SUMMARY Glioblastoma multiforme (GBM) displays cellular hierarchies harboring a subpopulation of stem-like cells (GSCs). Enhancer of Zeste Homolog 2 (EZH2), the lysine methyl transferase of Polycomb repressive complex 2, mediates transcriptional repression of pro-differentiation genes in both normal and neoplastic stem cells. An oncogenic role of EZH2 as a transcriptional silencer is well established; however, additional functions of EZH2 are incompletely understood. Here we show that EZH2 binds to and methylates STAT3, leading to enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3. The EZH2-STAT3 interaction preferentially occurs in GSCs relative to non-stem bulk tumor cells, and it requires a specific phosphorylation of EZH2. Inhibition of EZH2 reverses the silencing of Polycomb target genes and diminishes STAT3 activity, suggesting therapeutic strategies.

A number of recent reports have demonstrated that only CD133-positive cancer cells of glioblastoma multiforme (GBM) have tumor-initiating potential. These findings raise an attractive hypothesis that GBMs can be cured by eradicating CD133-positive cancer stem cells (CSCs), which are a small portion of GBM cells. However, as GBMs are known to possess various genetic alterations, GBMs might harbor heterogeneous CSCs with different genetic alterations. Here, we compared the clinical characteristics of two GBM patient groups divided according to CD133-positive cell ratios. The CD133-low GBMs showed more invasive growth and gene expression profiles characteristic of mesenchymal or proliferative subtypes, whereas the CD133-high GBMs showed features of cortical and well-demarcated tumors and gene expressions typical of proneuronal subtype. Both CD133-positive and CD133-negative cells purified from four out of six GBM patients produced typical GBM tumor masses in NOD-SCID brains, whereas brain mass from CD133-negative cells showed more proliferative and angiogenic features compared to that from CD133-positive cells. Our results suggest, in contrast to previous reports that only CD133-positive cells of GBMs can initiate tumor formation in vivo CD133-negative cells also possess tumor-initiating potential, which is indicative of complexity in the identification of cancer cells for therapeutic targeting. A recent concept in brain tumor biology is that brain tumors arise from cancer stem cells (CSCs) that are CD133 positive (CD133 ( þ ) ). It has been reported that a small number of CD133 ( þ ) glioblastoma multiforme (GBM) cells are able to recapitulate the original tumor in vivo, whereas millions of CD133-negative (CD133 (À) ) cells could not produce brain tumor masses. 1-6 However, accumulating evidence suggests that CD133 (À) GBM cells can also regenerate heterogenous tumors in vivo, 7,8 and generation of the huge and rapidly growing tumors by only CD133 ( þ ) CSCs would be difficult because more than 50% of GBM patients have few CD133 ( þ ) cells. 9 As a majority of neurogenic astrocytes in the adult brain are not recognized by a CD133 antibody, 8 it is likely that CD133 might be newly expressed in GBM CSCs that are derived from CD133 (À) adult neural stem cells (NSCs) or terminally differentiated brain cells, such as astrocytes, neurons, and oligodendrocytes. Given that the gene expression profile is changed when GBM recurs after treatments, 10 it is plausible that new CD133 expression may occur if the characteristics of CSCs are changed or if some CSCs are selected by treatment. Furthermore, the wide-range variation in CD133 ( þ ) cell ratio (0.1-50% in GBM patients) 1-6 also suggests the existence of other GBM CSCs that do not express CD133.Therefore, we hypothesize that there are several kinds of CSCs in the tumor mass of GMB, and these diverse CSCs

Tumor recurrence following treatment is the major cause of mortality for glioblastoma multiforme (GBM) patients. Thus, insights on the evolutionary process at recurrence are critical for improved patient care. Here, we describe our genomic analyses of the initial and recurrent tumor specimens from each of 38 GBM patients. A substantial divergence in the landscape of driver alterations was associated with distant appearance of a recurrent tumor from the initial tumor, suggesting that the genomic profile of the initial tumor can mislead targeted therapies for the distally recurred tumor. In addition, in contrast to IDH1-mutated gliomas, IDH1-wild-type primary GBMs rarely developed hypermutation following temozolomide (TMZ) treatment, indicating low risk for TMZ-induced hypermutation for these tumors under the standard regimen.

BackgroundIntra-tumoral genetic and functional heterogeneity correlates with cancer clinical prognoses. However, the mechanisms by which intra-tumoral heterogeneity impacts therapeutic outcome remain poorly understood. RNA sequencing (RNA-seq) of single tumor cells can provide comprehensive information about gene expression and single-nucleotide variations in individual tumor cells, which may allow for the translation of heterogeneous tumor cell functional responses into customized anti-cancer treatments.ResultsWe isolated 34 patient-derived xenograft (PDX) tumor cells from a lung adenocarcinoma patient tumor xenograft. Individual tumor cells were subjected to single cell RNA-seq for gene expression profiling and expressed mutation profiling. Fifty tumor-specific single-nucleotide variations, including KRASG12D, were observed to be heterogeneous in individual PDX cells. Semi-supervised clustering, based on KRASG12D mutant expression and a risk score representing expression of 69 lung adenocarcinoma-prognostic genes, classified PDX cells into four groups. PDX cells that survived in vitro anti-cancer drug treatment displayed transcriptome signatures consistent with the group characterized by KRASG12D and low risk score.ConclusionsSingle-cell RNA-seq on viable PDX cells identified a candidate tumor cell subgroup associated with anti-cancer drug resistance. Thus, single-cell RNA-seq is a powerful approach for identifying unique tumor cell-specific gene expression profiles which could facilitate the development of optimized clinical anti-cancer strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0692-3) contains supplementary material, which is available to authorized users.

Frequent discrepancies between preclinical and clinical results of anticancer agents demand a reliable translational platform that can precisely recapitulate the biology of human cancers. Another critical unmet need is the ability to predict therapeutic responses for individual patients. Toward this goal, we have established a library of orthotopic glioblastoma (GBM) xenograft models using surgical samples of GBM patients. These patient-specific GBM xenograft tumors recapitulate histopathological properties and maintain genomic characteristics of parental GBMs in situ. Furthermore, in vivo irradiation, chemotherapy, and targeted therapy of these xenograft tumors mimic the treatment response of parental GBMs. We also found that establishment of orthotopic xenograft models portends poor prognosis of GBM patients and identified the gene signatures and pathways signatures associated with the clinical aggressiveness of GBMs. Together, the patient-specific orthotopic GBM xenograft library represent the preclinically and clinically valuable "patient tumor's phenocopy" that represents molecular and functional heterogeneity of GBMs.

BackgroundIntratumoral heterogeneity hampers the success of marker-based anticancer treatment because the targeted therapy may eliminate a specific subpopulation of tumor cells while leaving others unharmed. Accordingly, a rational strategy minimizing survival of the drug-resistant subpopulation is essential to achieve long-term therapeutic efficacy.ResultsUsing single-cell RNA sequencing (RNA-seq), we examine the intratumoral heterogeneity of a pair of primary renal cell carcinoma and its lung metastasis. Activation of drug target pathways demonstrates considerable variability between the primary and metastatic sites, as well as among individual cancer cells within each site. Based on the prediction of multiple drug target pathway activation, we derive a combinatorial regimen co-targeting two mutually exclusive pathways for the metastatic cancer cells. This combinatorial strategy shows significant increase in the treatment efficacy over monotherapy in the experimental validation using patient-derived xenograft platforms in vitro and in vivo.ConclusionsOur findings demonstrate the investigational application of single-cell RNA-seq in the design of an anticancer regimen. The approach may overcome intratumoral heterogeneity which hampers the success of precision medicine.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0945-9) contains supplementary material, which is available to authorized users.

Glioblastomas multiforme (GBM) contain highly tumorigenic, self-renewing populations of stem/initiating cells [glioblastoma stem cells (GSC)] that contribute to tumor propagation and treatment resistance. However, our knowledge of the specific signaling pathways that regulate GSCs is limited. The MET tyrosine kinase is known to stimulate the survival, proliferation, and invasion of various cancers including GBM. Here, we identified a distinct fraction of cells expressing a high level of MET in human primary GBM specimens that were preferentially localized in perivascular regions of human GBM biopsy tissues and were found to be highly clonogenic, tumorigenic, and resistant to radiation. Inhibition of MET signaling in GSCs disrupted tumor growth and invasiveness both in vitro and in vivo, suggesting that MET activation is required for GSCs. Together, our findings indicate that MET activation in GBM is a functional requisite for the cancer stem cell phenotype and a promising therapeutic target. Cancer Res; 72(15); 3828-38. Ó2012 AACR.

Objective Human Lyme arthritis caused by Borrelia burgdorferi is characterized by an inflammatory infiltrate that consists mainly of neutrophils and T cells. This study was undertaken to evaluate the role of the innate and acquired immune responses elicited by the neutrophil‐activating protein A (NapA) of B burgdorferi in patients with Lyme arthritis. Methods Serum anti‐NapA antibodies were measured in 27 patients with Lyme arthritis and 30 healthy control subjects. The cytokine profile of synovial fluid T cells specific for NapA was investigated in 5 patients with Lyme arthritis. The cytokine profile induced by NapA in neutrophils and monocytes was also investigated. Results Serum anti‐NapA antibodies were found in 48% of the patients with Lyme arthritis but were undetectable in the healthy controls. T cells from the synovial fluid of patients with Lyme arthritis produced interleukin‐17 (IL‐17) in response to NapA. Moreover, NapA was able to induce the expression of IL‐23 in neutrophils and monocytes, as well as the expression of IL‐6, IL‐1β, and transforming growth factor β (TGFβ) in monocytes, via Toll‐like receptor 2. Conclusion These findings indicate that NapA of B burgdorferi is able to drive the expression of IL‐6, IL‐1β, IL‐23, and TGFβ by cells of the innate immune system and to elicit a synovial fluid Th17 cell response that might play a crucial role in the pathogenesis of Lyme arthritis.