The role of stress-activated RNA-protein granules in surviving adversity.

Severe environmental stress can trigger a plethora of physiological changes and, in the process, significant cytoplasmic reorganization. Stress-activated RNA-protein granules have been implicated in this cellular overhaul by sequestering pre-existing mRNAs and influencing their fates during and after stress acclimation. While the composition and dynamics of stress-activated granule formation has been well studied, their function and impact on RNA-cargo has remained murky. Several recent studies challenge the view that these granules degrade and silence mRNAs present at the onset of stress and instead suggest new roles for these structures in mRNA storage, transit, and inheritance. Here we discuss recent evidence for revised models of stress-activated granule functions and the role of these granules in stress survival and recovery.

The genetic basis of aneuploidy tolerance in wild yeast.

Aneuploidy is highly detrimental during development yet common in cancers and pathogenic fungi - what gives rise to differences in aneuploidy tolerance remains unclear. We previously showed that wild isolates of Saccharomyces cerevisiae tolerate chromosome amplification while laboratory strains used as a model for aneuploid syndromes do not. Here, we mapped the genetic basis to Ssd1, an RNA-binding translational regulator that is functional in wild aneuploids but defective in laboratory strain W303. Loss of SSD1 recapitulates myriad aneuploidy signatures previously taken as eukaryotic responses. We show that aneuploidy tolerance is enabled via a role for Ssd1 in mitochondrial physiology, including binding and regulating nuclear-encoded mitochondrial mRNAs, coupled with a role in mitigating proteostasis stress. Recapitulating ssd1Δ defects with combinatorial drug treatment selectively blocked proliferation of wild-type aneuploids compared to euploids. Our work adds to elegant studies in the sensitized laboratory strain to present a mechanistic understanding of eukaryotic aneuploidy tolerance.

Developmental and oncogenic programs in H3K27M gliomas dissected by single-cell RNA-seq.

Gliomas with histone H3 lysine27-to-methionine mutations (H3K27M-glioma) arise primarily in the midline of the central nervous system of young children, suggesting a cooperation between genetics and cellular context in tumorigenesis. Although the genetics of H3K27M-glioma are well characterized, their cellular architecture remains uncharted. We performed single-cell RNA sequencing in 3321 cells from six primary H3K27M-glioma and matched models. We found that H3K27M-glioma primarily contain cells that resemble oligodendrocyte precursor cells (OPC-like), whereas more differentiated malignant cells are a minority. OPC-like cells exhibit greater proliferation and tumor-propagating potential than their more differentiated counterparts and are at least in part sustained by PDGFRA signaling. Our study characterizes oncogenic and developmental programs in H3K27M-glioma at single-cell resolution and across genetic subclones, suggesting potential therapeutic targets in this disease.

Single-cell RNA sequencing reveals intrinsic and extrinsic regulatory heterogeneity in yeast responding to stress.

From bacteria to humans, individual cells within isogenic populations can show significant variation in stress tolerance, but the nature of this heterogeneity is not clear. To investigate this, we used single-cell RNA sequencing to quantify transcript heterogeneity in single Saccharomyces cerevisiae cells treated with and without salt stress to explore population variation and identify cellular covariates that influence the stress-responsive transcriptome. Leveraging the extensive knowledge of yeast transcriptional regulation, we uncovered significant regulatory variation in individual yeast cells, both before and after stress. We also discovered that a subset of cells appears to decouple expression of ribosomal protein genes from the environmental stress response in a manner partly correlated with the cell cycle but unrelated to the yeast ultradian metabolic cycle. Live-cell imaging of cells expressing pairs of fluorescent regulators, including the transcription factor Msn2 with Dot6, Sfp1, or MAP kinase Hog1, revealed both coordinated and decoupled nucleocytoplasmic shuttling. Together with transcriptomic analysis, our results suggest that cells maintain a cellular filter against decoupled bursts of transcription factor activation but mount a stress response upon coordinated regulation, even in a subset of unstressed cells.

Decoupling genetics, lineages, and microenvironment in IDH-mutant gliomas by single-cell RNA-seq.

Tumor subclasses differ according to the genotypes and phenotypes of malignant cells as well as the composition of the tumor microenvironment (TME). We dissected these influences in isocitrate dehydrogenase (IDH)-mutant gliomas by combining 14,226 single-cell RNA sequencing (RNA-seq) profiles from 16 patient samples with bulk RNA-seq profiles from 165 patient samples. Differences in bulk profiles between IDH-mutant astrocytoma and oligodendroglioma can be primarily explained by distinct TME and signature genetic events, whereas both tumor types share similar developmental hierarchies and lineages of glial differentiation. As tumor grade increases, we find enhanced proliferation of malignant cells, larger pools of undifferentiated glioma cells, and an increase in macrophage over microglia expression programs in TME. Our work provides a unifying model for IDH-mutant gliomas and a general framework for dissecting the differences among human tumor subclasses.

Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma.

Although human tumours are shaped by the genetic evolution of cancer cells, evidence also suggests that they display hierarchies related to developmental pathways and epigenetic programs in which cancer stem cells (CSCs) can drive tumour growth and give rise to differentiated progeny. Yet, unbiased evidence for CSCs in solid human malignancies remains elusive. Here we profile 4,347 single cells from six IDH1 or IDH2 mutant human oligodendrogliomas by RNA sequencing (RNA-seq) and reconstruct their developmental programs from genome-wide expression signatures. We infer that most cancer cells are differentiated along two specialized glial programs, whereas a rare subpopulation of cells is undifferentiated and associated with a neural stem cell expression program. Cells with expression signatures for proliferation are highly enriched in this rare subpopulation, consistent with a model in which CSCs are primarily responsible for fuelling the growth of oligodendroglioma in humans. Analysis of copy number variation (CNV) shows that distinct CNV sub-clones within tumours display similar cellular hierarchies, suggesting that the architecture of oligodendroglioma is primarily dictated by developmental programs. Subclonal point mutation analysis supports a similar model, although a full phylogenetic tree would be required to definitively determine the effect of genetic evolution on the inferred hierarchies. Our single-cell analyses provide insight into the cellular architecture of oligodendrogliomas at single-cell resolution and support the cancer stem cell model, with substantial implications for disease management.

Comparison of breast and abdominal adipose tissue mesenchymal stromal/stem cells in support of proliferation of breast cancer cells.

In this study, we compared MSCs from breast and abdominal tissue in terms of their expression of genes deemed important in the support of breast cancer growth and their effect on gene profile of macrophages after coculture. In addition, we investigated the role of MSCs, alone or in combination with macrophages, on proliferation of breast cancer cell lines. Our results show that MSCs derived from breast and abdominal adipose tissues have a comparable gene expression profile, have similar effect on gene expression of macrophages, and are comparable in supporting breast cancer cell line proliferation.

Macrophages and mesenchymal stromal cells support survival and proliferation of multiple myeloma cells.

Multiple myeloma (MM) is characterized by almost exclusive tropism of malignant cells for the bone marrow (BM) milieu. The survival and proliferation of malignant plasma cells have been shown to rely on interactions with nonmalignant stromal cells, in particular mesenchymal stromal cells (MSCs), in the BM microenvironment. However, the BM microenvironment is composed of a diverse array of cell types. This study examined the role of macrophages, an abundant component of BM stroma, as a potential niche component that supports malignant plasma cells. We investigated the proliferation of MM tumour cell lines when cultured alone or together with MSCs, macrophages, or a combination of MSCs and macrophages, using the carboxyfluorescein succinimidyl ester assay. Consistently, we observed increased proliferation of MM cell lines in the presence of either MSCs or macrophages compared to cell line-only control. Furthermore, the combined co-culture of MSCs plus macrophages induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, MSCs and macrophages decreased the rate of apoptosis of myeloma cells. Our in vitro studies provide evidence that highlights the role of macrophages as a key component of the BM microenvironment facilitating the growth of malignant plasma cells in MM.

Biologic and immunomodulatory properties of mesenchymal stromal cells derived from human pancreatic islets.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC. METHODS: We culture-expanded MSC from cadaveric human pancreatic islets and characterized them using flow cytometry, differentiation assays and nuclear magnetic resonance-based metabolomics. We also investigated the immunologic properties of pancreatic islet-derived MSC compared with bone marrow (BM) MSC. RESULTS: Pancreatic islet and BM-derived MSC expressed the same cell-surface markers by flow cytometry, and both could differentiate into bone, fat and cartilage. Metabolomics analysis of MSC from BM and pancreatic islets also showed a similar set of metabolic markers but quantitative polymerase chain reactions showed that pancreatic islet MSC expressed more interleukin(IL)-1b, IL-6, STAT3 and FGF9 compared with BM MSC, and less IL-10. However, similar to BM MSC, pancreatic islet MSC were able to suppress proliferation of allogeneic T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies. CONCLUSIONS: Our in vitro analysis shows pancreatic islet-derived MSC have phenotypic, biologic and immunomodulatory characteristics similar, but not identical, to BM-derived MSC. We propose that pancreatic islet-derived MSC could potentially play an important role in improving the outcome of pancreatic islet transplantation by promoting engraftment and creating a favorable immune environment for long-term survival of islet allografts.