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The breakthrough in derivation of human-induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells, including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy, we investigated in this study the tumor tropism of hiPSC-derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC-NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non-migratory hiPSC-NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down-regulated in migratory hiPSC-NSCs. Using nNOS inhibitors and nNOS siRNAs, we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC-NSCs toward cancer cells, and that inhibition of its activity or down-regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell-mediated cancer therapy.
Neural stem/progenitor cells (NSCs) are defined by their ability to self-renew and to differentiate into three fundamental neural lineages, neurons, astrocytes, and oligodendrocytes. These stem cells have found their way into many medical applications, especially in treating brain illnesses where tissues cannot ordinarily regenerate, such as Alzheimer's and Parkinson's diseases. Although endogenous and exogenous NSCs migrate through established migratory routes in a healthy brain, they can also move through atypical migratory routes to reach the tumor microenvironment in the brain of cancer patients (Aboody et al. 2000, 2006, 2008). In in vivo studies on mice, NSCs are observed to surround the tumor border as well as “chasing down” infiltrating tumor cells regardless of NSC injection location and its distance to the tumor. When administrated intravenously, NSCs are also able to migrate to various extra-cranial locations, such as the liver, ovaries, and bone marrow, to target disseminated tumor cells of both neural and non-neural origin (Aboody et al. 2000, 2006; Brown et al. 2003; Yang et al. 2012). These findings have suggested a powerful therapeutic approach of using NSCs to target not only the major tumor mass but also distant tumor outgrowths.
So far different NSCs have been tested in animal tumor models, including NSCs derived from fetal or adult human brain tissues, immortalized human NSC lines, and NSCs derived from human embryonic stem cells (hESCs) (Taupin 2007; Aboody et al. 2008; Kosztowski et al. 2009; Zhao et al. 2012). The great potential of NSCs in cancer therapy emphasizes the necessity for a renewable and steady supply of NSCs for future clinical use. Although NSCs derived from healthy areas of the patient's brain and expanded in vitro for use in autologous transplantation would eliminate a need for immunosuppressive drugs as well as concerns over donor and host compatibility, the risks associated with invasive surgical procedures, such as damaging healthy brain tissue, have limited the clinical application of this approach (Taupin 2007). Furthermore, NSCs derived from these human tissues vary in quality and have limited passaging capacity (Taupin 2007; Aboody et al. 2008; Kosztowski et al. 2009). Although oncogene-mediated immortalization provides a solution to the limited life span of NSCs, this method poses various safety concerns over the transforming and oncogenic potential of the oncogenes used (Taupin 2007; Aboody et al. 2008; Kosztowski et al. 2009).
An alternative cell source for NSC generation is induced pluripotent stem cells (iPSCs) (Takahashi et al. 2007). iPSCs are generated synthetically through the expression of specific transcription factors in differentiated adult somatic cells, creating cells that possess the ability to differentiate into any cell type in the body. Human-induced pluripotent stem cells (hiPSCs) express genes and surface proteins similar to hESCs (Takahashi et al. 2007; Patel and Yang 2010; Wu and Hochedlinger 2011). hESCs have been used to derive NSCs that are similar to naturally occurring NSCs in their genetic make-up, surface markers, and potential to differentiate into various neural cells, yet have the advantage of displaying a larger proliferative capacity (Zhao et al. 2012). hiPSCs could be even more advantageous than embryonic stem cells in generating NSCs as they possess similar characteristics but circumvent the bioethical controversies associated with isolating embryonic stem cells (Nishikawa et al. 2008; Patel and Yang 2010). Moreover, somatic cells isolated from patients could theoretically be reprogrammed into iPSCs that can be used to generate NSCs for autologous transplantation, hence minimizing host versus graft reaction. Likewise, human leukocyte antigen-typed iPSCs could be generated to make iPSC-derived cells that are more patient-specific than other cell sources (Nakatsuji 2010).
Regardless of the NSCs source used for cancer therapy, the migratory capacity of NSCs is crucial for the success in treating disseminated tumors. Cell migration is a highly complex process. The factors that regulate the procedure range from various extracellular environmental cues, cytoskeleton re-structuring after sensing the cues, dynamic regulation of cell attachment and detachment to extracellular matrix to signaling coordination between all these parts to control cell movement (Muller et al. 2006; Aboody et al. 2008; Jurvansuu et al. 2008). A proper understanding of the basic molecular mechanisms of NSC migration toward tumors, especially identification of key cellular regulators of the migration, will have important implications in improving the effectiveness of engineering and employing NSCs as tumor therapy agents. As an initial attempt to address the issue, this study used cDNA microarray technology to analyze gene expression patterns of NSCs collected from in vitro migration assays.
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- Materials and methods
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The ability of malignant tumors to infiltrate in the human body and the ineffectiveness of conventional therapies, such as surgery, radio- and chemotherapy, to eliminate disseminated tumors partially attribute to poor prognosis of these tumors. There is an urgent need to develop novel treatments to overcome the limitations of current cancer therapies. NSCs are able to home in on not just brain tumors but also solid tumors of a non-neural origin (Aboody et al. 2000, 2006; Brown et al. 2003; Yang et al. 2012). Especially, the tumor tropism of NSCs has been utilized for targeted delivery of anti-cancer genes to both major tumor mass and distant tumor outgrowths in animal tumor models. A phase 1 clinical trial of gene therapy for recurrent glioblastoma multiforme using a human NSC cell line to deliver a suicide gene is currently ongoing at the City of Hope Medical Center in Duarte, California. We have previously reported that mouse iPSC-NSCs, hESC-NSCs and hiPSC-NSCs can be used for targeted gene therapy for glioma and breast cancer (Lee et al. 2011; Yang et al. 2012; Zhao et al. 2012). We investigated in this study putative molecular mechanisms underlying tumor tropism of iPSC-NSCs and whether the tumor tropism can be enhanced by chemical drug manipulation.
This study is the first attempt to use microarray-based gene expression profiling technology, a technology that allows gene expression analysis with a high-throughput, to map gene expression profiles in hiPSC-NSCs. Given the fact that limited information is available so far on the molecular mechanisms underlying NSC migration toward tumor, we used Affymetrix high density array chips for analysis of up to 45,000 transcripts in migratory or non-migratory NSCs collected from migration assays toward either human glioma tumor cell-conditioned medium or mouse breast cancer cell-conditioned medium.
Comparing the up- and down-regulated genes in hiPSC-NSCs migrating toward 4T1 and U87 conditioned medium, we identified several commonly down-regulated genes associated with intracellular non-membrane-bounded organelles, suggesting that these genes may be significant in regulating the migration of NSCs toward tumors. Intracellular non-membrane-bounded organelles are organized cellular structures with distinctive morphology and functions but not bounded by lipid bilayer membranes (DAVID, Bioinformatics Resources 6.7). These organelles include chromosomes, ribosomes, and the cytoskeleton. Although general functions of chromosomes and ribosomes are not directly related to cell migration, cytoskeleton dynamics plays important roles in cell migration including NSC migration (Kasper et al. 2009). Of the genes associated with intracellular non-membrane-bounded organelles that were confirmed with qPCR analysis, NOS appears highly interesting, as it has been investigated in previous studies for its functions associated with cell migration.
Nitric oxide (NO) is a radical molecule involved in various physiological and pathophysiological processes. It is synthesized from L-arginine by NOS, which exists in three isoforms based on the tissue source, inducible-NOS, endothelial-NOS and finally nNOS (Moreno-Lopez et al. 2000; Su et al. 2005; Zhang et al. 2007). These three isoforms of NOS are co-localized with the cytoskeleton including microtubules, actin microfilaments, and intermediate filaments directly or indirectly. This association assists NOS in performing their function. Re-organization of cytoskeleton, which can be induced by extracellular signals, will indirectly affect NO production. Thus, altered NO production by cytoskeletal reorganization might be important in physiological and pathophysiological conditions. In the nervous system, nNOS takes part in various functions such as vascular homeostasis, neurotransmission, host defense and angiogenesis (Su et al. 2005).
Several studies have demonstrated a connection between NOS expression and migration of neuronal cells. One such study performed on rats subject to cerebral ischemia indicated a decline in nNOS expression post-ischemia in migrating cells originated from the ependymal and subventricular zone, the regions in the mammalian brain that retain the ability for neurogenesis in adulthood because of the presence of neural stem cells (Zhang et al. 2007). The study also noted a decreased level of nNOS expression in cerebral regions were the migrating cells passed through, suggesting that decreased nNOS expression in the migration routes could be related to the cell migration. These findings were consistent with an earlier study by Moreno-López et al. (2000), in which they found that neural precursor cells did not express nNOS either in proliferation or migration regions of adult mouse brains. Furthermore, endothelial-NOS was found to attenuate pulmonary leukocyte migration via inhibition of endothelial adhesion molecule expression (Kaminski et al. 2004).
Also reported previously, there exists a positive relationship between an increased level of NO and NSC differentiation (Moreno-Lopez et al. 2000; Luo et al. 2010) and NOS inhibition enhances NSC proliferation but decreases their differentiation (Maeda et al. 2000; Ciani et al. 2004; Luo et al. 2010). In our study, older passages of NSCs, which are more differentiated and hence might have a higher level of NO, have a decreased migratory capacity (data not shown). The findings from our NSCs migration study correlate well with our microarray results, supporting a hypothesis that up-regulation of nNOS increases NO production and thus differentiation, which in turn might reduce cytoskeletal dynamics, leading to reduced cell migration. Thus, down-regulation of nNOS would on the other hand, decrease intracellular NO level, acting as an effective way to enhance NSC migration toward tumors.
However, other studies performed on rats have indicated an increased proliferation followed by increased migration of neural precursor cells post-ischemia when NO donor was administrated (Zhang et al. 2001). Publications by Tegenge et al. (2011) have indicated that NO stimulates migration of human neural progenitor cells under healthy conditions via a cGMP-mediated signal transduction pathway by activating the soluble enzyme, guanylyl cyclase using NO (Tegenge and Bicker 2009; Tegenge et al. 2011). These studies also showed that the migration of these cells is blocked by the use of nNOS inhibitors and enhanced by the use of an NO donor. The discrepancies between these studies and those showing increased NSC migration associated with decreased nNOS expression could be explained by the difference between physiological and pathological conditions and/or different types of pathological conditions, the species of biological samples used, as well as the neural precursor cell type, age, and NO source. In a previous report, regulation of neurogenesis by nNOS was found to be bi-directional in a way that neuron-derived nNOS and exogenous NO donors decrease neurogenesis and neuronal differentiation, whereas NSC-derived nNOS enhances neurogenesis (Luo et al. 2010). Hence, altered nNOS source may result in altered functions. Since no previous studies have been conducted to investigate the effects of NO on the migration of human NSCs toward tumors, our findings on nNOS down-regulation in migratory NSCs deserve further scrutiny. An interesting topic for future research would be whether nNOS inhibitors can increase NSCs tumor tropism in vivo.
The generation of hiPSCs starts from adult cells, circumventing the bioethical controversies associated with the derivation of hESCs from human embryos (Narsinh et al. 2011). When somatic cells isolated from patients are re-programmed into iPS cells, transplantation of the differentiated progeny of the generated iPSCs will less likely elicit immune rejection responses in the patients. In the case that a hiPSC bank consisting of various human leukocyte antigen types is established (Tamaoki et al. 2010), iPSCs selected from the cell bank can be alternatively used to generate tumor-targeting NSCs with reduced likelihood of immune-mediated rejection. Furthermore, the approach of using iPSCs to derive NSCs is applicable for large-scale mass manufacture of uniform batches of cellular products that are sufficient for repeated patient treatments. Importantly, this approach will help eliminate variability in the quality of cellular products, thus facilitating reliable comparative analysis of clinical outcomes. Large-scale manufacturing of human cells from hiPSCs, instead of collecting and expanding primary cells from individual patients, will increase cost-effectiveness by reducing the laboriousness and simplifying the logistics of cell culture operations. These NSCs can be further prepared as commercial products in a cryopreserved, ready-to-go format. Hence, iPSC-NSCs hold great potential for cancer treatment.
It should be noted that hiPSCs used in this study were generated by transduction with a polycistronic lentiviral vector containing reprogramming factor genes, Oct4, Klf4, Sox2, and c-Myc genes. This expression cassette is excisable by cre-recombinase (Sommer et al. 2010). Although the presence of the reprogramming factor genes in hiPSCs did not impede neural differentiation to generate tumor-tropic NSCs as shown in this study, excision of the expression cassette could possibly improve the developmental potential of hiPSCs and significantly augments their capacity to undergo directed differentiation (Sommer et al. 2010). More importantly, since several re-programming factors, for example, c-Myc, Klf4, and Oct4, have the potential to induce genomic instability and possess oncogenic activity, great caution should be exercised by removing the expression cassette before therapeutic applications of hiPSC-derived cells.