We introduce CIBERSORT, a method for characterizing cell composition of complex tissues from their gene expression profiles. When applied to enumeration of hematopoietic subsets in RNA mixtures from fresh, frozen, and fixed tissues, including solid tumors, CIBERSORT outperformed other methods with respect to noise, unknown mixture content, and closely related cell types. CIBERSORT should enable large-scale analysis of RNA mixtures for cellular biomarkers and therapeutic targets (http://cibersort.stanford.edu).

Molecular profiles of tumors and tumor-associated cells hold great promise as biomarkers of clinical outcomes. However, existing datasets are fragmented and difficult to analyze systematically. Here we present a pan-cancer resource and meta-analysis of expression signatures from ~18,000 human tumors with overall survival outcomes across 39 malignancies. Using this resource, we identified a FOXM1 regulatory network as a major predictor of adverse outcomes, and found that expression of favorably prognostic genes, including KLRB1, largely reflect tumor-associated leukocytes. By applying CIBERSORT, a computational approach for inferring leukocyte representation in bulk tumor transcriptomes, we identified complex associations between 22 distinct leukocyte subsets and cancer survival. For example, tumor-associated neutrophil and plasma cell signatures emerged as significant but opposite predictors of survival for diverse solid tumors, including breast and lung adenocarcinomas. This resource and associated analytical tools (http://precog.stanford.edu) may help delineate prognostic genes and leukocyte subsets within and across cancers, shed light on the impact of tumor heterogeneity on cancer outcomes, and discover biomarkers and therapeutic targets.

Using a high-throughput chemical screen, we identified two small molecules that enhance the survival of human embryonic stem cells (hESCs). By characterizing their mechanisms of action, we discovered an essential role of E-cadherin signaling for ESC survival. Specifically, we showed that the primary cause of hESC death following enzymatic dissociation comes from an irreparable disruption of E-cadherin signaling, which then leads to a fatal perturbation of integrin signaling. Furthermore, we found that stability of E-cadherin and the resulting survival of ESCs were controlled by specific growth factor signaling. Finally, we generated mESC-like hESCs by culturing them in mESC conditions. And these converted hESCs rely more on E-cadherin signaling and significantly less on integrin signaling. Our data suggest that differential usage of cell adhesion systems by ESCs to maintain self-renewal may explain their profound differences in terms of morphology, growth factor requirement, and sensitivity to enzymatic cell dissociation.human embryonic stem cell survival | cell-cell adhesion | cell-ECM adhesion C onventional murine and human embryonic stem cells (hESCs) derived from blastocysts can be propagated indefinitely and have the ability to generate all cell types (1-3). They express pluripotency transcription factors, including the ones that can reprogram somatic cells back to pluripotent states: Oct4, Sox2, Nanog, and Klf4. However, murine and human ESCs respond very differently to several key signaling pathways in selfrenewal or differentiation. For example, murine ESCs (mESCs) self-renew under leukemia inhibitory factor (LIF) and bone morphogenic protein (BMP) (4, 5), whereas human ESCs (hESCs) appear dependent on FGF, and TGFβ/Activin/Nodal pathway activity for self-renewal (6-11). These studies clearly suggest that there exist two distinct self-renewal mechanisms. In addition, hESCs grow in vitro as large flattened 2D colonies, whereas mESCs display characteristic small-domed 3D appearances of compact colonies. Moreover, unlike mESCs, hESCs are very vulnerable to single-cell dissociation. Massive cell death occurs after complete single-cell dissociation, which has been a significant hurdle for rapid expansion and genetic manipulation of hESCs. To address this critical challenge and understand the molecular mechanisms that govern hESC survival, we used a high-throughput chemical screening approach and identified two small molecules with distinct mechanisms of action that significantly increase hESC survival after single-cell dissociation. In depth characterizations of compounds' mechanism of action revealed that hESC survival and self-renewal is regulated by the interplay between two cell adhesion systems: cell-cell adhesion and cell-ECM adhesion. Our studies also uncovered a common mechanism that underlies and integrates two seemingly distinct self-renewal states represented by conventional murine and human ESCs.

This radiogenomics strategy for identifying imaging biomarkers may enable a more rapid evaluation of novel imaging modalities, thereby accelerating their translation to personalized medicine.

An improved understanding of stem-cell and regenerative biology, as well as a better control of stem-cell fate, is likely to produce treatments for many devastating diseases and injuries. Chemical approaches are starting to have an increasingly important role in this young field. Attention has focused on chemical approaches that allow the precise manipulation of cells in vitro to obtain homogeneous cell types for cell-based therapies. Another promising approach is the development of conventional chemical and biological therapeutics to stimulate endogenous cells to regenerate. Such therapeutics can act on target cells or their niches in vivo to promote cell survival, proliferation, differentiation, reprogramming and homing.

Tumor-associated macrophages (TAM) are prominent components of tumor microenvironment (TME) and capable of promoting cancer progression. However, the mechanisms for the formation of M2-like TAMs remain enigmatic. Here, we show that lactate is a pivotal oncometabolite in the TME that drives macrophage M2-polarization to promote breast cancer proliferation, migration, and angiogenesis. In addition, we identified that the activation of ERK/STAT3, major signaling molecules in the lactate signaling pathway, deepens our molecular understanding of how lactate educates TAMs. Moreover, suppression of ERK/STAT3 signaling diminished tumor growth and angiogenesis by abolishing lactate-induced M2 macrophage polarization. Finally, research data of the natural compound withanolide D provide evidence for ERK/STAT3 signaling as a potential therapeutic strategy for the prevention and treatment of breast cancer. These findings suggest that the lactate-ERK/STAT3 signaling pathway is a driver of breast cancer progression by stimulating macrophage M2-like polarization and reveal potential new therapeutic targets for breast cancer treatment.

Poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation-induced necrosis has been implicated in several pathophysiological conditions. Although mitochondrial dysfunction and apoptosis-inducing factor translocation from the mitochondria to the nucleus have been suggested to play very important roles in PARP-1-mediated cell death, the signaling events downstream of PARP-1 activation in initiating mitochondria dysfunction are not clear. Here we used the DNA alkylating agent N-methyl-N-nitro-N-nitrosoguanidine, a potent PARP-1 activator, to study PARP-1 activation-mediated cell death. We found, based on genetic knockouts and pharmacological inhibition, that c-Jun N-terminal kinase (JNK), especially JNK1, but not the other groups of mitogen-activated protein kinase, is required for PARP-1-induced mitochondrial dysfunction, apoptosis-inducing factor translocation, and subsequent cell death. We reveal that receptor-interacting protein 1 (RIP1) and tumor necrosis factor receptor-associated factor 2 (TRAF2), are upstream of JNK in PARP-1 hyperactivated cells, because PARP-1-induced JNK activation was attenuated in RIP1؊/؊ and TRAF2؊/؊ mouse embryonic fibroblast cells. Consistently, knockouts of RIP1 and TRAF2 caused a resistance to PARP-1-induced cell death. Therefore, our study uncovers that RIP1, TRAF2, and JNK comprise a pathway to mediate the signaling from PARP-1 overactivation to mitochondrial dysfunction.Poly(ADP-ribose) polymerase-1 (PARP-1) 2 is a nuclear enzyme activated by DNA strand breaks that catalyzes the covalent attachment of long branched chains of poly(ADP-ribose) with NAD ϩ as its substrate to a variety of nuclear DNA-binding proteins, including PARP-1 itself (1). PARP-1 activation plays an essential role in DNA repair under moderate stress (2); however, in several pathological situations that involve massive DNA damage, extensive activation of PARP-1 depletes cellular NAD ϩ and its precursor ATP, leading to irreversible cellular energy failure and necrotic cell death (3-5). The pathophysiological importance of PARP-1-mediated cell death has been suggested by the observation that genetic ablation of PARP-1 and pharmacological inhibition of PARP-1 activity elicit strong protection in several disease models, including ischemia-reperfusion injury after cerebral ischemia and myocardial infarction, inflammatory injury, reactive oxygen species-induced injury, and glutamate excitotoxicity (6 -10). Apoptosis and necrosis are two major forms of cell death with distinct morphological features. Apoptosis is an ordered and regulated process in which the cell actively destroys itself while maintaining plasma membrane integrity, thus permitting non-inflammatory phagocytosis of the dying cell. Necrosis, on the other hand, has traditionally been regarded as a passive and unregulated form of cell death with morphology of cell swelling, loss of plasma membrane integrity, and the release of cellular contents into the extracellular environment, thus triggering an inflammatory response (11). PARP-1-mediated cell death is c...

Macrophage cell death plays a role in many physiological and pathophysiological conditions. Previous work has shown that macrophages can undergo caspase-independent cell death, and this process is associated with Nur77 induction, which is involved in inducing chromatin condensation and DNA fragmentation. Here we show that autophagy is a cytosolic event that controls caspase-independent macrophage cell death. Autophagy was induced in macrophages treated with lipopolysaccharides (LPSs) and the pan-caspase inhibitor benzyloxycarbonylVal-Ala-Asp (Z-VAD), and the inhibition of autophagy by either chemical inhibitors or by the RNA interference knockdown of beclin (a protein required for autophagic body formation) inhibited caspase-independent macrophage cell death. We also found an increase in poly(ADP-ribose) (PAR) polymerase (PARP) activation and reactive oxygen species (ROS) production in LPS ؉ Z-VAD-treated macrophages, and both are involved in caspase-independent macrophage cell death. We further determined that the formation of autophagic bodies in macrophages occurs downstream of PARP activation, and PARP activation occurs downstream of ROS production. Using macrophages in which receptor-interacting protein 1 (RIP1) was knocked down by small interfering RNA, and macrophages isolated from Toll/ interleukin-1 receptor-domain-containing adaptor inducing IFN-␤ (TRIF)-deficient mice, we found that TRIF and RIP1 function upstream of ROS production in LPS ؉ Z-VAD-treated macrophages. We also found that Z-VAD inhibits LPS-induced RIP1 cleavage, which may contribute to ROS over-production in macrophages. This paper reveals that TRIF, RIP1, and ROS production, as well as PARP activation, are involved in inducing autophagy, which contributes to caspase-independent macrophage cell death.