Oleg Suslov

Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro

Neural stem cells from neurogenic regions of mammalian CNS are clonogenic in an in vitro culture system exploiting serum and anchorage withdrawal in medium supplemented with methyl cellulose and the pleiotropic growth factors EGF, FGF2, and insulin. The aim of this study was to test whether cortical glial tumors contain stem‐like cells capable, under this culture system, of forming clones showing intraclonal heterogeneity in the expression of neural lineage‐specific proteins. The high frequencies of clone‐forming cells (about 0.1–10 × 10−3) in clinical tumor specimens with mutated p53, and in neurogenic regions of normal human CNS, suggest that the ability to form clones in this culture system is induced epigenetically. RT‐PCR analyses of populations of normal brain‐ and tumor‐derived sister clones revealed transcripts for nestin, neuron‐specific enolase, and glial fibrillary acidic protein (GFAP). However, the tumor‐derived clones were different from clones derived from neurogenic regions of normal brain in the expression of transcripts specific for genes associated with neural cell fate determination via the Notch‐signaling pathway (Delta and Jagged), and cell survival at G2 or mitotic phases (Survivin). Moreover, the individual glioma‐derived clones contain cells immunopositive separately for GFAP or neuronal β‐III tubulin, as well as single cells coexpressing both glial and neuronal markers. The data suggest that the latent critical stem cell characteristics can be epigenetically induced by growth conditions not only in cells from neurogenic regions of normal CNS but also in cells from cortical glial tumors. Moreover, tumor stem‐like cells with genetically defective responses to epigenetic stimuli may contribute to gliomagenesis and the developmental pathological heterogeneity of glial tumors. GLIA 39:193–206, 2002.

Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres

Neural stem cells (NSCs) in vitro are able to generate clonal structures, “neurospheres,” that exhibit intra-clonal neural cell-lineage diversity; i.e., they contain, in addition to NSCs, neuronal and glial progenitors in different states of differentiation. The present study focuses on a subset of neurospheres derived from fresh clinical specimens of human brain by using an in vitro system that relies on particular growth factors, serum, and anchorage withdrawal. Thirty individual and exemplary cDNA libraries from these neurosphere clones were clustered and rearranged within a panel after characterization of differentially expressed transcripts. The molecular phenotypes that were obtained indicate that clonogenic NSCs in our in vitro system are heterogeneous, with subsets reflecting distinct neural developmental commitments. This approach is useful for the sorting and expansion of NSCs and facilitates the discovery of genes involved in cell proliferation, communication, fate control, and differentiation.

The ZEB1 pathway links glioblastoma initiation, invasion and chemoresistance

Glioblastoma remains one of the most lethal types of cancer, and is the most common brain tumour in adults. In particular, tumour recurrence after surgical resection and radiation invariably occurs regardless of aggressive chemotherapy. Here, we provide evidence that the transcription factor ZEB1 (zinc finger E‐box binding homeobox 1) exerts simultaneous influence over invasion, chemoresistance and tumourigenesis in glioblastoma. ZEB1 is preferentially expressed in invasive glioblastoma cells, where the ZEB1‐miR‐200 feedback loop interconnects these processes through the downstream effectors ROBO1, c‐MYB and MGMT. Moreover, ZEB1 expression in glioblastoma patients is predictive of shorter survival and poor Temozolomide response. Our findings indicate that this regulator of epithelial‐mesenchymal transition orchestrates key features of cancer stem cells in malignant glioma and identify ROBO1, OLIG2, CD133 and MGMT as novel targets of the ZEB1 pathway. Thus, ZEB1 is an important candidate molecule for glioblastoma recurrence, a marker of invasive tumour cells and a potential therapeutic target, along with its downstream effectors.

PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency

This study addresses the problem of PCR inhibition by reverse transcriptase. It has been shown that the inhibition occurs mostly when a small amount of RNA is taken for RT reaction, and it is more visible for rarely expressed transcripts. We show here that the inhibition takes place regardless of what amount of template is utilized for RT. The inhibition possesses a global nature, i.e. the amplification of any given transcript may be compromised with different levels of inhibition. The process of inhibition also explains wrongfully derived PCR amplification efficiencies, sometimes more than 100%, when the sequential dilutions of unpurified RT sample are utilized to build the calibration curve. The RT influences PCR not only by inhibiting it. When microgram(s) of RNA are taken for RT reaction, reverse transcriptase may cause overamplification of some transcripts under certain PCR conditions. The possible mechanism of RT influence on PCR is presented, and a purification method is implemented to remove the effects of RT on PCR.

A Supplemented High-Fat Low-Carbohydrate Diet for the Treatment of Glioblastoma

Purpose: Dysregulated energetics coupled with uncontrolled proliferation has become a hallmark of cancer, leading to increased interest in metabolic therapies. Glioblastoma (GB) is highly malignant, very metabolically active, and typically resistant to current therapies. Dietary treatment options based on glucose deprivation have been explored using a restrictive ketogenic diet (KD), with positive anticancer reports. However, negative side effects and a lack of palatability make the KD difficult to implement in an adult population. Hence, we developed a less stringent, supplemented high-fat low-carbohydrate (sHFLC) diet that mimics the metabolic and antitumor effects of the KD, maintains a stable nutritional profile, and presents an alternative clinical option for diverse patient populations. Experimental Design: The dietary paradigm was tested in vitro and in vivo, utilizing multiple patient-derived gliomasphere lines. Cellular proliferation, clonogenic frequency, and tumor stem cell population effects were determined in vitro using the neurosphere assay (NSA). Antitumor efficacy was tested in vivo in preclinical xenograft models and mechanistic regulation via the mTOR pathway was explored. Results: Reducing glucose in vitro to physiologic levels, coupled with ketone supplementation, inhibits proliferation of GB cells and reduces tumor stem cell expansion. In vivo, while maintaining animal health, the sHFLC diet significantly reduces the growth of tumor cells in a subcutaneous model of tumor progression and increases survival in an orthotopic xenograft model. Dietary-mediated anticancer effects correlate with the reduction of mTOR effector expression. Conclusions: We demonstrate that the sHFLC diet is a viable treatment alternative to the KD, and should be considered for clinical testing. Clin Cancer Res; 22(10); 2482–95. ©2015 AACR.

Production of Neurospheres from CNS Tissue

The relatively recent discovery of persistent adult neurogenesis has led to the experimental isolation and characterization of central nervous system neural stem cell populations. Protocols for in vitro analysis and expansion of neural stem cells are crucial for understanding their properties and defining characteristics. The methods described here allow for cell and molecular analysis of individual clones of cells--neurospheres--derived from neural stem/progenitor cells. Neurospheres can be cultivated from a variety of normal, genetically altered, or pathological tissue specimens, even with protracted postmortem intervals, for studies of mechanisms underlying neurogenesis, cell fate decisions, and cell differentiation. Neurosphere-forming cells hold great promise for the development of cell and molecular therapeutics for a variety of neurological diseases.

Chapter 15 Boundary molecules during brain development, injury, and persistent neurogenesis - in vivo and in vitro studies

Publisher Summary In this chapter, three model systems have been described that exemplify the presence and potential biological functions of boundary extracellular matrix (ECM) molecules. Even though these developmentally regulated proteins, sometimes referred to as recognition molecules, are often uniformly expressed in low-levels throughout the developing neuraxis, their dense concentration in so-called boundary regions alerted to their possible functions as interfaces between different brain structures or units. This cordone hypothesis is also consistent with the observed upregulation of boundary molecules in association with the glial scar, and bioassays of such molecules in the astroglial scar reveal their complex interactions with numerous other molecules that can both encourage or deter neuronal adhesion and neurite growth. The persistent expression of boundary ECM molecules in the neurogenic subependymal zone (SEZ) may prove to be the most insightful model for studying the many roles of these molecules. Because cells are born, migrate, and die in relation to an enhanced ECM expression in vivo in the rostral migratory stream, as well as the inferred role of ECM in the generation of novel proliferative spheres in vitro , future studies need to focus on the patterns of gene expression of SEZ-derived stem and precursor cells in relation to ECM expression in order to establish their roles during the proliferation and specification of neurons and glia.

Abstract 4308: ZEB1 mediates invasion and chemoresistance of glioblastoma

Despite intense efforts in basic research and clinical medicine, glioblastoma (GBM) remains one of the most lethal types of cancer. In particular, tumor recurrence after surgical resection, aggressive chemotherapy, and targeted radiation remains an insurmountable obstacle. Recurrence has been attributed to residual cancer cells that re-initiate tumor growth after primary clinical intervention. Here, we provide new evidence that a subpopulation of invasive and chemoresistant cancer cells is maintained by the transcription factor ZEB1 (zinc finger E-box binding homeobox 1). ZEB1 is preferentially expressed in invasive glioblastoma cells, and its knockdown results in a dramatic reduction of tumor invasion as well as increased sensitivity to the chemotherapeutic agent Temozolomide (Temodar®, TMZ) in vitro and in vivo. We find that ZEB1 indirectly controls expression of the chemoresistance-mediating enzyme MGMT (O-6-Methylguanine DNA Methyltransferase), as well as cell-cell adhesion pathways, thus linking chemoresistance and brain tumor invasion. Moreover, ZEB1 expression in glioblastoma patients correlates with tumor grade and survival. These results indicate that invasive glioblastoma cells are particularly sheltered from current therapeutic approaches, rendering them likely candidates for tumor recurrence. This offers a potential novel model for GBM recurrence, and a potential new therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4308. doi:1538-7445.AM2012-4308

Abstract 3317: Single-cell invasion and chemoresistance of slowly proliferating brain-tumor stem cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Despite intense efforts in basic research and clinical medicine, glioblastoma (GBM) remains one of the most lethal types of cancer. In particular, tumor recurrence after surgical resection, aggressive chemotherapy, and targeted radiation remains an insurmountable obstacle. Recurrence may be attributed to residual cancer stem cells that re-initiate tumor growth after primary clinical intervention. Here, we provide new evidence for a subpopulation of relatively quiescent and chemoresistant cancer stem cells that invade deeply into the parenchyma. We have identified a critical transcription factor that is upregulated in these glioma stem cells, which regulates epithelial-mesenchymal transition (EMT) in other tumors. Knockdown of this transcription factor results in an increased sensitivity to chemotherapeutic agents and reduced tumor cell invasion. We hypothesize that single cell invasion of malignant gliomas is regulated by similar molecular pathways as EMT and metastasis in solid tissue tumors. These pathways allow slowly proliferating glioma stem cells to leave the site of the primary tumor, invade deeply into the surrounding parenchyma and, by virtue of their resistance to chemotherapeutic agents, evade all types of classical brain tumor therapy. This offers a potential novel model for GBM recurrence, and a potential new therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3317. doi:10.1158/1538-7445.AM2011-3317

Neural Stem/Progenitor Cell Clones or “Neurospheres”

Stem cell biology has contributed an impressive list of new and important findings in the last decade that are anticipated to lead to potentially powerful therapeutics for debilitating human diseases. The generation of human embryonic stem cell (ES cell) lines (1,2) is among this list of crucial technological breakthroughs, not to mention the applications of genetic, cell, and molecular biology to the first-time cloning of an entire organism (3) that has since been achieved in numerous species, including the mouse (4). Utilizing insights and approaches from these fields, as well as from developmental biology, the field of developmental neurobiology has been astonished in recent years by numerous “reversals of dogma” related to neurogenesis in the mature mammalian brain [defined as the generation of neurons, or the shortened form of “neuronogenesis,” versus “gliogenesis,” which is the production of astroglia and oligodendroglia; “neuromorphogenesis” is the combined events of neurogenesis and gliogenesis that ultimately generate a nervous system (5–8)]. In particular, despite the work of Allen (9), Altman and Das (10),and others supporting the existence of persistent neurogenesis in the adult rat olfactory and hippocampal systems, these were considered highly specialized cases that by no means supported a notion of neuropoiesis (persistent neurogenesis) in the adult central nervous system. The in vitro propagation of a putative stem cell population from the adult rat brain by the Weiss and Bartlett groups (11,12) suggested that there may be neuropoiesis; studies that followed have established the source of these stem/progenitor cells [a term used because it not only sidesteps the contentious issue of sternness (5,8,13) of these cells, but also because it encompasses the entire spectrum of proliferative neurogenic cells that can generate all cells in the nervous system] as the subependymal zone, ependyma, and hippocampus, even within the aged human brain (6, 14–16). It is possible to clone stem/progenitor cells from these adult brain regions (7) and even from cadaver specimens (17,18) with surprisingly long postmortem intervals [up to 5 d from cadaver specimens (17)]. For the purpose of discussion, these persistently neurogenic regions have been amalgamated under one term—“brain marrow” (8, 14,19). The analogy of a brain neuropoietic core to the hematopoietic bone marrow has been substantiated by the recent surprising finding that adult brain-derived stem/progenitor cells are considerably more pluripotent than ever expected [e.g., giving rise to blood cells after homing to bone marrow following systemic grafting (20), as well as to muscle (21,22) and even multiple organ systems (23)]; it also has recently been shown that non-neural stem cells can also be coaxed into nerve cell phenotypes following different neuralizing conditions (24–27). However, to date, most transplant or other in vivo studies of stem/progenitor cells derived from brain marrow suggest that these cells have a rather limited fate potential in the central nervous system (CNS) [e.g., glia (28)]—but also giving rise to granule cells and other interneuron populations although fetal neural stem cells and immortalized neural stem cell lines do appear to be more plastic and potent, incorporating into a variety of brain circuits (29–31).