Rona S. Carroll

αvβ3 and αvβ5 integrin expression in glioma periphery

Objective This study analyzed the expression of integrins alpha(v)beta3 and alpha(v)beta5 in glioma tissue and focused on the periphery of high-grade gliomas. Methods The analysis was performed with Western blot, immunohistochemistry, and immunofluorescence, by use of two monoclonal antibodies able to recognize the functional integrin heterodimer. The expression of integrin-related ligands and growth factors also was studied. Sections from the tumor periphery were classified as either tumor periphery (light tumor infiltrate or scant visible cells) or peritumor (heavy tumor infiltration). Results Our data on glioma tissues demonstrated that both integrins were expressed in glioma cells and vasculature and their expression correlated with the histological grade. Alpha(v)beta3 expression was prominent in astrocytic tumors. Both integrins were markers of tumor vasculature, particularly of endothelial proliferation. A high-grade glioma periphery demonstrated a prominent expression of integrin alpha(v)beta3. Cells demonstrating alpha(v)beta3 positivity were identified as tumor astrocytes and endothelial cells by double imaging. The same cells were surrounded by some alpha(v)beta3 ligands and co-localized fibroblast growth factor 2. Matrix metalloproteinase 2 also was found to be co-localized with alpha(v)beta3 in the same cells. Alpha(v)beta3 expression was more relevant in tumor astrocytes. Alpha(v)beta3 integrin and vascular endothelial growth factor expression increased from the periphery to the tumor center. Conclusion Our data support the role of integrins alpha(v)beta3 and alpha(v)beta5 in glioma-associated angiogenesis. In addition, they suggest a role for integrin alpha(v)beta3 in neoangiogenesis and cell migration in high-grade glioma periphery.

Central Precocious Puberty Caused by Mutations in the Imprinted Gene MKRN3

BACKGROUND The onset of puberty is first detected as an increase in pulsatile secretion of gonadotropin-releasing hormone (GnRH). Early activation of the hypothalamic-pituitary-gonadal axis results in central precocious puberty. The timing of pubertal development is driven in part by genetic factors, but only a few, rare molecular defects associated with central precocious puberty have been identified. METHODS We performed whole-exome sequencing in 40 members of 15 families with central precocious puberty. Candidate variants were confirmed with Sanger sequencing. We also performed quantitative real-time polymerase-chain-reaction assays to determine levels of messenger RNA (mRNA) in the hypothalami of mice at different ages. RESULTS We identified four novel heterozygous mutations in MKRN3, the gene encoding makorin RING-finger protein 3, in 5 of the 15 families; both sexes were affected. The mutations included three frameshift mutations, predicted to encode truncated proteins, and one missense mutation, predicted to disrupt protein function. MKRN3 is a paternally expressed, imprinted gene located in the Prader-Willi syndrome critical region (chromosome 15q11-q13). All affected persons inherited the mutations from their fathers, a finding that indicates perfect segregation with the mode of inheritance expected for an imprinted gene. Levels of Mkrn3 mRNA were high in the arcuate nucleus of prepubertal mice, decreased immediately before puberty, and remained low after puberty. CONCLUSIONS Deficiency of MKRN3 causes central precocious puberty in humans. (Funded by the National Institutes of Health and others.).

Intravascular Delivery of Neural Stem Cell Lines to Target Intracranial and Extracranial Tumors of Neural and Non-Neural Origin

The remarkable migratory and tumor-tropic capacities of neural stem cells (NSCs and/or neuroprogenitor cells) represent a potentially powerful approach to the treatment of invasive brain tumors, such as malignant gliomas. We have previously shown that whether implanted directly into or at distant sites from an experimental intracranial glioma, NSCs distributed efficiently throughout the main tumor mass and also tracked advancing tumor cells, while stably expressing a reporter transgene. As therapeutic proof-of-concept, NSCs genetically modified to produce the prodrug activating enzyme cytosine deaminase (CD), effected an 80% reduction in the resultant tumor mass, when tumor animals were treated with the systemic prodrug, 5-fluorocytosine. We now extend our findings of the tumor-tropic properties of NSCs (using a well-characterized, clonal NSC line C17.2), by investigating their capacity to target both intracranial and extracranial tumors, when administered into the peripheral vasculature. We furthermore demonstrate their capacity to target extracranial non-neural tumors such as prostate cancer and malignant melanoma. Well-characterized NSC lines (lacZ and/or CD-positive) were injected into the tail vein of adult nude mice with established experimental intracranial and/or subcutaneous flank tumors of neural and non-neural origin. The time course and distribution of NSCs within the tumor and internal organs was assessed in various models. Resulting data suggest that NSCs can localize to various tumor sites when injected via the peripheral vasculature, with little accumulation in normal tissues. Our findings suggest the novel use of intravascularly administered NSCs as an effective delivery vehicle to target and disseminate therapeutic agents to invasive tumors of neural and nonneural origin, both within and outside of the brain.

Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture

Time-released delivery of soluble growth factors (GFs) in engineered hydrogel tissue constructs promotes the migration and proliferation of embedded cells, which is an important factor for designing scaffolds that ultimately aim for neural tissue regeneration. We report a tissue engineering technique to print murine neural stem cells (C17.2), collagen hydrogel, and GF (vascular endothelial growth factor: VEGF)-releasing fibrin gel to construct an artificial neural tissue. We examined the morphological changes of the printed C17.2 cells embedded in the collagen and its migration toward the fibrin gel. The cells showed high viability (92.89+/-2.32%) after printing, which was equivalent to that of manually-plated cells. C17.2 cells printed within 1mm from the border of VEGF-releasing fibrin gel showed GF-induced changes in their morphology. The cells printed in this range also migrated toward the fibrin gel, with the total migration distance of 102.4+/-76.1microm over 3days. The cells in the control samples (fibrin without the VEGF or VEGF printed directly in collagen) neither proliferated nor migrated. The results demonstrated that bio-printing of VEGF-containing fibrin gel supported sustained release of the GF in the collagen scaffold. The presented method can be gainfully used in the development of three-dimensional (3D) artificial tissue assays and neural tissue regeneration applications.

Human Neural Stem Cells Target Experimental Intracranial Medulloblastoma and Deliver a Therapeutic Gene Leading to Tumor Regression

Purpose: Medulloblastoma, a malignant pediatric brain tumor, is incurable in about one third of patients despite multimodal treatments. In addition, current therapies can lead to long-term disabilities. Based on studies of the extensive tropism of neural stem cells (NSC) toward malignant gliomas and the secretion of growth factors common to glioma and medulloblastoma, we hypothesized that NSCs could target medulloblastoma and be used as a cellular therapeutic delivery system. Experimental Design: The migratory ability of HB1.F3 cells (an immortalized, clonal human NSC line) to medulloblastoma was studied both in vitro and in vivo. As proof-of-concept, we used HB1.F3 cells engineered to secrete the prodrug activating enzyme cytosine deaminase. We investigated the potential of human NSCs to deliver a therapeutic gene and reduce tumor growth. Results: The migratory capacity of HB1.F3 cells was confirmed by an in vitro migration assay, and corroborated in vivo by injecting chloromethylbenzamido-Dil–labeled HB1.F3 cells into the hemisphere contralateral to established medulloblastoma in nude mice. In vitro studies showed the therapeutic efficacy of HB1.F3-CD on Daoy cells in coculture experiments. In vitro therapeutic studies were conducted in which animals bearing intracranial medulloblastoma were injected ipsilaterally with HB1.F3-CD cells followed by systemic 5-flourocytosine treatment. Histologic analyses showed that human NSCs migrate to the tumor bed and its boundary, resulting in a 76% reduction of tumor volume in the treatment group (P < 0.01). Conclusion: These studies show for the first time the potential of human NSCs as an effective delivery system to target and disseminate therapeutic agents to medulloblastoma.

Human Bone Marrow-Derived Mesenchymal Stromal Cells Expressing S-TRAIL as a Cellular Delivery Vehicle for Human Glioma Therapy

Glioblastoma is among the most aggressive and treatment resistant of all human cancers. Conventional therapeutic approaches are unsuccessful because of diffuse infiltrative invasion of glioma tumor cells into normal brain parenchyma. Stem cell‐based therapies provide a promising approach for the treatment of malignant gliomas because of their migratory ability to invasive tumor cells. Our therapeutic strategy was to use human bone marrow‐derived mesenchymal stromal cells (hMSCs) as a cellular vehicle for the targeted delivery and local production of the biologic agent tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) at the glioma tumor site. hMSCs were transduced with a lentivirus expressing secretable TRAIL (S‐TRAIL) and mCherry (red fluorescent protein). Our results clearly demonstrate the retention of tumor tropic ability of hMSC S‐TRAIL cells by in vitro and in vivo migration assays. In vitro assays confirmed the expression, release, and biological activity of S‐TRAIL produced by hMSC S‐TRAIL cells. For the in vivo assessment of therapeutic efficacy, hMSCs were injected ipsilateral to an established intracranial glioma tumor in a mouse xenograft model. Genetically engineered hMSC S‐TRAIL cells were effective in inhibiting intracranial U87 glioma tumor growth (81.6%) in vivo and resulted in significantly longer animal survival. Immunohistochemical studies demonstrated significant, eight fold greater tumor cell apoptosis in the hMSC S‐TRAIL‐treated group than in controls. Our study demonstrates the therapeutic efficacy of hMSC S‐TRAIL cells and confirms that hMSCs can serve as a powerful cell‐based delivery vehicle for the site‐specific release of therapeutic proteins. STEM CELLS 2009;27:2320–2330

Targeting of melanoma brain metastases using engineered neural stem/progenitor cells

Brain metastases are an increasingly frequent and serious clinical problem for cancer patients, especially those with advanced melanoma. Given the extensive tropism of neural stem/progenitor cells (NSPCs) for pathological areas in the central nervous system, we expanded investigations to determine whether NSPCs could also target multiple sites of brain metastases in a syngeneic experimental melanoma model. Using cytosine deaminase-expressing NSPCs (CD-NSPCs) and systemic 5-fluorocytosine (5-FC) pro-drug administration, we explored their potential as a cell-based targeted drug delivery system to disseminated brain metastases. Our results indicate a strong tropism of NSPCs for intracerebral melanoma metastases. Furthermore, in our therapeutic paradigm, animals with established melanoma brain metastasis received intracranial implantation of CD-NSPCs followed by systemic 5-FC treatment, resulting in a significant (71%) reduction in tumor burden. These data provide proof of principle for the use of NSPCs for targeted delivery of therapeutic gene products to melanoma brain metastases.

Platelet-derived growth factor and its receptor expression in human oligodendrogliomas.

OBJECTIVE Platelet-derived growth factor (PDGF) induces cellular proliferation and differentiation by activating intracellular signaling mechanisms via their cognate receptors. In previous studies, we demonstrated that human brain tumors such as meningiomas, astrocytomas, medulloblastomas, and ependymomas expressed the messenger ribonucleic acid for the PDGF subunits and their receptors. In the present study, we investigated the expression of the messenger ribonucleic acid PDGF A and B chains and the PDGF alpha and beta receptors in 17 cases of oligodendrogliomas. METHODS Measurements of messenger ribonucleic acid levels were obtained using radioactive complementary deoxyribonucleic acid probes. Protein expression was analyzed with specific antibodies. RESULTS Sixteen of 17 tumors expressed the PDGF A subunit and all the PDGF alpha receptors. Furthermore, all the tumors expressed PDGF B and PDGF beta receptor subunits. CONCLUSION The results of this study suggest that oligodendrogliomas may have an autocrine loop stimulated by the interaction of PDGF and its receptor simultaneously produced by these tumors.

PEX-Producing Human Neural Stem Cells Inhibit Tumor Growth in a Mouse Glioma Model

A unique characteristic of neural stem cells is their capacity to track glioma cells that have migrated away from the main tumor mass into the normal brain parenchyma. PEX, a naturally occurring fragment of human metalloproteinase-2, acts as an inhibitor of glioma and endothelial cell proliferation, migration, and angiogenesis. In the present study, we evaluated the antitumor activity of PEX-producing human neural stem cells against malignant glioma. The HB1.F3 cell line (immortalized human neural stem cell) was transfected by a pTracer vector with PEX. The retention of the antiproliferative activity and migratory ability of PEX-producing HB1.F3 cells (HB1.F3-PEX) was confirmed in vitro. For the in vivo studies, DiI-labeled HB1.F3-PEX cells were stereotactically injected into established glioma tumor in nude mice. Tumor size was subsequently measured by magnetic resonance imaging and at the termination of the studies by histologic analysis including tumor volume, microvessel density, proliferation, and apoptosis rate. Histologic analysis showed that DiI-labeled HB1.F3-PEX cells migrate at the tumor boundary and cause a 90% reduction of tumor volume (P < 0.03). This reduction in tumor volume in animals treated with HB1.F3-PEX was associated with a significant decrease in angiogenesis (44.8%, P < 0.03) and proliferation (23.6%, P < 0.03). These results support the use of neural stem cells as delivery vehicle for targeting therapeutic genes against human glioma.

Targeting Rat Brainstem Glioma Using Human Neural Stem Cells and Human Mesenchymal Stem Cells

Purpose: Brainstem gliomas are usually inoperable and have a dismal prognosis. Based on the robust tropisms of neural stem cells (NSC) and mesenchymal stem cells (MSC) to brain tumors, we compared the tumor-tropic migratory capacities of these stem cells and evaluated the therapeutic potential of genetically engineered human NSCs encoding cytosine deaminase (CD) and IFNβ against brainstem gliomas. Experimental Design: The directed migratory capacities of NSCs and MSCs to brainstem glioma (F98) were evaluated both in vitro and in vivo. The human NSCs (HB1.F3) and various human MSCs, such as bone marrow–derived MSCs (HM3.B10), adipose tissue–derived MSCs, and umbilical cord blood–derived MSCs, were tested. Human fibroblast cells (HFF-1) were used as the negative control. As a proof of concept, the bioactivity of HB1.F3-CD-IFNβ was analyzed with a cell viability assay, and animals with brainstem gliomas were injected with HB1.F3-CD-IFNβ cells followed by systemic 5-fluorocytosine treatment. Results: In an in vitro modified Transwell migration assay and in vivo stem cell injection into established brainstem gliomas in rats, all the stem cells showed a significant migratory capacity compared with that of the control (P < 0.01). Histologic analysis showed a 59% reduction in tumor volume in the HB1.F3-CD-IFNβ–treated group (P < 0.05). Apoptotic cells were increased 2.33-fold in animals treated with HB1.F3-CD-IFNβ compared with the respective control groups (P < 0.01). Conclusion: The brainstem glioma-tropic migratory capacities of MSCs from various sources were similar to those of NSCs. Genetically engineered NSCs show therapeutic efficacy against brainstem gliomas.