In Vitro Blood-Brain Barrier Permeability and Cytotoxicity of an Atorvastatin-Loaded Nanoformulation Against Glioblastoma in 2D and 3D Models.

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the family of statins have been suggested as therapeutic options in various tumors. Atorvastatin is a statin with the potential to cross the blood-brain barrier; however, the concentrations necessary for a cytotoxic effect against cancer cells exceed the concentrations achievable via oral administration, which made the development of a novel atorvastatin formulation necessary. We characterized the drug loading and basic physicochemical characteristics of micellar atorvastatin formulations and tested their cytotoxicity against a panel of different glioblastoma cell lines. In addition, activity against tumor spheroids formed from mouse glioma and mouse cancer stem cells, respectively, was evaluated. Our results show good activity of atorvastatin against all tested cell lines. Interestingly, in the three-dimensional (3D) models, growth inhibition was more pronounced for the micellar formulation compared to free atorvastatin. Finally, atorvastatin penetration across a blood-brain barrier model obtained from human induced-pluripotent stem cells was evaluated. Our results suggest that the presented micelles may enable much higher serum concentrations than possible by oral administration; however, if transport across the blood-brain barrier is sufficient to reach the therapeutic atorvastatin concentration for the treatment of glioblastoma via intravenous administration remains unclear.

Modeling phenotypes of malignant gliomas.

Malignant gliomas are primary tumors of the central nervous system characterized by diffuse infiltration into the brain and a high recurrence rate. Advances in comprehensive genomic studies have provided unprecedented insight into the genetic and molecular heterogeneity of these tumors and refined our understanding of their evolution from low to high grade. However, similar levels of phenotypic characterization are indispensable to understanding the complexity of malignant gliomas. Experimental glioma models have also achieved great progress in recent years. Advances in transgenic technologies and cell culture have allowed the establishment of mouse models that mirror the human disease with increasing fidelity and which support single-cell resolution for phenotypic analyses. Here we review the major types of preclinical glioma models, with an emphasis on how recent developments in experimental modeling have shed new light on two fundamental aspects of glioma phenotype, their cell of origin and their invasive potential.

Epidermal growth factor receptor promotes glioma progression by regulating xCT and GluN2B-containing N-methyl-d-aspartate-sensitive glutamate receptor signaling.

Autocrine and paracrine factors, including glutamate and epidermal growth factor (EGF), are potent inducers of brain tumor cell invasion, a pathological hallmark of malignant gliomas. System xc(-) consists of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. We previously showed that the EGF receptor (EGFR) interacts with xCT and thereby promotes the activity of system xc(-) in a kinase-independent manner, resulting in enhanced glutamate release in glioma cells. However, the molecular mechanism underlying EGFR-mediated glioma progression in a glutamate-rich microenvironment has remained unclear. Here we show that the GluN2B subunit of the N-methyl-d-aspartate-sensitive glutamate receptor (NMDAR) is a substrate of EGFR in glioma cells. In response to EGF stimulation, EGFR phosphorylated the COOH-terminal domain of GluN2B and thereby enhanced glutamate-NMDAR signaling and consequent cell migration in EGFR-overexpressing glioma cells. Treatment with the NMDAR inhibitor MK-801 or the system xc(-) inhibitor sulfasalazine suppressed EGF-elicited glioma cell migration. The administration of sulfasalazine and MK-801 also synergistically suppressed the growth of subcutaneous tumors formed by EGFR-overexpressing glioma cells. Furthermore, shRNA-mediated knockdown of xCT and GluN2B cooperatively prolonged the survival of mice injected intracerebrally with such glioma cells. Our findings thus establish a central role for EGFR in the signaling crosstalk between xCT and GluN2B-containing NMDAR in glioma cells.

Convection-enhanced delivery of sulfasalazine prolongs survival in a glioma stem cell brain tumor model.

Expression of CD44 in glioma cells was previously correlated with tumor grade and is considered a stem cell marker. CD44 stabilizes the cystine-glutamate transporter (xCT) and inhibits apoptosis in cancer stem cells (CSCs). Recently it was found that Sulfasalazine (SSZ), an anti-inflammatory drug, acts as an inhibitor of xCT and therefore has potential as a targeted therapy for CSCs. In this study, we tested an efficacy of SSZ against glioma stem cell model developed in rats. As poor penetration of blood-brain barrier resulted in insufficient efficacy of systemic SSZ treatment, SSZ was delivered locally with convection-enhanced delivery (CED). In vitro, expression of CD44 in glioma cells and efficacy of SSZ against glioma cells and glioma stem cells were confirmed. SSZ demonstrated anti-proliferative activity in a dose dependent manner against these cells. This activity was partially reversible with the addition of antioxidant, N-acetyl-L-cysteine, to the medium. In vivo, CED successfully delivered SSZ into the rat brain parenchyma. When delivered at 5 mM concentration, which was the highest possible concentration when SSZ was dissolved in water, CED of SSZ resulted in almost no tissue damage. Against highly malignant bRiTs-G3 brain tumor xenografted rat model; the glioma stem cell model, CED of SSZ at 5 mM concentration induced apoptosis and prolonged survival. Consequently, CED of SSZ induced glioma stem cell death without evidence of tissue damage to normal brain parenchyma. This strategy may be a promising targeted treatment against glioma stem cells.

Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression.

The available evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells that self-renew and exhibit tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells or as a dynamically acquired property. We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary glioblastoma lines. Our results indicate that glioblastoma tumorigenicity is largely deterministic, yet the property can be acquired spontaneously at low frequencies. Further, these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD depletion increases trimethylation of histone 3 lysine 4 at the avian myelocytomatosis viral oncogene homolog (MYC) locus, which elevates MYC expression. MYC, in turn, regulates oligodendrocyte lineage transcription factor 2 (OLIG2), SRY (sex determining region Y)-box 2 (SOX2), and POU class 3 homeobox 2 (POU3F2), a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development.

HER2 Heterogeneity Is Associated with Poor Survival in HER2-Positive Breast Cancer.

Intratumoral human epidermal growth factor receptor 2 (HER2) heterogeneity has been reported in 16⁻36% of HER2-positive breast cancer and its clinical impact is under discussion. We examined the biological effects of HER2-heterogeneity on mouse models and analyzed metastatic brains by RNA sequence analysis. A metastatic mouse model was developed using 231-Luc (triple negative cells) and 2 HER2-positive cell lines, namely, HER2-60 and HER2-90 which showed heterogeneous and monotonous HER2 expressions, respectively. Metastatic lesions developed in 3 weeks in all the mice injected with HER2-60 cells, and in 69% of the mice injected with HER2-90 and 87.5% of the mice injected with 231-Luc. The median survival days of mice injected with 231-Luc, HER2-60, and HER2-90 cells were 29 (n = 24), 24 (n = 22) and 30 (n = 13) days, respectively. RNA sequence analysis showed that CASP-1 and its related genes were significantly downregulated in metastatic brain tumors with HER2-60 cells. The low expression of caspase-1 could be a new prognostic biomarker for early relapse in HER2-positive breast cancer.

Functional analysis of the DEPDC1 oncoantigen in malignant glioma and brain tumor initiating cells.

DEP domain containing 1 (DEPDC1) is a novel oncoantigen expressed in cancer cells, which presents oncogenic activity and high immunogenicity. Although DEPDC1 has been predicted to be a useful antigen for the development of a cancer vaccine, its pathophysiological roles in glioma have not been investigated. Here, we analyzed the expression and function of DEPDC1 in malignant glioma. DEPDC1 expression in glioma cell lines, glioma tissues, and brain tumor initiating cells (BTICs) was assessed by western blot and quantitative polymerase chain reaction (PCR). The effect of DEPDC1 downregulation on cell growth and nuclear factor kappa B (NFκB) signaling in glioma cells was investigated. Overall survival was assessed in mouse glioma models using human glioma cells and induced mouse brain tumor stem cells (imBTSCs) to determine the effect of DEPDC1 suppression in vivo. DEPDC1 expression was increased in glioma cell lines, tissues, and BTICs. Suppression of endogenous DEPDC1 expression by small interfering RNA (siRNA) inhibited glioma cell viability and induced apoptosis through NFκB signaling. In mouse glioma models using human glioma cells and imBTSCs, downregulation of DEPDC1 expression prolonged overall survival. These results suggest that DEPDC1 represents a target molecule for the treatment of glioma.

Blocking COX-2 induces apoptosis and inhibits cell proliferation via the Akt/survivin- and Akt/ID3 pathway in low-grade-glioma.

Approximately half of surgically-treated patients with low-grade-glioma (LGG) suffer recurrence or metastasis. Currently there is no effective drug treatment. While the selective COX-2 inhibitor celecoxib showed anti-neoplastic activity against several malignant tumors, its effects against LGG remain to be elucidated. Ours is the first report that the expression level of COX-2 in brain tissue samples from patients with LGG and in LGG cell lines is higher than in the non-neoplastic region and in normal brain cells. We found that celecoxib attenuated LGG cell proliferation in a dose-dependent manner. It inhibited the generation of prostaglandin E2 and induced apoptosis and cell-cycle arrest. We also show that celecoxib hampered the activation of the Akt/survivin- and the Akt/ID3 pathway in LGGs. These findings suggest that celecoxib may have a promising therapeutic potential and that the early treatment of LGG patients with the drug may be beneficial.

IGF1 receptor signaling regulates adaptive radioprotection in glioma stem cells.

Cancer stem cells (CSCs) play an important role in disease recurrence after radiation treatment as a result of intrinsic properties such as high DNA repair capability and antioxidative capacity. It is unclear, however, how CSCs further adapt to escape the toxicity of the repeated irradiation regimens used in clinical practice. Here, we have exposed a population of murine glioma stem cells (GSCs) to fractionated radiation in order to investigate the associated adaptive changes, with the ultimate goal of identifying a targetable factor that regulates acquired radioresistance. We have shown that fractionated radiation induces an increase in IGF1 secretion and a gradual upregulation of the IGF type 1 receptor (IGF1R) in GSCs. Interestingly, IGF1R upregulation exerts a dual radioprotective effect. In the resting state, continuous IGF1 stimulation ultimately induces downregulation of Akt/extracellular-signal-regulated kinases (ERK) and FoxO3a activation, which results in slower proliferation and enhanced self-renewal. In contrast, after acute radiation, the abundance of IGF1R and increased secretion of IGF1 promote a rapid shift from a latent state toward activation of Akt survival signaling, protecting GSCs from radiation toxicity. Treatment of tumors formed by the radioresistant GSCs with an IGF1R inhibitor resulted in a marked increase in radiosensitivity, suggesting that blockade of IGF1R signaling is an effective strategy to reverse radioresistance. Together, our results show that GSCs evade the damage of repeated radiation not only through innate properties but also through gradual inducement of resistance pathways and identify the dynamic regulation of GSCs by IGF1R signaling as a novel mechanism of adaptive radioprotection.

Berberine as a potential enhancer for 5-ALA-mediated fluorescence in glioblastoma: increasing detectability of infiltrating glioma stem cells to optimize 5-ALA-guided surgery.

OBJECTIVE: The prognosis of glioblastoma (GBM) correlates with residual tumor volume after surgery. In fluorescence-guided surgery, 5-aminolevulinic acid (ALA) has been used to maximize resection while avoiding neurological morbidity. However, not all tumor cells, particularly glioma stem cells (GSCs), display 5-ALA-mediated protoporphyrin IX (PpIX) fluorescence (5-ALA fluorescence). The authors searched for repositioned drugs that affect mitochondrial functions and energy metabolism, identifying berberine (BBR) as a potential enhancer of 5-ALA fluorescence. In this study, they investigated whether BBR can enhance 5-ALA fluorescence in GSCs and whether BBR can be applied to clinical practice as a 5-ALA fluorescence enhancer. METHODS: The effects of BBR on 5-ALA fluorescence in glioma and GSCs were evaluated by flow cytometry (fluorescence-activated cell sorting [FACS]) analysis. As 5-ALA is metabolized for heme synthesis, the effects of BBR on mRNA expressions of 7 enzymes in the heme-synthesis pathway were analyzed. Enzymes showing significantly higher expression than control in all cells were identified and protein analysis was performed. To examine clinical availability, the detectability and cytotoxicity of BBR in tumor-transplanted mice were analyzed. RESULTS: Fluorescence microscopy revealed much more intense 5-ALA fluorescence in both GSCs and non-stem cells with 5-ALA and BBR than with 5-ALA alone. FACS showed that BBR greatly enhanced 5-ALA fluorescence compared with 5-ALA alone, and enhancement was much higher for GSCs than for glioma cells. Among the 7 enzymes examined, BBR upregulated mRNA expressions of ALA synthetase 1 (ALAS1) more highly in all cells, and activated ALAS1 through deregulating ALAS1 activity inhibited by the negative feedback of heme. An in vivo study showed that 5-ALA fluorescence with 5-ALA and BBR was significantly stronger than with 5-ALA alone, and the sensitivity and specificity of BBR-enhanced fluorescence were both 100%. In addition, BBR did not show any cytotoxicity for normal brain tissue surrounding the tumor mass. CONCLUSIONS: BBR enhanced 5-ALA-mediated PpIX fluorescence by upregulating and activating ALAS1 through deregulation of negative feedback inhibition by heme. BBR is a clinically used drug with no side effects. BBR is expected to significantly augment fluorescence-guided surgery and photodynamic therapy.

Dual-radionuclide in vivo imaging of micro-metastasis and lymph tract with submillimetre resolution.

Multi-radionuclide in vivo imaging with submillimetre resolution can be a potent tool for biomedical research. While high-resolution radionuclide imaging faces challenges in sensitivity, multi-radionuclide imaging encounters difficulty due to radiation contamination, stemming from crosstalk between radionuclides and Compton scattering. Addressing these challenges simultaneously is imperative for multi-radionuclide high-resolution imaging. To tackle this, we developed a high-spatial-resolution and high-energy-resolution small animal single-photon emission computed tomography (SPECT) scanner, named CdTe-DSD SPECT-I. We first assessed the feasibility of multi-tracer SPECT imaging of submillimetre targets. Using the CdTe-DSD SPECT-I, we performed SPECT imaging of submillimetre zeolite spheres absorbed with 125I- and subsequently imaged 125I-accumulated spheroids of 200-400 µm in size within an hour, achieving clear and quantitative images. Furthermore, dual-radionuclide phantom imaging revealed a distinct image of the submillimetre sphere absorbed with 125I- immersed in a 99mTc-pertechnetate solution, and provided a fair quantification of each radionuclide. Lastly, in vivo imaging was conducted on a cancer-bearing mouse with lymph node micro-metastasis using dual-tracers. The results displayed dual-tracer images of lymph tract by 99mTc-phytic acid and the submillimetre metastatic lesion by 125I-, shown to align with the immunofluorescence image.

5-Aminolevulinic acid increases boronophenylalanine uptake into glioma stem cells and may sensitize malignant glioma to boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a high-LET particle radiotherapy clinically tested for treating malignant gliomas. Boronophenylalanine (BPA), a boron-containing phenylalanine derivative, is selectively transported into tumor cells by amino acid transporters, making it an ideal agent for BNCT. In this study, we investigated whether the amino acid 5-aminolevulinic acid (ALA) could sensitize glioma stem cells (GSCs) to BNCT by enhancing the uptake of BPA. Using human and mouse GSC lines, pre-incubation with ALA increased the intracellular accumulation of BPA dose-dependent. We also conducted in vivo experiments by intracerebrally implanting HGG13 cells in mice and administering ALA orally 24 h before BPA administration (ALA + BPA-BNCT). The ALA preloading group increased the tumor boron concentration and improved the tumor/blood boron concentration ratio, resulting in improved survival compared to the BPA-BNCT group. Furthermore, we found that the expression of amino acid transporters was upregulated following ALA treatment both in vitro and in vivo, particularly for ATB0,+. This suggests that ALA may sensitize GSCs to BNCT by upregulating the expression of amino acid transporters, thereby enhancing the uptake of BPA and improving the effectiveness of BNCT. These findings have important implications for strategies to improve the sensitivity of malignant gliomas to BPA-BNCT.

Gene therapy using genome-edited iPS cells for targeting malignant glioma.

Glioblastoma is characterized by diffuse infiltration into the normal brain. Invasive glioma stem cells (GSCs) are an underlying cause of treatment failure. Despite the use of multimodal therapies, the prognosis remains dismal. New therapeutic approach targeting invasive GSCs is required. Here, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited human-induced pluripotent stem cell (hiPSC) expressing a suicide gene had higher tumor-trophic migratory capacity compared with mesenchymal stem cells (MSCs), leading to marked in vivo antitumor effects. High migratory capacity in iPSC-NSCs was related to self-repulsive action and pathotropism involved in EphB-ephrinB and CXCL12-CXCR4 signaling. The gene insertion to ACTB provided higher and stable transgene expression than other common insertion sites, such as GAPDH or AAVS1. Ferroptosis was associated with enhanced antitumor immune responses. The thymidylate synthase and dihydroprimidine dehydrogenase expressions predicted the treatment efficacy of therapeutic hiPSC-NSCs. Our results indicate the potential benefit of genome-edited iPS cells based gene therapy for invasive GSCs. Furthermore, the present research concept may become a platform to promote clinical studies using hiPSC.

Spatial Heterogeneity of Invading Glioblastoma Cells Regulated by Paracrine Factors.

Glioblastoma (GBM) is the most common and lethal type of malignant primary brain tumor in adults. GBM displays heterogeneous tumor cell population comprising glioma-initiating cells (GICs) with stem cell-like characteristics and differentiated glioma cells. During GBM cell invasion into normal brain tissues, which is the hallmark characteristic of GBM, GICs at the invasion front retain stemness, while cells at the tumor core display cellular differentiation. However, the mechanism of cellular differentiation underlying the formation of spatial cellular heterogeneity in GBM remains unknown. In the present study, we first observed spatially heterogeneous GBM cell populations emerged from an isogenic clonal population of GICs during invasion into a 3D collagen hydrogel in a microfluidic device. Specifically, GICs at the invasion front maintained stemness, while trailing cells displayed astrocytic differentiation. The spatial cellular heterogeneity resulted from the difference in cell density between GICs at the invasion front and trailing cells. Trailing GICs at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they secreted, while cells at the invasion front of low cell density retained stemness due to the lack of paracrine factors. In addition, we demonstrated that interstitial flow suppressed astrocytic differentiation of trailing GICs by the clearance of paracrine factors. Our findings suggest that intercellular crosstalk between tumor cells is an essential factor in developing the spatial cellular heterogeneity of GBM cells with various differentiation statuses. It also provides insights into the development of novel therapeutic strategies targeting GBM cells with stem cell characteristics at the invasion front. Impact Statement We elucidated the mechanism of cellular differentiation underlying the spatial cellular heterogeneity of glioblastoma composed of glioma-initiating cells (GICs) and differentiated glioma cells during invasion in a microfluidic device. Trailing cells at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they produced, while cells at the invasion front of low cell density were shown to retain stemness due to the lack of paracrine factors. Our findings provide valuable knowledge for the development of effective therapeutic strategies targeting GICs at the invasion front.

Regulatory roles of fibronectin and integrin α5 in reorganization of the actin cytoskeleton and completion of adipogenesis.

Cellular differentiation is characterized by changes in cell morphology that are largely determined by actin dynamics. We previously showed that depolymerization of the actin cytoskeleton triggers the differentiation of preadipocytes into mature adipocytes as a result of inhibition of the transcriptional coactivator activity of megakaryoblastic leukemia 1 (MKL1). The extracellular matrix (ECM) influences cell morphology via interaction with integrins, and reorganization of the ECM is associated with cell differentiation. Here we show that interaction between actin dynamics and ECM rearrangement plays a key role in adipocyte differentiation. We found that depolymerization of the actin cytoskeleton precedes disruption and degradation of fibrillar fibronectin (FN) structures at the cell surface after the induction of adipogenesis in cultured preadipocytes. A FN matrix suppressed both reorganization of the actin cytoskeleton into the pattern characteristic of adipocytes and terminal adipocyte differentiation, and these inhibitory effects were overcome by knockdown of integrin α5 (ITGα5). Peroxisome proliferator-activated receptor γ was required for down-regulation of FN during adipocyte differentiation, and MKL1 was necessary for the expression of ITGα5. Our findings suggest that cell-autonomous down-regulation of FN-ITGα5 interaction contributes to reorganization of the actin cytoskeleton and completion of adipocyte differentiation.

Heterogeneous Glioma Cell Invasion Under Interstitial Flow Depending on Their Differentiation Status.

Glioblastoma (GBM) is the most common and lethal type of malignant brain tumor. A deeper mechanistic understanding of the invasion of heterogeneous GBM cell populations is crucial to develop therapeutic strategies. A key regulator of GBM cell invasion is interstitial flow. However, the effect of an interstitial flow on the invasion of heterogeneous GBM cell populations composed of glioma initiating cells (GICs) and relatively differentiated progeny cells remains unclear. In the present study, we investigated how GICs invade three-dimensional (3D) hydrogels in response to an interstitial flow with respect to their differentiation status. Microfluidic culture systems were used to apply an interstitial flow to the cells migrating from the cell aggregates into the 3D hydrogel. Phase-contrast microscopy revealed that the invasion and protrusion formation of the GICs in differentiated cell conditions were significantly enhanced by a forward interstitial flow, whose direction was the same as that of the cell invasion, whereas those in stem cell conditions were not enhanced by the interstitial flow. The mechanism of flow-induced invasion was further investigated by focusing on differentiated cell conditions. Immunofluorescence images revealed that the expression of cell-extracellular matrix adhesion-associated molecules, such as integrin ß1, focal adhesion kinase, and phosphorylated Src, was upregulated in forward interstitial flow conditions. We then confirmed that cell invasion and protrusion formation were significantly inhibited by PP2, a Src inhibitor. Finally, we observed that the flow-induced cell invasion was preceded by nestin-positive immature GICs at the invasion front and followed by tubulin ß3-positive differentiated cells. Our findings provide insights into the development of novel therapeutic strategies to inhibit flow-induced glioma invasion. Impact statement A mechanistic understanding of heterogeneous glioblastoma cell invasion is crucial for developing therapeutic strategies. We observed that the invasion and protrusion formation of glioma initiating cells (GICs) were significantly enhanced by forward interstitial flow in differentiated cell conditions. The expression of integrin ß1, focal adhesion kinase, and phosphorylated Src was upregulated, and the flow-induced invasion was significantly inhibited by a Src inhibitor. The flow-induced heterogeneous cell invasion was preceded by nestin-positive GICs at the invasion front and followed by tubulin ß3-positive differentiated cells. Our findings provide insights into the development of novel therapeutic strategies to inhibit flow-induced glioma invasion.

Lactate Reprograms Energy and Lipid Metabolism in Glucose-Deprived Oxidative Glioma Stem Cells.

Fast-growing tumors satisfy their bioenergetic needs by supplementing glucose with alternative carbon sources. Cancer stem cells are the most versatile and robust cells within malignant tumors. They avoid potentially lethal metabolic and other types of stress through flexible reprogramming of relevant pathways, but it has remained unclear whether alternative carbon sources are important for the maintenance of their tumor-propagating ability. Here we assessed the ability of glycolytic and oxidative murine glioma stem cells (GSCs) to grow in an ultralow glucose medium. Sphere formation assays revealed that exogenous lactate and acetate reversed the growth impairment of oxidative GSCs in such medium. Extracellular flux analysis showed that lactate supported oxygen consumption in these cells, whereas metabolomics analysis revealed that it increased the intracellular levels of tricarboxylic acid cycle intermediates, ATP, and GTP as well as increased adenylate and guanylate charge. Lactate also reversed the depletion of choline apparent in the glucose-deprived cells as well as reprogrammed phospholipid and fatty acid biosynthesis. This metabolic reprogramming was associated with a more aggressive phenotype of intracranial tumors formed by lactate-treated GSCs. Our results thus suggest that lactate is an important alternative energetic and biosynthetic substrate for oxidative GSCs, and that it sustains their growth under conditions of glucose deprivation.

MEK inhibition preferentially suppresses anchorage-independent growth in osteosarcoma cells and decreases tumors in vivo.

Osteosarcoma is the most common high-grade malignancy of bone, and novel therapeutic options are urgently required. Previously, we developed mouse osteosarcoma AXT cells that can proliferate both under adherent and nonadherent conditions. Based on metabolite levels, nonadherent conditions were more similar to the in vivo environment than adherent conditions. A drug screen identified MEK inhibitors, including trametinib, that preferentially decreased the viability of nonadherent AXT cells. Trametinib inhibited the cell cycle and induced apoptosis in AXT cells, and both effects were stronger under nonadherent conditions. Trametinib also potently decreased viability in U2OS cells, but its effects were less prominent in MG63 or Saos2 cells. By contrast, MG63 and Saos2 cells were more sensitive to PI3K inhibition than AXT or U2OS cells. Notably, the combination of MAPK/ERK kinase (MEK) and PI3K inhibition synergistically decreased viability in U2OS and AXT cells, but this effect was less pronounced in MG63 or Saos2 cells. Therefore, signal dependence for cell survival and crosstalk between MEK-ERK and PI3K-AKT pathways in osteosarcoma are cell context-dependent. The activation status of other kinases including CREB varied in a cell context-dependent manner, which might determine the response to MEK inhibition. A single dose of trametinib was sufficient to decrease the size of the primary tumor and circulating tumor cells in vivo. Moreover, combined administration of trametinib and rapamycin or conventional anticancer drugs further increased antitumor activity. Thus, given optimal biomarkers for predicting its effects, trametinib holds therapeutic potential for the treatment of osteosarcoma.

Elevated cellular PpIX potentiates sonodynamic therapy in a mouse glioma stem cell-bearing glioma model by downregulating the Akt/NF-κB/MDR1 pathway.

Glioblastoma (GBM) has high mortality rates because of extreme therapeutic resistance. During surgical resection for GBM, 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is conventionally applied to distinguish GBM. However, surgical intervention is insufficient for high invasive GBM. Sonodynamic therapy (SDT) combined with low-intensity ultrasonication (US) and PpIX, as a sonosensitizer, is an emerging and promising approach, although its efficacy is limited. Based on our previous study that down-regulation of multidrug resistant protein (MDR1) in GBM augmented the anti-tumor effects of chemotherapy, we hypothesized that elevation of cellular PpIX levels by down-regulation of MDR1 enhances anti-tumor effects by SDT. In high invasive progeny cells from mouse glioma stem cells (GSCs) and a GSC-bearing mouse glioma model, we assessed the anti-tumor effects of SDT with a COX-2 inhibitor, celecoxib. Down-regulation of MDR1 by celecoxib increased cellular PpIX levels, as well as valspodar, an MDR1 inhibitor, and augmented anti-tumor effects of SDT. MDR1 down-regulation via the Akt/NF-κB pathway by celecoxib was confirmed, using an NF-κB inhibitor, CAPÉ. Thus, elevation of cellar PpIX by down-regulation of MDR1 via the Akt/NF-κB pathway may be crucial to potentiate the efficacy of SDT in a site-directed manner and provide a promising new therapeutic strategy for GBM.