The tyrosine kinase c-Met promotes the formation and malignant progression of multiple cancers. It is well known that c-Met hyperactivation increases tumorigenicity and tumor cell resistance to DNA damaging agents, properties associated with tumor-initiating stem cells. However, a link between c-Met signaling and the formation and/or maintenance of neoplastic stem cells has not been previously identified. Here, we show that c-Met is activated and functional in glioblastoma (GBM) neurospheres enriched for glioblastoma tumor-initiating stem cells and that c-Met expression/function correlates with stem cell marker expression and the neoplastic stem cell phenotype in glioblastoma neurospheres and clinical glioblastoma specimens. c-Met activation was found to induce the expression of reprogramming transcription factors (RFs) known to support embryonic stem cells and induce differentiated cells to form pluripotent stem (iPS) cells, and c-Met activation counteracted the effects of forced differentiation in glioblastoma neurospheres. Expression of the reprogramming transcription factor Nanog by glioblastoma cells is shown to mediate the ability of c-Met to induce the stem cell characteristics of neurosphere formation and neurosphere cell self-renewal. These findings show that c-Met enhances the population of glioblastoma stem cells (GBM SCs) via a mechanism requiring Nanog and potentially other c-Met–responsive reprogramming transcription factors.

The origins of tumor-propagating neoplastic stem-like cells [cancer stem cells (CSC)] and their relationship to the bulk population of tumor cells that lack stem-like tumor-propagating features (i.e., transit-amplifying cancer progenitor cells) remain unclear. Recent findings from multiple laboratories show that cancer progenitor cells have the capacity to dedifferentiate and acquire a stem-like phenotype in response to either genetic manipulation or environmental cues. These findings suggest that CSCs and relatively differentiated progenitors coexist in dynamic equilibrium and are subject to bidirectional conversion. In this review, we discuss emerging concepts regarding the stem-like phenotype, its acquisition by cancer progenitor cells, and the molecular mechanisms involved. Understanding the dynamic equilibrium between CSCs and cancer progenitor cells is critical for the development of therapeutic strategies to deplete tumors of their tumor-propagating and treatment-resistant cell subpopulations. Cancer Res; 72(3); 576–80. ©2012 AACR.

A subset of stem-like cells in glioblastoma (GBM; GSC) underlies tumor propagation, therapeutic resistance, and tumor recurrence. Immune evasion is critical for GSCs to carry out these functions. However, the molecular mechanisms employed by GSCs to escape antitumor immunity remain largely unknown. The reprogramming transcription factors Oct4 and Sox2 function as core multipotency factors and play an essential role in the formation and maintenance of GSCs, but the roles of these transcription factors in GSC immune escape have not been well explored. Here we examine how Oct4/Sox2 coexpression contributes to the immunosuppressive phenotype of GSCs. Combined transcription profiling and functional studies of Oct4/Sox2 coexpressing GSCs and differentiated GBM cells demonstrated that Oct4 and Sox2 cooperatively induce an immunosuppressive transcriptome consisting of multiple immunosuppressive checkpoints (i.e., PD-L1, CD70, A2aR, TDO) and dysregulation of cytokines and chemokines that are associated with an immunosuppressive tumor microenvironment. Mechanistically, induction and function of BRD/H3k27Ac-dependent immunosuppressive genes played a role in the immunosuppressive phenotype of GSCs. Pan-BET bromodomain inhibitors (e.g., JQ1) and shBRD4 constructs significantly inhibited the immunosuppressive transcriptome and immunosuppressive biological responses induced by Oct4/Sox2. Our findings identify targetable mechanisms by which tumor-propagating GSCs contribute to the immunosuppressive microenvironment in GBM. Significance: This report identifies mechanisms by which the reprogramming transcription factors Oct4 and Sox2 function to drive the immunomodulatory transcriptome of GSCs and contribute to the immunosuppressive microenvironment in GBM.

ATRX inactivation occurs with IDH1^R132H and p53 mutations in over 80% of Grades II/III astrocytomas. It is believed that ATRX loss contributes to oncogenesis by dysregulating epigenetic and telomere mechanisms but effects on anti-glioma immunity have not been explored. This paper examines how ATRX loss contributes to the malignant and immunosuppressive phenotypes of IDH1^R132H/p53_mut glioma cells and xenografts. Isogenic astrocytoma cells (+/−IDH1^R132H/+/−ATRX_loss) were established in p53mut astrocytoma cell lines using lentivirus encoding doxycycline-inducible IDH1^R132H, ATRX shRNA, or Lenti-CRISPR/Cas9 ATRX. Effects of IDH1^R132H+/−ATRX_loss on cell migration, growth, DNA repair, and tumorigenicity were evaluated by clonal growth, transwell and scratch assays, MTT, immunofluorence and immunoblotting assays, and xenograft growth. Effects on the expression and function of modulators of the immune microenvironment were quantified by qRT-PCR, immunoblot, T-cell function, macrophage polarization, and flow cytometry assays. Pharmacologic inhibitors were used to examine epigenetic drivers of the immunosuppressive transcriptome of IDH1^R132H/p53_mut/ATRX_loss cells. Adding ATRX loss to the IDH1^R132H/p53_mut background promoted astrocytoma cell aggressiveness, induced expression of BET proteins BRD3/4 and an immune-suppressive transcriptome consisting of up-regulated immune checkpoints (e.g., PD-L1, PD-L2) and altered cytokine/chemokine profiles (e.g., IL33, CXCL8, CSF2, IL6, CXCL9). ATRX loss enhanced the capacity of IDH1^R132H/p53_mut cells to induce T-cell apoptosis, tumorigenic/anti-inflammatory macrophage polarization and Treg infiltration. The transcriptional and biological immune-suppressive responses to ATRX loss were enhanced by temozolomide and radiation and abrogated by pharmacologic BET inhibition. ATRX loss activates a BRD-dependent immune-suppressive transcriptome and immune escape mechanism in IDH1^R132H/p53_mut astrocytoma cells.

The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350–400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy (SMLM). In a single labeling step, antibodies conjugated with short DNA oligonucleotides visualized multiple targets by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. This approach avoids potential effects on structural integrity when using multiple rounds of immunolabeling and eliminates chromatic aberration, because all targets are imaged using a single excitation laser wavelength. This method proved robust for multi-target imaging in semi-thin tissue sections with a lateral resolution better than 25 nm, paving the way toward structural cell biology with single-molecule SRM.