Glioblastoma (GBM) tumor microenvironment (TME) is a highly heterogeneous and complex system, which in addition to cancer cells, consists of various resident brain and immune cells as well as cells in transit through the tumor such as marrow-derived immune cells. The TME is a dynamic environment which is heavily influenced by alterations in cellular composition, cell-to-cell contact and cellular metabolic products as well as other chemical factors, such as pH and oxygen levels. Emerging evidence suggests that GBM cells appear to reprogram their the TME, and hijack microenvironmental elements to facilitate rapid proliferation, invasion, migration, and survival thus generating treatment resistance. GBM cells interact with their microenvironment directly through cell-to-cell by interaction mediated by cell-surface molecules, or indirectly through apocrine or paracrine signaling via cytokines, growth factors, and extracellular vehicles. The recent discovery of neuron–glioma interfaces and neurotransmitter-based interactions has uncovered novel mechanisms that favor tumor cell survival and growth. Here, we review the known and emerging evidence related to the communication between GBM cells and various components of its TME, discuss models for studying the TME and outline current studies targeting components of the TME for therapeutic purposes.

Purpose: Proficient DNA repair by homologous recombination (HR) facilitates resistance to chemoradiation in glioma stem cells (GSC). We evaluated whether compromising HR by targeting HSP90, a molecular chaperone required for the function of key HR proteins, using onalespib, a long-acting, brain-penetrant HSP90 inhibitor, would sensitize high-grade gliomas to chemoradiation in vitro and in vivo. Experimental Design: The ability of onalespib to deplete HR client proteins, impair HR repair capacity, and sensitize glioblastoma (GBM) to chemoradiation was evaluated in vitro in GSCs, and in vivo using zebrafish and mouse intracranial glioma xenograft models. The effects of HSP90 inhibition on the transcriptome and cytoplasmic proteins was assessed in GSCs and in ex vivo organotypic human glioma slice cultures. Results: Treatment with onalespib depleted CHK1 and RAD51, two key proteins of the HR pathway, and attenuated HR repair, sensitizing GSCs to the combination of radiation and temozolomide (TMZ). HSP90 inhibition reprogrammed the transcriptome of GSCs and broadly altered expression of cytoplasmic proteins including known and novel client proteins relevant to GSCs. The combination of onalespib with radiation and TMZ extended survival in a zebrafish and a mouse xenograft model of GBM compared with the standard of care (radiation and TMZ) or onalespib with radiation. Conclusions: The results of this study demonstrate that targeting HR by HSP90 inhibition sensitizes GSCs to radiation and chemotherapy and extends survival in zebrafish and mouse intracranial models of GBM. These results provide a preclinical rationale for assessment of HSP90 inhibitors in combination with chemoradiation in patients with GBM.

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria’s ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

Background: The most prevalent cancer treatments cause cell death through DNA damage. However, DNA damage response (DDR) repair pathways, initiated by tumor cells, can withstand the effects of anticancer drugs, providing justification for combining DDR inhibitors with DNA-damaging anticancer treatments.Methods: Cell viability assays were performed with CellTiter-Glo assay. DNA damage was evaluated using Western blotting analysis. RNA-seq and single-cell level expression were used to identify the DDR signatures. In vivo, studies were conducted in mice to determine the effect of ATris on TMZ sensitization.Results: We found a subpopulation of glioma sphere-forming cells (GSCs) with substantial synergism with temozolomide (TMZ) using a panel of 3 clinical-grade ataxia-telangiectasia- and Rad3-related kinase inhibitors (ATRis), (elimusertib, berzosertib, and ceralasertib). Interestingly, most synergistic cell lines had O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, indicating that ATRi mainly benefits tumors with no MGMT repair. Further, TMZ activated the ATR-checkpoint kinase 1 (Chk1) axis in an MGMT-dependent way. TMZ caused ATR-dependent Chk1 phosphorylation and DNA double-strand breaks as shown by increased γH2AX. Increased DNA damage and decreased Chk1 phosphorylation were observed upon the addition of ATRis to TMZ in MGMT-methylated (MGMT-) GSCs. TMZ also improved sensitivity to ATRis in vivo, as shown by increased mouse survival with the TMZ and ATRi combination treatment.Conclusions: This research provides a rationale for selectively targeting MGMT-methylated cells using ATRis and TMZ combination. Overall, we believe that MGMT methylation status in GBM could serve as a robust biomarker for patient selection for ATRi combined with TMZ.

Neorickettsia (formerly Ehrlichia ) risticii is an obligatory intracellular bacterium of digenetic trematodes. When a horse accidentally ingests aquatic insects containing encysted trematodes infected with N. risticii , the bacterium is transmitted from trematodes to horse cells and causes an acute and often fatal disease called Potomac horse fever (PHF). Since the discovery of N. risticii in the United States in 1984, using immunofluorescence and PCR assays, PHF has been increasingly recognized throughout North America and South America. However, so far, there exist only a few stable N. risticii culture isolates, all of which are from horses within the United States, and the strain diversity and environmental spreading and distribution of pathogenic N. risticii strains remain poorly understood. This paper reports the isolation of N. risticii from the blood of a horse with acute PHF in Ontario, Canada. Intracellular N. risticii colonies were detected in P388D ₁ cells after 47 days of culturing and 8 days after the addition of rapamycin. Molecular phylogenetic analysis based on amino acid sequences of major surface proteins P51 and Ssa1 showed that this isolate is distinct from any previously sequenced strains but closely related to midwestern U.S. strains. This is the first Canadian strain cultured, and a new method was developed to reactivate dormant N. risticii to improve culture isolation. IMPORTANCE Neorickettsia risticii is an environmental bacterium that lives inside flukes that are parasitic to aquatic snails, insects, and bats. When a horse accidentally ingests insects harboring flukes infected with N. risticii , the bacterium is transmitted to the horse and causes an acute and often fatal disease called Potomac horse fever. Although the disease has been increasingly recognized throughout North and South America, N. risticii has not been cultured outside the United States. This paper reports the first Canadian strain cultured and a new method to effectively culture isolate N. risticii from the horse blood sample. Molecular analysis showed that the genotype of this Canadian strain is distinct from previously sequenced strains but closely related to midwestern U.S. strains. Culture isolation of N. risticii strains would confirm the geographic presence of pathogenic N. risticii , help elucidate N. risticii strain diversity and environmental spreading and distribution, and improve diagnosis and development of vaccines for this dreadful disease.