-held paradigm that cell fusion reprograms gene expression but the extent of reprogramming and whether it is affected by the cell types employed remain unknown. We recently showed that the silencing of somatic genes is attributable to either trans-acting cellular environment or cis-acting chromatin context. Here, we examine how trans- versus cis-silenced genes in a somatic cell type behave in fusions to another somatic cell type or to embryonic stem cells (ESCs). We demonstrate that while reprogramming of trans-silenced somatic genes occurs in both cases, reprogramming of cis-silenced somatic genes occurs only in somatic-ESC fusions. Importantly, ESCs reprogram the somatic genome in two distinct phases: trans-reprogramming occurs rapidly, independent of DNA replication, whereas cis-reprogramming occurs with slow kinetics requiring DNA replication. We also show that pluripotency genes Oct4 and Nanog are cis-silenced in somatic cells. We conclude that cis-reprogramming capacity is a fundamental feature distinguishing ESCs from somatic cells.

Evidence for a critical role of gene occlusion in cell fate restriction.

The progressive restriction of cell fate during lineage differentiation is a poorly understood phenomenon despite its ubiquity in multicellular organisms. We recently used a cell fusion assay to define a mode of epigenetic silencing that we termed "occlusion", wherein affected genes are silenced by cis-acting chromatin mechanisms irrespective of whether trans-acting transcriptional activators are present. We hypothesized that occlusion of lineage-inappropriate genes could contribute to cell fate restriction. Here, we test this hypothesis by introducing bacterial artificial chromosomes (BACs), which are devoid of chromatin modifications necessary for occlusion, into mouse fibroblasts. We found that BAC transgenes corresponding to occluded endogenous genes are expressed in most cases, whereas BAC transgenes corresponding to silent but non-occluded endogenous genes are not expressed. This indicates that the cellular milieu in trans supports the expression of most occluded genes in fibroblasts, and that the silent state of these genes is solely the consequence of occlusion in cis. For the BAC corresponding to the occluded myogenic master regulator Myf5, expression of the Myf5 transgene on the BAC triggered fibroblasts to acquire a muscle-like phenotype. These results provide compelling evidence for a critical role of gene occlusion in cell fate restriction.

miR-17 family miRNAs are expressed during early mammalian development and regulate stem cell differentiation.

MicroRNAs are small non-coding RNAs that regulate protein expression by binding 3'UTRs of target mRNAs, thereby inhibiting translation. Similar to siRNAs, miRNAs are cleaved by Dicer. Mouse and ES cell Dicer mutants demonstrate that microRNAs are necessary for embryonic development and cellular differentiation. However, technical obstacles and the relative infancy of this field have resulted in few data on the functional significance of individual microRNAs. We present evidence that miR-17 family members, miR-17-5p, miR-20a, miR-93, and miR-106a, are differentially expressed in developing mouse embryos and function to control differentiation of stem cells. Specifically, miR-93 localizes to differentiating primitive endoderm and trophectoderm of the blastocyst. We also observe high miR-93 and miR-17-5p expression within the mesoderm of gastrulating embryos. Using an ES cell model system, we demonstrate that modulation of these miRNAs delays or enhances differentiation into the germ layers. Additionally, we demonstrate that these miRNAs regulate STAT3 mRNA in vitro. We suggest that STAT3, a known ES cell regulator, is one target mRNA responsible for the effects of these miRNAs on cellular differentiation.

Regulation of Sox2 by STAT3 initiates commitment to the neural precursor cell fate.

STAT3, a member of the signal transducer and activator or transcription (STAT) family of proteins, plays a major role in gliogenesis; however, its functions during differentiation of neural precursor cells (NPCs) are unclear. Our data demonstrate that STAT3 is present and active in the developing mouse central nervous system (CNS) as early as E7.5, several days prior to gliogenesis. We hypothesize that STAT3 is functioning very early in neural development to regulate NPC differentiation. To test this hypothesis, STAT3 dominant negative embryonic stem (ES) cells were generated and subjected to neural differentiation. The loss of STAT3 resulted in production of significantly fewer NPCs along with decreased expression of the neural stem cell marker nestin. Further investigation revealed the existence of a novel signaling pathway during early neural development in which STAT3 directly regulates the Sox2 promoter leading to Sox2 expression and subsequent nestin expression and commitment to the NPC fate.

Small RNAs, big potential: the role of MicroRNAs in stem cell function.

MicroRNAs (miRNAs) are a newly discovered, yet powerful mechanism for regulating protein expression via mRNA translational inhibition. Loss of all miRNA function within mice leads to embryonic lethality with a loss of the stem cell population in the epiblast and failure to form a primitive streak. These data suggest that miRNAs play a major role in embryonic development. As critical regulation of protein expression is also important for controlling the balance between self-renewal and differentiation in stem cells, the study of miRNAs within this model system is rapidly expanding. New data suggest that stem cells have discrete miRNA expression profiles, which may account for, or contribute to, the intrinsic stem cell properties of self-renewal and pluripotency. Specifically, miRNAs have been implicated in downregulation of cell cycle checkpoint proteins during germ stem cell division. Other data demonstrate that changes in miRNA expression can promote or inhibit stem or progenitor cell differentiation within different cell lineages, including hematopoietic cells, cardiomyocytes, myoblasts, and neural cells. In this review we detail the established functional roles of miRNAs in the embryonic and adult stem cell model systems. Finally, we explore new techniques that exploit endogenous miRNA processing and function for applications in basic and clinical research.