Paul A. Clark

Significant Association of Multiple Human Cytomegalovirus Genomic Loci with Glioblastoma Multiforme Samples

ABSTRACT Viruses are appreciated as etiological agents of certain human tumors, but the number of different cancer types induced or exacerbated by viral infections is unknown. Glioblastoma multiforme (GBM)/astrocytoma grade IV is a malignant and lethal brain cancer of unknown origin. Over the past decade, several studies have searched for the presence of a prominent herpesvirus, human cytomegalovirus (HCMV), in GBM samples. While some have detected HCMV DNA, RNA, and proteins in GBM tissues, others have not. Therefore, any purported association of HCMV with GBM remains controversial. In most of the previous studies, only one or a select few viral targets were analyzed. Thus, it remains unclear the extent to which the entire viral genome was present when detected. Here we report the results of a survey of GBM specimens for as many as 20 different regions of the HCMV genome. Our findings indicate that multiple HCMV loci are statistically more likely to be found in GBM samples than in other brain tumors or epileptic brain specimens and that the viral genome was more often detected in frozen samples than in paraffin-embedded archival tissue samples. Finally, our experimental results indicate that cellular genomes substantially outnumber viral genomes in HCMV-positive GBM specimens, likely indicating that only a minority of the cells found in such samples harbor viral DNA. These data argue for the association of HCMV with GBM, defining the virus as oncoaccessory. Furthermore, they imply that, were HCMV to enhance the growth or survival of a tumor (i.e., if it is oncomodulatory), it would likely do so through mechanisms distinct from classic tumor viruses that express transforming viral oncoproteins in the overwhelming majority of tumor cells.

Ex vivo adipose tissue engineering by human marrow stromal cell seeded gelatin sponge.

The limitation of current clinical treatment for restoration extended defects of soft tissue associated with trauma, tumor resections, and congenital deformities are well known. This study demonstrates that human bone marrow stromal cells (MSCs) can be utilized to tissue engineer adipose tissue for therapeutic purposes. Adipogenic potentials of monolayer-cultured human MSCs were evaluated by biochemical measurement of an adipogenic differentiation marker (glycerol-3-phosphate dehydrogenase, G-3-PDH) and cellular morphology. After preparation by seeding human MSCs on a 3-dimensional gelatin sponge and exposure to adipogenic differentiation medium, the ex vivo tissue-engineered adipose constructs were assessed histomorphologically and biochemically. Lipid droplets accumulated and expanded within the constructs accompanied by a significant increase of G-3-PDH activity. The present study indicates that bone MSCs could be a cell resource in tissue engineering adipose tissue, while gelatin sponge could be a good scaffold in this approach to improve the outcome of clinical treatment.

Synergistic actions of hematopoietic and mesenchymal stem/progenitor cells in vascularizing bioengineered tissues

Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs) and mesenchymal stem/progenitor cells (MSCs) were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP) scaffolds, followed by infusion of gel-suspended CD34+ hematopoietic cells. Co-transplantation of CD34+ HSCs and CD34− MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromized mice yielded vascularized tissue. The average vascular number of co-transplanted CD34+ and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34+ cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34+ cells. Based on additional in vitro results of endothelial differentiation of CD34+ cells by vascular endothelial growth factor (VEGF), we adsorbed VEGF with co-transplanted CD34+ and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34+ cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone, adipose, muscle and dermal grafts, and may have implications in the regeneration of internal organs.

Developmental signaling pathways in brain tumor-derived stem-like cells.

Recently, a subpopulation of cells highly efficient in tumor initiation and growth has been isolated from brain tumors. Of interest, these brain tumor initiating cells exhibit many stem‐like properties, including self‐renewal, extended proliferation, and multipotency, and are both phenotypically and genetically similar to normal neural stem cells (NSCs). Aberrant expression of developmental pathways, such as WNT, Hedgehog, Notch, and transforming growth factor‐β/bone morphogenetic protein, have been demonstrated in brain tumors, and extrinsic regulation of these pathways may be used to target brain tumor stem‐like cells (BTSCs) and form the basis of novel biological therapies. Because of regulatory redundancy during normal development, future therapeutic strategies to inhibit BTSC‐mediated tumor growth and minimize NSC‐related deleterious effects may require detailed understanding and regulation of multiple cellular mechanisms. This review analyzes the role developmental pathways play in brain tumors, focusing on the potential effects of pathway regulation on BTSC‐driven tumorigenesis. Developmental Dynamics 236:3297–3308, 2007.

The cancer stem cell paradigm: a new understanding of tumor development and treatment

Importance of the field: Cancer is the second leading cause of death in the United States, and therefore remains a central focus of modern medical research. Accumulating evidence supports a ‘cancer stem cell’ (CSC) model – where cancer growth and/or recurrence is driven by a small subset of tumor cells that exhibit properties similar to stem cells. This model may provide a conceptual framework for developing more effective cancer therapies that target cells propelling cancer growth. Areas covered in this review: We review evidence supporting the CSC model and associated implications for understanding cancer biology and developing novel therapeutic strategies. Current controversies and unanswered questions of the CSC model are also discussed. What the reader will gain: This review aims to describe how the CSC model is key to developing novel treatments and discusses associated shortcomings and unanswered questions. Take home message: A fresh look at cancer biology and treatment is needed for many incurable cancers to improve clinical prognosis for patients. The CSC model posits a hierarchy in cancer where only a subset of cells drive malignancy, and if features of this model are correct, has implications for development of novel and hopefully more successful approaches to cancer therapy.

MicroRNAs in Cancer: Glioblastoma and Glioblastoma Cancer Stem Cells

MicroRNAs represent an abundant class of endogenously expressed 18-25 nucleotide non-coding RNA molecules that function to silence gene expression through a process of post-transcriptional modification. They exhibit varied and widespread functions during normal development and tissue homeostasis, and accordingly their dysregulation plays major roles in many cancer types. Gliomas are cancers arising from the central nervous system. The most malignant and common glioma is glioblastoma multiforme (GBM), and even with aggressive treatment (surgical resection, chemotherapy, and radiation), average patient survival remains less than 2 years. In this review we will summarize the current findings regarding microRNAs in GBM and the biological and clinical implications of this data.

Introduction to Induced Pluripotent Stem Cells: Advancing the Potential for Personalized Medicine

Induced pluripotent stem (iPS) cell technology has enormous potential to advance medical therapy by personalizing regenerative medicine and creating novel human disease models for research and therapeutic testing. Before this technology is broadly used in the clinic, we must realistically evaluate its disease modeling and therapeutic potential. Recent advances including the use of iPS cells to successfully model spinal muscular atrophy in vitro, as well as new techniques in generating iPS cells with recombinant proteins have accelerated the prospects of iPS cells for clinical use in regenerative therapy. This review explores the development and limitations of iPS cell technology, presents a critical comparison of iPS cells and embryonic stem cells, and discusses potential clinical applications and future research directions.

Glioblastoma cancer stem cells: Biomarker and therapeutic advances

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans. It accounts for fifty-two percent of primary brain malignancies in the United States and twenty percent of all primary intracranial tumors. Despite the current standard therapies of maximal safe surgical resection followed by temozolomide and radiotherapy, the median patient survival is still less than 2 years due to inevitable tumor recurrence. Glioblastoma cancer stem cells (GSCs) are a subgroup of tumor cells that are radiation and chemotherapy resistant and likely contribute to rapid tumor recurrence. In order to gain a better understanding of the many GBM-associated mutations, analysis of the GBM cancer genome is on-going; however, innovative strategies to target GSCs and overcome tumor resistance are needed to improve patient survival. Cancer stem cell biology studies reveal basic understandings of GSC resistance patterns and therapeutic responses. Membrane proteomics using phage and yeast display libraries provides a method to identify novel antibodies and surface antigens to better recognize, isolate, and target GSCs. Altogether, basic GBM and GSC genetics and proteomics studies combined with strategies to discover GSC-targeting agents could lead to novel treatments that significantly improve patient survival and quality of life.

Alkylphosphocholine Analogs for Broad-Spectrum Cancer Imaging and Therapy

Tumor-specific alkylphosphocholine analogs were evaluated as imaging and therapy agents in patients and in animal models of human cancer. A Broad View of Cancer Many consider targeted or molecular imaging to be the optimal way to image cancer. Weichert and colleagues feel differently: Uptake of certain small molecules by all cancer cells can give a broad view of cancer, and perhaps also treat it. These small molecules are alkylphosphocholine (APC) analogs, which are taken up preferentially by cancer cells—as compared to, for example, fibroblasts—via plasma membranes and transported into the cells by lipid rafts. The authors tested the uptake of radiolabeled APC analogs in vitro and in vivo in animals in 57 different spontaneous and transgenic tumors, of both human and rodent origin. Because of the well-established efficacy of radiotherapy, the authors demonstrated that the APC analogs could be used to not only visualize tumors but also kill them. Translating this to cancer patients, Weichert et al. showed preliminary preferential uptake of a radiolabeled APC analog in brain tumors. These broadly applicable imaging and therapeutic APC-based agents have been tested in dozens of different human cancers, and preliminarily in people, and are now well poised for further translation to clinical trials. Many solid tumors contain an overabundance of phospholipid ethers relative to normal cells. Capitalizing on this difference, we created cancer-targeted alkylphosphocholine (APC) analogs through structure-activity analyses. Depending on the iodine isotope used, radioiodinated APC analog CLR1404 was used as either a positron emission tomography (PET) imaging (124I) or molecular radiotherapeutic (131I) agent. CLR1404 analogs displayed prolonged tumor-selective retention in 55 in vivo rodent and human cancer and cancer stem cell models. 131I-CLR1404 also displayed efficacy (tumor growth suppression and survival extension) in a wide range of human tumor xenograft models. Human PET/CT (computed tomography) and SPECT (single-photon emission computed tomography)/CT imaging in advanced-cancer patients with 124I-CLR1404 or 131I-CLR1404, respectively, demonstrated selective uptake and prolonged retention in both primary and metastatic malignant tumors. Combined application of these chemically identical APC-based radioisosteres will enable personalized dual modality cancer therapy of using molecular 124I-CLR1404 tumor imaging for planning 131I-CLR1404 therapy.

Inhibition of Na+-K+-2Cl− cotransporter isoform 1 accelerates temozolomide-mediated apoptosis in glioblastoma cancer cells

The hallmark of apoptosis is a significant reduction in cell volume (AVD) resulting from loss of K⁺_iand Cl^-_i. Loss of cell volume and lowering of ionic strength of intracellular K⁺ and Cl^-occur before any other detectable characteristics of apoptosis. In the present study, temozolomide (TMZ) triggered loss of K⁺_iand Cl^–_iand AVD in primary glioblastoma multiforme (GBM) cancer cells (GC) and GC cancer stem cells (GSC). We hypothesize that Na⁺-K⁺-2Cl^–cotransporter isoform 1 (NKCC1) counteracts AVD during apoptosis in GBM cancer cells by regulating cell volume and Cl^-homeostasis. NKCC1 protein was expressed in both GC and GSC and played an essential role in regulatory volume increase (RVI) in response to hypertonic cell shrinkage and isotonic cell shrinkage. Blocking NKCC1 activity with its potent inhibitor bumetanide abolished RVI. These cells maintained a basal [Cl^–]_i( ∼ 68 mM) above the electrochemical equilibrium for Cl^–_i. NKCC1 also functioned to replenish Cl^–_ilevels following the loss of Cl^–_i. TMZ-treated cells exhibited increased phosphorylation of NKCC1 and its up-stream novel Cl^–/volume-sensitive regulatory kinase WNK1. Inhibition of NKCC1 activity with bumetanide accelerated AVD, early apoptosis, as well as activation of caspase-3 and caspase-8. Taken together, this study strongly suggests that NKCC1 is an essential mechanism in GBM cells to maintain K⁺, Cl^–, and volume homeostasis to counteract TMZ-induced loss of K⁺, Cl^– and AVD. Therefore, blocking NKCC1 function augments TMZ-induced apoptosis in glioma cells.