Targeting ROR1 inhibits the self‐renewal and invasive ability of glioblastoma stem cells

Glioblastoma is the most malignant of brain tumours and is difficult to cure because of interruption of drug delivery by the blood–brain barrier system, its high metastatic capacity and the existence of cancer stem cells (CSCs). Although CSCs are present as a small population in malignant tumours, CSCs have been studied as they are responsible for causing recurrence, metastasis and resistance to chemotherapy and radiotherapy for cancer. CSCs have self‐renewal characteristics like normal stem cells. The aim of this study was to investigate whether receptor tyrosine kinase‐like orphan receptor 1 (ROR1) is involved in stem cell maintenance and malignant properties in human glioblastoma. Knockdown of ROR1 caused reduction of stemness and sphere formation capacity. Moreover, down‐regulation of ROR1 suppressed the expression of epithelial‐mesenchymal transition‐related genes and the tumour migratory and invasive abilities. The results of this study indicate that targeting ROR1 can induce differentiation of CSCs and inhibit metastasis in glioblastoma. In addition, ROR1 may be used as a potential marker for glioblastoma stem cells as well as a potential target for glioblastoma stem cell therapy. Copyright © 2016 John Wiley & Sons, Ltd.     

Maintenance of stem cell viability and differentiation potential following cryopreservation within 3-dimensional hyaluronic acid hydrogels

While significant progress has been made in directing the behavior of cells encapsulated within three-dimensional (3D) covalently crosslinked hydrogels, the capacity of these materials to support in situ cryopreservation of cells directly within the gels has not been assessed. Here, we demonstrate the retention of human mesenchymal stem cell (hMSC) viability within hyaluronic acid (HA) and polyethylene glycol based hydrogels via a facile gradual cooling and freezing protocol. Encapsulated cell viability was retained at similar rates in both materials systems regardless of initial duration in culture or adhesive ligand incorporation, indicating the versatility of the approach. Additionally, the cryopreservation protocol maintains stem cell differentiation potential; incubation in adipogenic differentiation media induced equal rates of hMSC adipogenesis in freeze-thawed and non-frozen HA based hydrogels on a per-cell basis. Collectively, these findings highlight the cryopreservation protocol as a platform technology that, in addition to contributing to an increased understanding of three-dimensional cell-matrix interactions, could enable the long-term preservation of tissue engineering constructs for clinical applications.     

Differentiation potential of human adipose stem cells bioprinted with hyaluronic acid/gelatin‐based bioink through microextrusion and visible light‐initiated crosslinking

Bioprinting has a great potential to fabricate three‐dimensional (3D) functional tissues and organs. In particular, the technique enables fabrication of 3D constructs containing stem cells while maintaining cell proliferation and differentiation abilities, which is believed to be promising in the fields of tissue engineering and regenerative medicine. We aimed to demonstrate the utility of the bioprinting technique to create hydrogel constructs consisting of hyaluronic acid (HA) and gelatin derivatives through irradiation by visible light to fabricate 3D constructs containing human adipose stem cells (hADSCs). The hydrogel was obtained from a solution of HA and gelatin derivatives possessing phenolic hydroxyl moieties in the presence of ruthenium(II) tris‐bipyridyl dication and sodium ammonium persulfate. hADSCs enclosed in the bioprinted hydrogel construct elongated and proliferated in the hydrogel. In addition, their differentiation potential was confirmed by examining the expression of pluripotency marker genes and cell surface marker proteins, and differentiation to adipocytes in adipogenic differentiation medium. Our results demonstrate the great potential of the bioprinting method and the resultant hADSC‐laden HA/gelatin constructs for applications in tissue engineering and regenerative medicine.     

Photocrosslinked hyaluronic acid hydrogels: natural,biodegradable tissue engineering scaffolds

Ideally, rationally designed tissue engineering scaffolds promote natural wound healing and regeneration. Therefore, we sought to synthesize a biomimetic hydrogel specifically designed to promote tissue repair and chose hyaluronic acid (HA; also called hyaluronan) as our initial material. Hyaluronic acid is a naturally occurring polymer associated with various cellular processes involved in wound healing, such as angiogenesis. Hyaluronic acid also presents unique advantages: it is easy to produce and modify, hydrophilic and nonadhesive, and naturally biodegradable. We prepared a range of glycidyl methacrylate-HA (GMHA) conjugates, which were subsequently photopolymerized to form crosslinked GMHA hydrogels. A range of hydrogel degradation rates was achieved as well as a corresponding, modest range of material properties (e.g., swelling, mesh size). Increased amounts of conjugated methacrylate groups corresponded with increased crosslink densities and decreased degradation rates and yet had an insignificant effect on human aortic endothelial cell cytocompatibility and proliferation. Rat subcutaneous implants of the GMHA hydrogels showed good biocompatibility, little inflammatory response, and similar levels of vascularization at the implant edge compared with those of fibrin positive controls. Therefore, these novel GMHA hydrogels are suitable for modification with adhesive peptide sequences (e.g., RGD) and use in a variety of wound-healing applications.     

Suberoylanilide hydroxamic acid (SAHA) causes tumor growth slowdown and triggers autophagy in glioblastoma stem cells

Although suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been used in clinical trials for cancer therapies, its pharmacological effects occur through a poorly understood mechanism. Here, we report that SAHA specifically triggers autophagy and reduces cell viability via promotion of apoptosis in the late phase of glioblastoma stem cells (GSCs). Using a cell line cultured from a glioblastoma biopsy, we investigated the properties and effects of GSCs under SAHA treatment in vitro. In vivo xenograft assays revealed that SAHA effectively caused tumor growth slowdown and the induction of autophagy. SAHA was sufficient to increase formation of intracellular acidic vesicle organelles, recruitment of LC3-II to the autophagosomes, potentiation of BECN1 protein levels and reduced SQSTM1 levels. We determined that SAHA triggered autophagy through the downregulation of AKT-MTOR signaling, a major suppressive cascade of autophagy. Interestingly, upon depletion or pharmacological inhibition of autophagy, SAHA facilitates apoptosis and results in cell death at the early phase, suggesting that SAHA-induced autophagy functions probably act as a prosurvival mechanism. Furthermore, our results also indicated that the inhibition of SAHA-induced autophagy using chloroquine has synergistic effects that further increase apoptosis. Moreover, we found that a reduced dose of SAHA functioned as a potent modulator of differentiation and senescence. Taken together, our results provide a new perspective on the treatment of GSCs, indicating that SAHA is a promising agent for targeting GSCs through the induction of autophagy.     

Pancreatic cancer stem cell proliferation is strongly inhibited by diethyldithiocarbamate-copper complex loaded into hyaluronic acid decorated liposomes

Background

Pancreatic cancer stem cells (CSCs) are responsible for resistance to standard therapy, metastatic potential, and disease relapse following treatments. The current therapy for pancreatic ductal adenocarcinoma (PDAC) preferentially targets the more differentiated cancer cell population, leaving CSCs as a cell source for tumor mass formation and recurrence. For this reason, there is an urgent need to improve current therapies and develop novel CSC-targeted therapeutic approaches.

A solid-phase immunoassay for the binding of cartilage proteoglycan to hyaluronic acid

A solid-phase assay for detecting the binding of cartilage proteoglycan (PG) to hyaluronic acid (HA) is described. In the assay, HA is immobilized on protamine-treated microtiter wells, the wells are incubated with PG monomer and antibody to PG monomer, and then an ELISA system is used to detect binding of the PG to HA. The specificity of the assay is indicated by the failure to detect PG binding to chondroitin sulfate or albumin-coated microtiter wells, the absence of binding with tryptic fragments of PG monomer other than the HA-binding segment, the loss of binding after reduction and alkylation of PG monomer, and the inhibition of binding by preincubation of PG monomer with small amounts of HA. In contrast to the HA-PG interaction in solution, hyaluronidase digestion of HA does not affect its ability to inhibit the reaction of PG monomer with immobilized HA. The microtiter well-based assay appears to be a rapid, simple, and potentially versatile method for studying interactions with HA.     

Therapy targets in glioblastoma and cancer stem cells: lessons from haematopoietic neoplasms

Despite intense efforts to identify cancer‐initiating cells in malignant brain tumours, markers linked to the function of these cells have only very recently begun to be uncovered. The notion of cancer stem cell gained prominence, several molecules and signalling pathways becoming relevant for diagnosis and treatment. Whether a substantial fraction or only a tiny minority of cells in a tumor can initiate and perpetuate cancer, is still debated. The paradigm of cancer‐initiating stem cells has initially been developed with respect to blood cancers where chronic conditions such as myeloproliferative neoplasms are due to mutations acquired in a haematopoietic stem cell (HSC), which maintains the normal hierarchy to neoplastic haematopoiesis. In contrast, acute leukaemia transformation of such blood neoplasms appears to derive not only from HSCs but also from committed progenitors that cannot differentiate. This review will focus on putative novel therapy targets represented by markers described to define cancer stem/initiating cells in malignant gliomas, which have been called ‘leukaemia of the brain’, given their rapid migration and evolution. Parallels are drawn with other cancers, especially haematopoietic, given the similar rampant proliferation and treatment resistance of glioblastoma multiforme and secondary acute leukaemias. Genes associated with the malignant conditions and especially expressed in glioma cancer stem cells are intensively searched. Although many such molecules might only coincidentally be expressed in cancer‐initiating cells, some may function in the oncogenic process, and those would be the prime candidates for diagnostic and targeted therapy. For the latter, combination therapies are likely to be envisaged, given the robust and plastic signalling networks supporting malignant proliferation.     

Sorafenib selectively depletes human glioblastoma tumor-initiating cells from primary cultures

Glioblastomas are grade IV brain tumors characterized by high aggressiveness and invasiveness, giving patients a poor prognosis. We investigated the effects of the multi-kinase inhibitor sorafenib on six cultures isolated from human glioblastomas and maintained in tumor initiating cells-enriching conditions. These cell subpopulations are thought to be responsible for tumor recurrence and radio- and chemo-resistance, representing the perfect target for glioblastoma therapy. Sorafenib reduces proliferation of glioblastoma cultures, and this effect depends, at least in part, on the inhibition of PI3K/Akt and MAPK pathways, both involved in gliomagenesis. Sorafenib significantly induces apoptosis/cell death via downregulation of the survival factor Mcl-1. We provide evidence that sorafenib has a selective action on glioblastoma stem cells, causing enrichment of cultures in differentiated cells, downregulation of the expression of stemness markers required to maintain malignancy (nestin, Olig2 and Sox2) and reducing cell clonogenic ability in vitro and tumorigenic potential in vivo. The selectivity of sorafenib effects on glioblastoma stem cells is confirmed by the lower sensitivity of glioblastoma cultures after differentiation as compared with the undifferentiated counterpart. Since current GBM therapy enriches the tumor in cancer stem cells, the evidence of a selective action of sorafenib on these cells is therapeutically relevant, even if, so far, results from first phase II clinical trials did not demonstrate its efficacy.     

MicroRNA-4731-5p delivered by AD-mesenchymal stem cells induces cell cycle arrest and apoptosis in glioblastoma

Glioblastoma multiforme (GBM) exhibits the most malignant brain tumor with very poor prognosis. MicroRNAs (miRNAs) are regulatory factors that can downregulate the expression of multiple genes. Several miRNAs acting as tumor-suppressor genes have been identified so far. The delivery of miRNA by mesenchymal stem cell (MSC) due to their ability to specifically target tumors is a new, hopeful therapeutic approach for glioblastoma. The objective of our study is the investigation of the effect of lentivirus-mediated microRNA-4731 (miR-4731) genetic manipulated adipose-derived (AD)-MSC on GBM. The downregulation of miR-4731 in human GBM tumor was detected using the GEO dataset. To evaluate the function of miR-4731, we overexpressed miR-4731 using lentiviral vectors in U-87 and U-251 GBM cell lines. The effects of miR-4731 on cell proliferation and cell cycle of glioma cells were analyzed by wound test and flow-cytometry assay. miR-4731 inhibited the proliferation of GBM cancer cells. Coculturing was used to study the antiproliferative effect of miR-4731-AD-MSCs on GBM cell lines. Direct and indirect coculture of GBM cell lines with miR-4731-AD-MSCs induced cell cycle arrest and apoptosis. Our findings suggest that AD-MSCs expressing miR-4731 have favorable antitumor characteristics and should be further explored in future glioma therapy.     

离子交换层析纯化透明质酸

考察6种离子交换树脂的静态吸附解析效果,选出201＊7阴离子交换树脂填柱,确定洗脱流速为0.6mL/min,40mL0.3mol/LNaCl和50mL0.5mol/L NaCl双浓度洗脱,实现透明质酸和杂蛋白的分离。制得透明质酸产品蛋白含量为0.057%,葡萄糖醛酸含量为43%,平均相对分子质量大于1.1×10^6,收率为54%,符合医用级透明质酸行业标准的要求。     

The DEAD-box helicase DDX56 is a conserved stemness regulator in normal and cancer stem cells

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Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis

Hyaluronic acid (HA) is a high‐value biopolymer used in the biomedical, pharmaceutical, cosmetic, and food industries. Current methods of HA production, including extraction from animal sources and streptococcal cultivations, are associated with high costs and health risks. Accordingly, the development of bioprocesses for HA production centered on robust “Generally Recognized as Safe (GRAS)” organisms such as Bacillus subtilis is highly attractive. Here, we report the development of novel strains of B. subtilis in which the membrane cardiolipin (CL) content and distribution has been engineered to enhance the functional expression of heterologously expressed hyaluronan synthase (HAS) of Streptococcus equisimilis (SeHAS), in turn, improving the culture performance for HA production. Elevation of membrane CL levels via overexpressing components involved in the CL biosynthesis pathway, and redistribution of CL along the lateral membrane via repression of the cell division initiator protein FtsZ resulted in increases to the HA titer of up to 204% and peak molecular weight of up to 2.2 MDa. Moreover, removal of phosphatidylethanolamine and neutral glycolipids from the membrane of HA‐producing B. subtilis via inactivation of pssA and ugtP, respectively, has suggested the lipid dependence for functional expression of SeHAS. Our study demonstrates successful application of membrane engineering strategies to develop an effective platform for biomanufacturing of HA with B. subtilis strains expressing Class I streptococcal HAS.     

BET bromodomain proteins are required for glioblastoma cell proliferation

Epigenetic proteins have recently emerged as novel anticancer targets. Among these, bromodomain and extra terminal domain (BET) proteins recognize lysine-acetylated histones, thereby regulating gene expression. Newly described small molecules that inhibit BET proteins BRD2, BRD3, and BRD4 reduce proliferation of NUT (nuclear protein in testis)-midline carcinoma, multiple myeloma, and leukemia cells in vitro and in vivo. These findings prompted us to determine whether BET proteins may be therapeutic targets in the most common primary adult brain tumor, glioblastoma (GBM). We performed NanoString analysis of GBM tumor samples and controls to identify novel therapeutic targets. Several cell proliferation assays of GBM cell lines and stem cells were used to analyze the efficacy of the drug I-BET151 relative to temozolomide (TMZ) or cell cycle inhibitors. Lastly, we performed xenograft experiments to determine the efficacy of I-BET151 in vivo. We demonstrate that BRD2 and BRD4 RNA are significantly overexpressed in GBM, suggesting that BET protein inhibition may be an effective means of reducing GBM cell proliferation. Disruption of BRD4 expression in glioblastoma cells reduced cell cycle progression. Similarly, treatment with the BET protein inhibitor I-BET151 reduced GBM cell proliferation in vitro and in vivo. I-BET151 treatment enriched cells at the G1/S cell cycle transition. Importantly, I-BET151 is as potent at inhibiting GBM cell proliferation as TMZ, the current chemotherapy treatment administered to GBM patients. Since I-BET151 inhibits GBM cell proliferation by arresting cell cycle progression, we propose that BET protein inhibition may be a viable therapeutic option for GBM patients suffering from TMZ resistant tumors.