Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9) technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO) cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro´5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properti

Consumer Intravenous Vitamin Therapy: Wellness Boost or Toxicity

Intravenous vitamin therapy (IVVT) has become increasingly popular in recent years promising to cure or improve a variety of health problems or infuse “wellness�. Patients and consumers have IV vitamins or other nutrients and fluids infused into their arms outside the hospital setting in medical spas, hydration rooms, integrative medicine, and concierge primary care practices. The IVVT “menu� options include but are not limited to mixes containing Vitamin C, B12, glutathione, electrolytes, and saline. In the U.S., the IV administration of nutrients is considered drug or parenteral nutrition. In this paper, we describe what we learned while trying to answer a question of a patient contemplating an IVVT treatment at a retail store. Discussion of the regulatory issues and pharmacokinetics associated with IVVT is complex and beyond the scope of this paper. There is insufficient evidence to conclude there is benefit from these expensive services provided often without the knowledge of the person’s primary care physician but believe there is possibility of harm

Consumer Intravenous Vitamin Therapy: Wellness Boost or Toxicity

Intravenous vitamin therapy (IVVT) has become increasingly popular in recent years promising to cure or improve a variety of health problems or infuse “wellness”. Patients and consumers have IV vitamins or other nutrients and fluids infused into their arms outside the hospital setting in medical spas, hydration rooms, integrative medicine, and concierge primary care practices. The IVVT “menu” options include but are not limited to mixes containing Vitamin C, B12, glutathione, electrolytes, and saline. In the U.S., the IV administration of nutrients is considered drug or parenteral nutrition. In this paper, we describe what we learned while trying to answer a question of a patient contemplating an IVVT treatment at a retail store. Discussion of the regulatory issues and pharmacokinetics associated with IVVT is complex and beyond the scope of this paper. There is insufficient evidence to conclude there is benefit from these expensive services provided often without the knowledge of the person’s primary care physician but believe there is possibility of harm

Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

Glycosylation modulates growth, maintenance, and stress signaling processes.Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore,expanding our understanding of N-glycans in altering biological processes is of utmost interest.Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9)technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO) cell line,K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabledus to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-establishedPro´5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively.Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Celldissociation and migration assays demonstrated the greatest strength of cell--cell adhesion and fastestmigratory rates for oligomannose N-glycans, and these properties decreased as oligomannose typewere converted to hybrid type, and further decreased upon conversion to complex type. Next,we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin,a cell--cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell--cell borderwhen N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans wereprocessed to hybrid and then to complex. Thus, we provide evidence that all three general types ofN-glycans impact plasma membrane architecture and cellular properti

CNSC-15. MITOCHONDRIA TRANSFER VIA GLIOMA-ASTROCYTE NETWORK MICROTUBES REPROGRAMS TUMOR CELLS FOR ENHANCED TUMORIGENICITY

Abstract Glioblastoma (GBM) interaction with neural cells is critical to its pathobiology. Emerging evidence suggests that GBM cells form an interconnected network with astrocytes, facilitating tumor persistence. Given reports of intercellular transfer of mitochondria in ischemic stroke and other pathologic disease states outside the CNS, we hypothesized that this network facilitates mitochondria transfer from astrocytes to GBM with protumorigenic sequelae. Employing transgenic mice and intracranial viral vector transductions in rats, we found that mitochondria transfer from the TME to GBM occurs in intracranial mouse and patient-derived xenograft models (in nude rats) of GBM. Mitochondria transfer from bone marrow-derived immune cells was minimal in bone marrow chimera mouse models of orthotopic GBM, suggesting that neural cells were the primary mitochondria donors. We confirmed this in vitro, where mouse astrocytes were the major mitochondria donors, followed by microglia and to a much smaller extent bone marrow-derived macrophages. Immortalized human astrocytes transduced with mitochondria-localized mCherry (mito-mCherry) also transferred their mitochondria to numerous patient-derived glioma stem cell (GSC) models at rates of ~5-20%, assessed by flow cytometry and confocal microscopy. Mitochondria were visualized along intercellular actin bridges, structurally resembling tumor microtubes. Blocking actin polymerization or knocking down GAP43 (previously linked to microtube formation) decreased mitochondria transfer from astrocytes to GBM in vitro. Functionally, sorted mito-mCherry+ patient-derived GSCs displayed higher mitochondrial respiration, metabolomic reprogramming and proliferation-promoting phospho-signaling. Mito-mCherry+ GBM cells were more likely to be in the proliferative G2/M phases of the cell cycle, and when sorted from co-cultures had high self-renewal (in vitro) and tumor-initiating capacity (in vivo xenograft mouse model). In ongoing work, we are investigating the role of retrograde GBM to astrocyte transfer of mitochondria by dual-color labeling of the organelle, as well as further delineating the protein machinery involved in this fundamental protumorigenic process, with the goal of identifying novel therapeutic targets

GAP43-dependent mitochondria transfer from astrocytes enhances glioblastoma tumorigenicity

The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host-tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development