CMV-Independent Lysis of Glioblastoma by Ex Vivo Expanded/Activated Vδ1+ γδ T Cells

Vδ2^neg γδ T cells, of which Vδ1+ γδ T cells are by far the largest subset, are important effectors against CMV infection. Malignant gliomas often contain CMV genetic material and proteins, and evidence exists that CMV infection may be associated with initiation and/or progression of glioblastoma multiforme (GBM). We sought to determine if Vδ1+ γδ T cells were cytotoxic to GBM and the extent to which their cytotoxicity was CMV dependent. We examined the cytotoxic effect of ex vivo expanded/activated Vδ1+ γδ T cells from healthy CMV seropositive and CMV seronegative donors on unmanipulated and CMV-infected established GBM cell lines and cell lines developed from short- term culture of primary tumors. Expanded/activated Vδ1+ T cells killed CMV-negative U251, U87, and U373 GBM cell lines and two primary tumor explants regardless of the serologic status of the donor. Experimental CMV infection did not increase Vδ1+ T cell - mediated cytotoxicity and in some cases the cell lines were more resistant to lysis when infected with CMV. Flow cytometry analysis of CMV-infected cell lines revealed down-regulation of the NKG2D ligands ULBP-2, and ULBP-3 as well as MICA/B in CMV-infected cells. These studies show that ex vivo expanded/activated Vδ1+ γδ T cells readily recognize and kill established GBM cell lines and primary tumor-derived GBM cells regardless of whether CMV infection is present, however, CMV may enhance the resistance GBM cell lines to innate recognition possibly contributing to the poor immunogenicity of GBM.     

Inhibition of TRPM7 with waixenicin A reduces glioblastoma cellular functions

Glioblastoma (GBM) is the most common malignant primary brain tumour originating in the CNS. Median patient survival is <15 months with standard treatment which consists of surgery alongside radiation therapy and temozolomide chemotherapy. However, because of the aggressive nature of GBM, and the significant toxicity of these adjuvant therapies, long-term therapeutic effects are unsatisfactory. Thus, there is urgency to identify new drug targets for GBM. Recent evidence shows that the transient receptor potential melastatin 7 (TRPM7) cation channel is aberrantly upregulated in GBM and its inhibition leads to reduction of GBM cellular functions. This suggests that TRPM7 may be a potential drug target for GBM treatment. In this study, we assessed the effects of the specific TRPM7 antagonist waixenicin A on human GBM cell lines U87 or U251 both in vitro and in vivo. First, we demonstrated in vitro that application of waixenicin A reduced TRPM7 protein expression and inhibited the TRPM7-like currents in GBM cells. We also observed reduction of GBM cell viability, migration, and invasion. Using an intracranial xenograft GBM mouse model, we showed that with treatment of waixenicin A, there was increased cleaved caspase 3 activity, alongside reduction in Ki-67, cofilin, and Akt activity in vivo. Together, these data demonstrate higher GBM cell apoptosis, and lower proliferation, migration, invasion and survivability following treatment with waixenicin A.     

Improving Antigenic Peptide Vaccines for Cancer Immunotherapy Using a Dominant Tumor-specific T Cell Receptor

Vaccines that incorporate peptide mimics of tumor antigens, or mimotope vaccines, are commonly used in cancer immunotherapy and function by eliciting increased numbers of T cells that cross-react with the native tumor antigen. Unfortunately, they often elicit T cells that do not cross-react with or that have low affinity for the tumor antigen. Using a high affinity tumor-specific T cell clone, we identified a panel of mimotope vaccines for the dominant peptide antigen from a mouse colon tumor that elicits a range of tumor protection following vaccination. The TCR from this high affinity T cell clone was rarely identified in ex vivo evaluation of tumor-specific T cells elicited by mimotope vaccination. Conversely, a low affinity clone found in the tumor and following immunization was frequently identified. Using peptide libraries, we determined if this frequently identified TCR improved the discovery of efficacious mimotopes. We demonstrated that the representative TCR identified more protective mimotopes than the high affinity TCR. These results suggest that targeting a dominant fraction of tumor-specific T cells generates potent immunity and that consideration of the available T cell repertoire is necessary for targeted T cell therapy. These results have important implications when optimizing mimotope vaccines for cancer immunotherapy.     

Saponin 1 Induces Apoptosis and Suppresses NF-κB-Mediated Survival Signaling in Glioblastoma Multiforme (GBM)

Saponin 1 is a triterpeniod saponin extracted from Anemone taipaiensis, a traditional Chinese medicine against rheumatism and phlebitis. It has also been shown to exhibit significant anti-tumor activity against human leukemia (HL-60 cells) and human hepatocellular carcinoma (Hep-G2 cells). Herein we investigated the effect of saponin 1 in human glioblastoma multiforme (GBM) U251MG and U87MG cells. Saponin 1 induced significant growth inhibition in both glioblastoma cell lines, with a 50% inhibitory concentration at 24 h of 7.4 µg/ml in U251MG cells and 8.6 µg/ml in U87MG cells, respectively. Nuclear fluorescent staining and electron microscopy showed that saponin 1 caused characteristic apoptotic morphological changes in the GBM cell lines. Saponin 1-induced apoptosis was also verified by DNA ladder electrophoresis and flow cytometry. Additionally, immunocytochemistry and western blotting analyses revealed a time-dependent decrease in the expression and nuclear location of NF-κB following saponin 1 treatment. Western blotting data indicated a significant decreased expression of inhibitors of apoptosis (IAP) family members,(e.g., survivin and XIAP) by saponin 1. Moreover, saponin 1 caused a decrease in the Bcl-2/Bax ratio and initiated apoptosis by activating caspase-9 and caspase-3 in the GBM cell lines. These findings indicate that saponin 1 inhibits cell growth of GBM cells at least partially by inducing apoptosis and inhibiting survival signaling mediated by NF-κB. In addition, in vivo study also demonstrated an obvious inhibition of saponin 1 treatment on the tumor growth of U251MG and U87MG cells-produced xenograft tumors in nude mice. Given the minimal toxicities of saponin 1 in non-neoplastic astrocytes, our results suggest that saponin 1 exhibits significant in vitro and in vivo anti-tumor efficacy and merits further investigation as a potential therapeutic agent for GBM.     

In Vivo c-Met Pathway Inhibition Depletes Human Glioma Xenografts of Tumor-Propagating Stem-Like Cells

Solid malignancies contain sphere-forming stem-like cells that are particularly efficient in propagating tumors. Identifying agents that target these cells will advance the development of more effective therapies. Recent converging evidence shows that c-Met expression marks tumor-initiating stem-like cells and that c-Met signaling drives human glioblastoma multiforme (GBM) cell stemness in vitro. However, the degree to which tumor-propagating stem-like cells depend on c-Met signaling in histologically complex cancers remains unknown. We examined the effects of in vivo c-Met pathway inhibitor therapy on tumor-propagating stem-like cells in human GBM xenografts. Animals bearing pre-established tumor xenografts expressing activated c-Met were treated with either neutralizing anti- hepatocyte growth factor (HGF) monoclonal antibody L2G7 or with the c-Met kinase inhibitor PF2341066 (Crizotinib). c-Met pathway inhibition inhibited tumor growth, depleted tumors of sphere-forming cells, and inhibited tumor expression of stem cell markers CD133, Sox2, Nanog, and Musashi. Withdrawing c-Met pathway inhibitor therapy resulted in a substantial rebound in stem cell marker expression concurrent with tumor recurrence. Cells derived from xenografts treated with anti-HGF in vivo were depleted of tumor-propagating potential as determined by in vivo serial dilution tumor-propagating assay. Furthermore, daughter xenografts that did form were 12-fold smaller than controls. These findings show that stem-like tumor-initiating cells are dynamically regulated by c-Met signaling in vivo and that c-Met pathway inhibitors can deplete tumors of their tumor-propagating stem-like cells.     

Lyn Facilitates Glioblastoma Cell Survival under Conditions of Nutrient Deprivation by Promoting Autophagy

Members of the Src family kinases (SFK) can modulate diverse cellular processes, including division, death and survival, but their role in autophagy has been minimally explored. Here, we investigated the roles of Lyn, a SFK, in promoting the survival of human glioblastoma tumor (GBM) cells in vitro and in vivo using lentiviral vector-mediated expression of constitutively-active Lyn (CA-Lyn) or dominant-negative Lyn (DN-Lyn). Expression of either CA-Lyn or DN-Lyn had no effect on the survival of U87 GBM cells grown under nutrient-rich conditions. In contrast, under nutrient-deprived conditions (absence of supplementation with L-glutamine, which is essential for growth of GBM cells, and FBS) CA-Lyn expression enhanced survival and promoted autophagy as well as inhibiting cell death and promoting proliferation. Expression of DN-Lyn promoted cell death. In the nutrient-deprived GBM cells, CA-Lyn expression enhanced AMPK activity and reduced the levels of pS6 kinase whereas DN-Lyn enhanced the levels of pS6 kinase. Similar results were obtained in vitro using another cultured GBM cell line and primary glioma stem cells. On propagation of the transduced GBM cells in the brains of nude mice, the CA-Lyn xenografts formed larger tumors than control cells and autophagosomes were detectable in the tumor cells. The DN-Lyn xenografts formed smaller tumors and contained more apoptotic cells. Our findings suggest that on nutrient deprivation in vitro Lyn acts to enhance the survival of GBM cells by promoting autophagy and proliferation as well as inhibiting cell death, and Lyn promotes the same effects in vivo in xenograft tumors. As the levels of Lyn protein or its activity are elevated in several cancers these findings may be of broad relevance to cancer biology.     

EGFRvIII-Specific Chimeric Antigen Receptor T Cells Migrate to and Kill Tumor Deposits Infiltrating the Brain Parenchyma in an Invasive Xenograft Model of Glioblastoma

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and is uniformly lethal. T-cell-based immunotherapy offers a promising platform for treatment given its potential to specifically target tumor tissue while sparing the normal brain. However, the diffuse and infiltrative nature of these tumors in the brain parenchyma may pose an exceptional hurdle to successful immunotherapy in patients. Areas of invasive tumor are thought to reside behind an intact blood brain barrier, isolating them from effective immunosurveillance and thereby predisposing the development of "immunologically silent" tumor peninsulas. Therefore, it remains unclear if adoptively transferred T cells can migrate to and mediate regression in areas of invasive GBM. One barrier has been the lack of a preclinical mouse model that accurately recapitulates the growth patterns of human GBM in vivo. Here, we demonstrate that D-270 MG xenografts exhibit the classical features of GBM and produce the diffuse and invasive tumors seen in patients. Using this model, we designed experiments to assess whether T cells expressing third-generation chimeric antigen receptors (CARs) targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, would localize to and treat invasive intracerebral GBM. EGFRvIII-targeted CAR (EGFRvIII⁺ CAR) T cells demonstrated in vitro EGFRvIII antigen-specific recognition and reactivity to the D-270 MG cell line, which naturally expresses EGFRvIII. Moreover, when administered systemically, EGFRvIII⁺ CAR T cells localized to areas of invasive tumor, suppressed tumor growth, and enhanced survival of mice with established intracranial D-270 MG tumors. Together, these data demonstrate that systemically administered T cells are capable of migrating to the invasive edges of GBM to mediate antitumor efficacy and tumor regression.     

Dendritic Cell Based PSMA Immunotherapy for Prostate Cancer Using a CD40-Targeted Adenovirus Vector

Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.     

Decrease of circARID1A retards glioblastoma invasion by modulating miR-370-3p/ TGFBR2 pathway

Increasing evidence suggests that circular RNAs (circRNAs) are involved in regulating tumor biological activity. Glioblastoma (GBM) is one of the most lethal diseases characterized by highly aggressive proliferative and invasive behaviors. We aimed to explore how circRNAs influenced GBM biological activity. By circRNA array analysis we found that circARID1A was significantly up-regulated in GBM. Next, we found that circARID1A was up-regulated in GBM tissues and cell lines. Interfering with circARID1A inhibited the migration and invasion of a human GBM cell line U87. By performing dual-luciferase reporter assays, RNA pull-down and fluorescent in situ hybridization (FISH), we determined that circARID1A directly bound to miR-370-3p. Moreover, we confirmed that transforming growth factor beta receptor 2 (TGFBR2) was the target gene of miR-370-3p by performing RNA pull-down, dual-luciferase reporter assays and western blotting. Further experiments verified that circARID1A promoted GBM cell migration and invasion by modulating miR-370-3p/ TGFBR2 pathway. In addition, we demonstrated that silencing circARID1A restrain the growth of GBM in vivo. Finally, we showed that circARID1A was abundant in GBM cell derived exosomes. In conclusion, circARID1A participated in regulating migration and invasion of GBM via modulation of miR-370-3p/ TGFBR2 and thus may be a potential serum biomarker of GBM.     

Expansion of tumor-specific cytolytic T lymphocytes using in vitro restimulation with tumor-specific transplantation antigen

Tumor-specific T lymphocytes (CTL) induced by in vivo immunization of C3H/HeJ mice with the syngeneic methylcholanthrene (MCA)-induced fibrosarcoma MCA-F were expanded in vitro by restimulation with 1-butanol-extracted, isoelectrophoretically purified, tumor-specific transplantation antigen (TSTA) in combination with purified rat interleukin-2 (IL-2) and fresh, syngeneic, 2000-R-irradiated, adherent splenic antigen-presenting cells (APC). The cultured immune T-cell population, containing 40–55% Lyt 2⁺ and 40–60% L3T4⁺ cells, displayed TSTA-specific proliferative and cytotoxic activities in vitro. The expanded T cells appear to recognize butanol-extracted TSTA in association with specific H-2 class I antigens, as revealed by the benefit of syngeneic over allogeneic cells as APC and by the adverse effect of depletion using anti-H-2K, but not anti-Ia, monoclonal antibodies. In adoptive transfer assays in vitro, expanded T cells specifically neutralize homotypic, but not heterotypic, tumor growth in vivo. Based upon the effects of depletion of T-lymphocyte subpopulations using monoclonal antibodies, the Lyt 2⁺ cytotoxic T lymphocytes (CTL) appear to display greater in vivo neutralizing activity than L3T4⁺ T cells. Thus in vitro stimulation of in vivo-immunized T cells, using butanol-extracted TSTA in combination with IL-2 and syngeneic APC, expands tumor-specific CTL.     

Mesenchymal stromal/stem cells markers in the human bone marrow

Background aimsMesenchymal stromal/stem cells (MSCs) can be isolated from human bone marrow (BM), expanded ex vivo and identified via numerous surface antigens. Despite the importance of these cells in regenerative therapy programs, it is unclear whether the cell membrane signature defining MSC preparations ex vivo is determined during culture or may reflect an in vivo counterpart. BM-MSC phenotype in vivo requires further investigation.MethodsTo characterize cells in their natural BM environment, we performed multi-parametric immunohistochemistry on trabecular bone biopsy specimens from multiple donors and described cells by different morphology and micro-anatomic localization in relationship to a precise pattern of MSC antigen expression.ResultsMicroscopically examined high-power field marrow sections revealed an overlapping in vivo expression of antigens characterizing ex vivo expanded BM-MSCs, including CD10, CD73, CD140b, CD146, GD2 and CD271. Expanding this panel to proteins associated with pluripotency, such as Oct4, Nanog and SSEA-4, we were able to identify different cellular populations in the human trabecular bone and BM expressing different progenitor cell markers.ConclusionsTargeting several multipotency and pluripotency markers, we found that the BM contains identifiable and distinct progenitor cells further justifying their introduction for a wide range of applications in regenerative medicine.     

Expansion of Human and Murine Hematopoietic Stem and Progenitor Cells Ex Vivo without Genetic Modification Using MYC and Bcl-2 Fusion Proteins

The long-term repopulating hematopoietic stem cell (HSC) population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs). We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC) population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat) and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use.     

Mesenchymal stromal cell supported umbilical cord blood ex vivo expansion enhances regulatory T cells and reduces graft versus host disease

Background aimsDouble cord blood transplantation (DCBT) may shorten neutrophil and platelet recovery times compared with standard umbilical cord blood transplantation. However, DCBT may be associated with a higher incidence of graft versus host disease (GVHD). In this study, we explored the effect of ex vivo expansion of a single cord blood unit (CBU) in a DCBT setting on GVHD and engraftment.MethodsPost-thaw cryopreserved CBUs from cord blood banks, hereinafter termed “banked” CBUs, were co-cultured with confluent bone marrow mesenchymal stromal cells (MSCs) supplemented with a cytokine cocktail comprising 100 ng/mL stem cell factor, 50 ng/mL flt3-ligand, 100 ng/mL thrombopoietin and 20 ng/mL insulin-like growth factor binding protein 2 for 12 days.ResultsWhen DCBT of one unexpanded and one expanded CBU was performed in non-obese diabetic/severe combined immunodeficient-IL2Rgamma^null (NOD/SCID-IL2γ^?/?, NSG) mice, the expanded CBU significantly boosted in vivo hematopoiesis of the unexpanded CBU. The median survival of NSG mice was significantly improved from 63.4% (range, 60.0–66.7%) for mice receiving only unexpanded units to 86.5% (range, 80.0–92.9%) for mice receiving an expanded unit (P < 0.001). The difference in survival appeared to be due to a lower incidence of GVHD in the mice receiving expanded cells. This effect on GVHD was mediated by a significant increase in regulatory T cells seen in the presence of MSC co-culture.ConclusionsMSC-supported ex vivo expansion of “banked” CBU boosted unexpanded CBU hematopoiesis in vivo, increased regulatory T cell content and decreased the incidence of GVHD.     

Non-invasive monitoring of the kinetic infiltration and therapeutic efficacy of nanoparticle-labeled chimeric antigen receptor T cells in glioblastoma via 7.0-Tesla magnetic resonance imaging

Background aimsChimeric antigen receptor (CAR) T-cell therapy is a promising treatment strategy in solid tumors. In vivo cell tracking techniques can help us better understand the infiltration, persistence and therapeutic efficacy of CAR T cells. In this field, magnetic resonance imaging (MRI) can achieve high-resolution images of cells by using cellular imaging probes. MRI can also provide various biological information on solid tumors.MethodsThe authors adopted the amino alcohol derivatives of glucose-coated nanoparticles, ultra-small superparamagnetic particles of iron oxide (USPIOs), to label CAR T cells for non-invasive monitoring of kinetic infiltration and persistence in glioblastoma (GBM). The specific targeting CARs included anti-human epidermal growth factor receptor variant III and IL13 receptor subunit alpha 2 CARs.ResultsWhen using an appropriate concentration, USPIO labeling exerted no negative effects on the biological characteristics and killing efficiency of CAR T cells. Increasing hypointensity signals could be detected in GBM models by susceptibility-weighted imaging MRI ranging from 3 days to 14 days following the injection of USPIO-labeled CAR T cells. In addition, nanoparticles and CAR T cells were found on consecutive histopathological sections. Moreover, diffusion and perfusion MRI revealed significantly increased water diffusion and decreased vascular permeability on day 3 after treatment, which was simultaneously accompanied by a significant decrease in tumor cell proliferation and increase in intercellular tight junction on immunostaining sections.ConclusionThese results establish an effective imaging technique that can track CAR T cells in GBM models and validate their early therapeutic effects, which may guide the evaluation of CAR T-cell therapies in solid tumors.     

Dendritic Cell-Specific Delivery of Flt3L by Coronavirus Vectors Secures Induction of Therapeutic Antitumor Immunity

Efficacy of antitumor vaccination depends to a large extent on antigen targeting to dendritic cells (DCs). Here, we assessed antitumor immunity induced by attenuated coronavirus vectors which exclusively target DCs in vivo and express either lymphocyte- or DC-activating cytokines in combination with a GFP-tagged model antigen. Tracking of in vivo transduced DCs revealed that vectors encoding for Fms-like tyrosine kinase 3 ligand (Flt3L) exhibited a higher capacity to induce DC maturation compared to vectors delivering IL-2 or IL-15. Moreover, Flt3L vectors more efficiently induced tumor-specific CD8⁺ T cells, expanded the epitope repertoire, and provided both prophylactic and therapeutic tumor immunity. In contrast, IL-2- or IL-15-encoding vectors showed a substantially lower efficacy in CD8⁺ T cell priming and failed to protect the host once tumors had been established. Thus, specific in vivo targeting of DCs with coronavirus vectors in conjunction with appropriate conditioning of the microenvironment through Flt3L represents an efficient strategy for the generation of therapeutic antitumor immunity.     

Presence of osteoclast precursor cells during ex vivo expansion of bone marrow-derived mesenchymal stem cells for autologous use in cell therapy

Background aimsTo obtain a cell product competent for clinical use in terms of cell dose and biologic properties, bone marrow-derived mesenchymal stem cells (MSCs) must be expanded ex vivo.MethodsA retrospective analysis was performed of records of 76 autologous MSC products used in phase I or II clinical studies performed in a cohort of cardiovascular patients. In all cases, native MSCs present in patient bone marrow aspirates were separated and expanded ex vivo.ResultsThe cell products were classified in two groups (A and B), according to biologic properties and expansion time (ex vivo passages) to reach the protocol-established cell dose. In group A, the population of adherent cells obtained during the expansion period (2 ± 1 passages) was composed entirely of MSCs and met the requirements of cell number and biologic features as established in the respective clinical protocol. In group B, in addition to MSCs, we observed during expansion a high proportion of ancillary cells, characterized as osteoclast precursor cells. In this case, although the biologic properties of the resulting MSC product were not affected, the yield of MSCs was significantly lower. The expansion cycles had to be increased (3 ± 1 passages).ConclusionsThese results suggest that the presence of osteoclast precursor cells in bone marrow aspirates may impose a limit for the proper clinical use of ex vivo expanded autologous bone marrow-derived MSCs.     

Swelling‐induced chloride current in glioblastoma proliferation,migration, and invasion

Glioblastoma (GBM) remains as the most common and aggressive brain tumor. The survival of GBM has been linked to the aberrant activation of swelling‐induced chloride current I_Cl,swell. In this study, we investigated the effects of I_Cl,swell on cell viability, proliferation, and migration in the human GBM cell lines, U251 and U87, using a combination of patch clamp electrophysiology, MTT, colony formation, wound healing assays and Western immunoblotting. First, we showed that the specific inhibitor of I_Cl,swell, DCPIB, potently reduced the I_Cl,swell in U87 cells. Next, in both U87 and U251 cells, we found that DCPIB reduced GBM viability, proliferation, colony formation, migration, and invasion. In addition, our Western immunoblot assay showed that DCPIB‐treated U251 cells had a reduction in JAK2, STAT3, and Akt phosphorylation, thus, suggesting that DCPIB potentially suppresses GBM functions through inhibition of the JAK2/STAT3 and PI3K/Akt signaling pathways. Therefore, the I_Cl,swell may be a potential drug target for GBM.     

A stromal-free,serum-free system to expand ex vivo hematopoietic stem cells from mobilized peripheral blood of patients with hematologic malignancies and healthy donors

Background aimsThe number of hematopoietic stem cells (HSCs) is critical for transplantation. The ex vivo expansion of mobilized peripheral blood (MPB) HSCs is of clinical value for reconstitution to meet clinical need.MethodsThis study proposed a simple, defined, stromal-free and serum-free culture system (SF-HSC medium) for clinical use, which is composed of Iscove's modified Dulbecco's medium, cytokine cocktails and serum substitutes. This study also characterized the cellular properties of expanded MPB CD133⁺ HSCs from patients with hematologic malignancies and healthy donors by surface antigen, colony-forming cell, long-term culture-initiating cell, gene expression and in vivo engraftment assays.ResultsThe expanded fold values of CD45⁺ white blood cells and CD34⁺, CD133⁺, CD34⁺CD38^?, CD133⁺CD38^?, CD34⁺CD133⁺, colony-forming and long-term culture-initiating cells at the end of 7-day culture from CD133⁺ MPB of hematologic malignancies were 9.4-fold, 5.9-fold, 4.0-fold, 35.8-fold, 21.9-fold, 3.8-fold, 11.8-fold and 6.7-fold, and values from healthy donor CD133⁺ MPB were 20.7-fold, 14.5-fold, 8.5-fold, 83.8-fold, 37.3-fold, 6.2-fold, 19.1-fold and 14.6-fold. The high enrichment of CD38^? cells, which were either CD34⁺ or CD133⁺, sustained the proliferation of early uncommitted HSCs. The expanded cells showed high levels of messenger RNA expression of HOBX4, ABCG2 and HTERT and had the in vivo ability to re-populate NOD/SCID mice.ConclusionsOur results demonstrated that an initial, limited number of MPB CD133⁺ HSCs could be expanded functionally in SF-HSC medium. We believe that this serum-free expansion technique can be employed in both basic research and clinical transplantation.