TAMI-09. ENERGY METABOLISM AND THERAPEUTIC T CELL EFFICACY IN THE GLIOBLASTOMA MICROENVIRONMENT

Abstract Glioblastoma (GBM), like most cancers, undergo metabolic alterations to primarily utilize aerobic glycolysis in the hypoxic tumor microenvironment (TME). Similarly, activated T cells switch to glycolysis upon antigen recognition to cope with proliferation needs but are not equally equipped to survive in the hypoxic TME. Metabolic reprogramming within GBM TME contributes to therapeutic resistance and tumor progression, but the effects of metabolic alterations on therapeutic T cell survival and efficacy have not been fully elucidated. We hypothesized that hypoxia in GBM/T-cell co-cultures will significantly impair T cell proliferation and function. We conducted in vitro co-culture assays and nuclear magnetic resonance (NMR)–based assessments in hypoxic (1%O2) or normoxic conditions to detect metabolic changes in real-time. Imaging cytometry for cell cycle assessment demonstrated that GSCs were unaffected by hypoxia, but roughly 90% of healthy T cells arrested in G0/G1 along with significant reduction in glycan precursor UDP-GlcNAc presence. Media samples over 96h in normal and hypoxic oxygen conditions from cells in solitary or co-cultures were analyzed using a Bruker Avance III HD spectrometer at 600 MHz for comparison over time using PCA analysis of metabolic intermediate differences. After 16h, there was observable differences in produced metabolites between the T cells cultured alone or co-culture with GSCs, compared to the GSCs alone or media alone controls. Quantifiable changes in glucose, lactate, fumarate, acetate and pyruvate, among others, indicated a large shift in T cell metabolism dependent on oxygen conditions and co-culture interactions, while GSCs are less metabolically responsive to culture conditions. Ongoing experiments will examine precise changes in UDP-GlcNAc and glycosylation precursors in T cells and CAR-T cells via targeted NMR analysis, which we expect will help us understand energy dependent mechanisms of T cell exhaustion and lead to development of novel strategies to sustain T cell function in the hostile TME.</jats:p

Kinetics and potency of T Cell-mediated cytolysis of glioblastoma

Abstract Glioblastoma is an aggressive brain cancer with no effective treatments and a prognosis of 12–15 months. Immune system effector T cells are a promising therapy due to their high specificity and innate cytotoxicity. Assessing the efficacy and potency of T cell therapies in vitro and with high throughput is vital for development of this promising therapy. Cellular impedance-based assays offer sensitive, label-free, and continuous monitoring of cell proliferation and immune cell-mediated cytotoxicity, revealing both the potency and kinetics of T cell killing. Here, we investigated the targeting potency of activated human T cells on human U87MG glioma and patient-derived glioma cells. U87MG and patient-derived glioma cells were plated at densities ranging from 10k to 50k cells per well on a PDL-coated CytoView-Z 96-well plate (n=12 wells per density). After 24 hours, activated patient-derived T Cells (ImmunoCult CD3/CD28 activation media) were added in a 10:1 effector:target ratio. Impedance and cytolysis were continuously monitored for over 120 hours. As expected, higher densities of glioma cells proliferated faster and reached a higher impedance level at 24 hours. Addition of activated human T cells resulted in a decrease in impedance consistent with T cell-mediated lysis of the glioma cells. The highest density of T cells (500k) exhibited the fastest rate of U87 cytolysis, with a Kill Time 50 of 35 hours. All densities reached 100% cytolysis by 100 hours. Overall, activated human T cells were highly effective for cytolysis of U87MG and patient-derived glioma cells. The cellular impedance assay revealed high potency, with kinetics that varied across cell densities despite the same effector:target ratios.</jats:p

Label-free ferrohydrodynamic separation of exosome-like nanoparticles

Particle ferrohydrodynamics and its device (FerroChip) enables label-free and size-dependent separation of exosome-like nanoparticles with high recovery rate and purity.</p

Fully Synthetic Heparan Sulfate-Based Neural Tissue Construct That Maintains the Undifferentiated State of Neural Stem Cells

Heparin and heparan sulfate (HS) are attractive components for constructing biomaterials due to their ability to recruit and regulate the activity of growth factors. The structural and functional heterogeneity of naturally derived heparin and HS is, however, an impediment for the preparation of biomaterials for regenerative medicine. To address this problem, we have prepared hydrogels modified by well-defined synthetic HS-derived disaccharides. Human induced pluripotent cell-derived neural stem cells (HIP-NSCs) encapsulated in a polyethylene glycol-based hydrogel modified by a pendent HS disaccharide that is a known ligand for fibroblast growth factor-2 (FGF-2) exhibited a significant increase in proliferation and self-renewal. This observation is important because evidence is emerging that undifferentiated stems cells can yield significant therapeutic benefits via their paracrine signaling mechanisms. Our data indicate that the HS disaccharide protects FGF-2, which has a very short biological half-live, from degradation. It is anticipated that, by careful selection of a synthetic HS oligosaccharide, it will be possible to control retention and release of specific growth factor, which in turn will provide control over cell fate

GD2 CAR-T Cells exhibit strong cytolytic potency against glioma stem cells

Abstract Glioblastoma (GBM) is an aggressive brain cancer without effective treatments. CAR-T cells targeted to tumor-associated antigens offer promise for treating GBM. Here, we used cellular impedance assays to compare the cytolytic potency and kinetics of conventional viral vs non-viral CRISPR engineered GD2 CAR-T cells against glioma stem cells (GSC), a subpopulation of glioblastoma cells. Patient-derived N08 GSCs were plated at 50k cells/well on 96-well plates, and impedance was continuously monitored on the Maestro Z impedance platform (Axion BioSystems). GD2 CAR-T cells were engineered using either retroviral transduction (RV) or non-viral CRISPR editing (NV). At 48 hours, GD2 CAR-T cells were added at Effector:Target ratios of 0.1:1, 1:1, and 10:1. Comparisons were made to mCherry T cells (mCh) as a control. Impedance and cytolysis were monitored up to 7 days. RV and NV GD2 CAR-T cells caused decreases in impedance consistent with T cell-mediated lysis of GSCs, whereas mCh T cells induced little change. NV CAR-T cells exhibited faster killing kinetics compared to RV CAR-T cells. The time to 50% cytolysis (KT50) was significantly shorter for NV vs RV CAR-T cells at 1:1 and 10:1 E:T ratios. Cytotoxic function was validated with flow cytometry and cytokine analysis at 7 days. All T cells exhibited chronic activation measured by CD69 and CD137 upregulation. Importantly, NV CAR-T cells exhibited less exhaustion, as measured by PD1 and LAG3 expression. Both RV and NV GD2 CAR-T cells effectively cytolyzed GSCs, with NV CAR-T cells exhibiting more potent and efficient killing. The high potency, fast kinetics, and reduced exhaustion of NV CRISPR GD2 CAR-T cells offer great clinical promise for treating GBM.</jats:p