Ceramide induces cell death in response to many stimuli. Its mechanism of action, however, is not completely understood. Ceramide induces autophagy in mammalian cells maintained in rich media and nutrient permease downregulation in yeast. These observations suggested to us that ceramide might kill mammalian cells by limiting cellular access to extracellular nutrients. Consistent with this proposal, physiologically relevant concentrations of ceramide produced a profound and specific downregulation of nutrient transporter proteins in mammalian cells. Blocking ceramide-induced nutrient transporter loss or supplementation with the cell-permeable nutrient, methyl pyruvate, reversed ceramide-dependent toxicity. Conversely, cells became more sensitive to ceramide when nutrient stress was increased by acutely limiting extracellular nutrients, inhibiting autophagy, or deleting AMP-activated protein kinase (AMPK). Observations that ceramide can trigger either apoptosis or caspase-independent cell death may be explained by this model. We found that methyl pyruvate (MP) also protected cells from ceramide-induced, nonapoptotic death consistent with the idea that severe bioenergetic stress was responsible. Taken together, these studies suggest that the cellular metabolic state is an important arbiter of the cellular response to ceramide. In fact, increasing nutrient demand by incubating cells in high levels of growth factor sensitized cells to ceramide. On the other hand, gradually adapting cells to tolerate low levels of extracellular nutrients completely blocked ceramide-induced death. In sum, these results support a model where ceramide kills cells by inducing intracellular nutrient limitation subsequent to nutrient transporter downregulation.autophagy ͉ caspase-independent cell death ͉ daunorubicin ͉ bioenergetics ͉ sphingolipid C eramide and related sphingolipids play an evolutionarily conserved role in the cellular response to stress by regulating cell growth, differentiation, senescence, and survival (1). Uncovering the mechanisms by which ceramide regulates these processes is of paramount importance given the wide range of human diseases that result from altered ceramide metabolism including cancer, type II diabetes, and neurodegenerative disease (2-4). Ceramide plays a particularly well-established role in cancer. Decreasing cellular ceramide levels increases tumor growth and metastasis and can lead to multidrug resistance, a major cause of cancer treatment failure (2, 5, 6). The ability of ceramide to trigger programmed cell death in response to growth factor withdrawal, death receptor ligation, hypoxia, and chemotherapeutic drugs is likely integral to its role in suppressing cancer initiation and progression. Although many of the downstream, executioner pathways that are activated by ceramide are known, how ceramide triggers these pathways is not completely understood.Autophagy, a process by which cells catabolize their own components, is induced in ceramide-treated cells (7-9). Autophagy has been conserved through...

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A growing understanding of the molecular interactions between immune effector cells and target tumor cells, coupled with refined gene therapy approaches, are giving rise to novel cancer immunotherapeutics with remarkable efficacy in the clinic against both solid and liquid tumors. While immunotherapy holds tremendous promise for treatment of certain cancers, significant challenges remain in the clinical translation to many other types of cancers and also in minimizing adverse effects. Therefore, there is an urgent need for functional potency assays, in vitro and in vivo, that could model the complex interaction of immune cells with tumor cells and can be used to rapidly test the efficacy of different immunotherapy approaches, whether it is small molecule, biologics, cell therapies or combinations thereof. Herein we report the development of an xCELLigence real-time cytolytic in vitro potency assay that uses cellular impedance to continuously monitor the viability of target tumor cells while they are being subjected to different types of treatments. Specialized microtiter plates containing integrated gold microelectrodes enable the number, size, and surface attachment strength of adherent target tumor cells to be selectively monitored within a heterogeneous mixture that includes effector cells, antibodies, small molecules, etc. Through surface-tethering approach, the killing of liquid cancers can also be monitored. Using NK92 effector cells as example, results from RTCA potency assay are very well correlated with end point data from image-based assays as well as flow cytometry. Several effector cells, i.e., PBMC, NK, CAR-T were tested and validated as well as biological molecules such as Bi-specific T cell Engagers (BiTEs) targeting the EpCAM protein expressed on tumor cells and blocking antibodies against the immune checkpoint inhibitor PD-1. Using the specifically designed xCELLigence immunotherapy software, quantitative parameters such as KT50 (the amount of time it takes to kill 50% of the target tumor cells) and % cytolysis are calculated and used for comparing the relative efficacy of different reagents. In summary, our results demonstrate the xCELLigence platform to be well suited for potency assays, providing quantitative assessment with high reproducibility and a greatly simplified work flow.

The Rab7 GTPase promotes membrane fusion reactions between late endosomes and lysosomes. In previous studies, we demonstrated that Rab7 inactivation blocks growth factor withdrawal-induced cell death. These results led us to hypothesize that growth factor withdrawal activates Rab7. Here, we show that growth factor deprivation increased both the fraction of Rab7 that was associated with cellular membranes and the percentage of Rab7 bound to guanosine triphosphate (GTP). Moreover, expressing a constitutively GTP-bound mutant of Rab7, Rab7-Q67L, was sufficient to trigger cell death even in the presence of growth factors. This activated Rab7 mutant was also able to reverse the growth factor-independent cell survival conferred by protein kinase C (PKC) delta inhibition. PKCdelta is one of the most highly induced proteins after growth factor withdrawal and contributes to the induction of apoptosis. To evaluate whether PKCdelta regulates Rab7, we first examined lysosomal morphology in cells with reduced PKCdelta activity. Consistent with a potential role as a Rab7 activator, blocking PKCdelta function caused profound lysosomal fragmentation comparable to that observed when Rab7 was directly inhibited. Interestingly, PKCdelta inhibition fragmented the lysosome without decreasing Rab7-GTP levels. Taken together, these results suggest that Rab7 activation by growth factor withdrawal contributes to the induction of apoptosis and that Rab7-dependent fusion reactions may be targeted by signaling pathways that limit growth factor-independent cell survival.

Nutrient stress that produces quiescence and catabolism in normal cells is lethal to cancer cells because oncogenic mutations constitutively drive anabolism. One driver of biosynthesis in cancer cells is the mTORC1 signaling complex. Activating mTORC1 by deleting its negative regulator TSC2 leads to hypersensitivity to glucose deprivation. We have previously shown that ceramide kills cells in part by triggering nutrient transporter loss and restricting access to extracellular amino acids and glucose suggesting that TSC2-deficient cells would be hypersensitive to ceramide. However, murine embryonic fibroblasts (MEFs) lacking TSC2 were highly resistant to ceramide-induced death. Consistent with the observation that ceramide limits access to both amino acids and glucose, TSC2−/− MEFs also had a survival advantage when extracellular amino acids and glucose were both reduced. As TSC2−/− MEFs were resistant to nutrient stress despite sustained mTORC1 activity, we assessed whether mTORC1 signaling might be beneficial under these conditions. In low amino acid and glucose medium and following ceramide-induced nutrient transporter loss, elevated mTORC1 activity significantly enhanced the adaptive up-regulation of new transporter proteins for amino acids and glucose. Strikingly, the introduction of oncogenic Ras abrogated the survival advantage of TSC2−/− MEFs upon ceramide treatment most likely by increasing nutrient demand. These results suggest that, in the absence of oncogene-driven biosynthetic demand, mTORC1 dependent translation facilitates the adaptive cellular response to nutrient stress.

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The human immune system is highly complex and immune status is associated with disease status, treatment efficiency, and response to external stimuli such as vaccines. For example, the immunophenotyping of leukemia has become indispensable for diagnosis of hematological malignancies. Monitoring T lymphocytes has gained diagnostic importance in the prognosis and prediction of therapies. In this study, a series of T-cell markers previously used in the HIPC (Human Immunology Phenotyping Consortium) project were assessed. Through comprehensive consideration and analysis of the expression of each marker, fluorescence intensity, spectral overlap, and NovoCyte configuration, a 13-color T-cell phenotyping panel was designed. Next, peripheral blood mononuclear cells (PBMCs) were tested on a NovoCyte. Live T cells (CD3+/Aqua−) were identified as either T helper cells (Th) or cytotoxic T cells (Tc) cells by expression of CD4 and CD8 antigens. Next, further T cell subtype was determined by CCR7 and CD45RA: naïve (CD45RA+/CCR7+), effector (TEF, CD45RA+/CCR7−), effector memory (TEM, CD45RA−/CCR7−) and central memory (TCM, CD45RA−/CCR7+). Activation of T cells was assessed by the activation marker HLA-DR. Identification of Th subtypes (Th1, Th2 and Th17) was determined by CXCR3 and CCR6. Regulatory T cells (Treg) were identified as CD4+/CD25+/CD127lo/CCR4+, and memory and naïve Treg cells were further differentiated by CD45RO. With this panel, the phenotype and activation of T cell subtypes can be determined in one run which allows quickly and in-depth analysis of human T lymphocyte subsets. NovoCyte flow cytometer features high resolution of dim signals and flexible configuration and is particularly suitable for multicolor analysis.