Joseph Najbauer

The origin of the cancer stem cell: current controversies and new insights

Most tumours are derived from a single cell that is transformed into a cancer-initiating cell (cancer stem cell) that has the capacity to proliferate and form tumours in vivo. However, the origin of the cancer stem cell remains elusive. Interestingly, during development and tissue repair the fusion of genetic and cytoplasmic material between cells of different origins is an important physiological process. Such cell fusion and horizontal gene-transfer events have also been linked to several fundamental features of cancer and could be important in the development of the cancer stem cell.

Stem and progenitor cell-mediated tumor selective gene therapy

The poor prognosis for patients with aggressive or metastatic tumors and the toxic side effects of currently available treatments necessitate the development of more effective tumor-selective therapies. Stem/progenitor cells display inherent tumor-tropic properties that can be exploited for targeted delivery of anticancer genes to invasive and metastatic tumors. Therapeutic genes that have been inserted into stem cells and delivered to tumors with high selectivity include prodrug-activating enzymes (cytosine deaminase, carboxylesterase, thymidine kinase), interleukins (IL-2, IL-4, IL-12, IL-23), interferon-β, apoptosis-promoting genes (tumor necrosis factor-related apoptosis-inducing ligand) and metalloproteinases (PEX). We and others have demonstrated that neural and mesenchymal stem cells can deliver therapeutic genes to elicit a significant antitumor response in animal models of intracranial glioma, medulloblastoma, melanoma brain metastasis, disseminated neuroblastoma and breast cancer lung metastasis. Most studies reported reduction in tumor volume (up to 90%) and increased survival of tumor-bearing animals. Complete cures have also been achieved (90% disease-free survival for >1 year of mice bearing disseminated neuroblastoma tumors). As we learn more about the biology of stem cells and the molecular mechanisms that mediate their tumor-tropism and we identify efficacious gene products for specific tumor types, the clinical utility of cell-based delivery strategies becomes increasingly evident.

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.

Neural stem cell tropism to glioma: critical role of tumor hypoxia.

Hypoxia is a critical aspect of the microenvironment in glioma and generally signifies unfavorable clinical outcome. Effective targeting of hypoxic areas in gliomas remains a significant therapeutic challenge. New therapeutic platforms using neural stem cells (NSC) for tumor-targeted drug delivery show promise in treatment of cancers that are refractory to traditional therapies. However, the molecular mechanisms of NSC targeting to hypoxic tumor areas are not well understood. Therefore, we investigated the role of hypoxia in directed migration of NSCs to glioma and identified the specific signaling molecules involved. Our data showed that hypoxia caused increased migration of human HB1.F3 NSCs to U251 human glioma-conditioned medium in vitro. In HB1.F3 NSCs, hypoxia led to up-regulation of CXCR4, urokinase-type plasminogen activator receptor (uPAR), vascular endothelial growth factor receptor 2 (VEGFR2), and c-Met receptors. Function-inhibiting antibodies to these receptors inhibited the migration of HB1.F3 cells to glioma-conditioned medium. Small interfering RNA knockdown of hypoxia-inducible factor-1α in glioma cells blocked the hypoxia-induced migration of NSCs, which was due to decreased expression of stromal cell–derived factor-1 (SDF-1), uPA, and VEGF in glioma cells. Our in vivo data provided direct evidence that NSCs preferentially distributed to hypoxic areas inside intracranial glioma xenografts, as detected by pimonidazole hypoxia probe, as well as to the tumor edge, and that both areas displayed high SDF-1 expression. These observations indicate that hypoxia is a key factor in determining NSC tropism to glioma and that SDF-1/CXCR4, uPA/uPAR, VEGF/VEGFR2, and hepatocyte growth factor/c-Met signaling pathways mediate increased NSC-to-glioma tropism under hypoxia. These results have significant implications for development of stem cell–mediated tumor-selective gene therapies. (Mol Cancer Res 2008;6(12):1819–29)

Tumor-Targeted Enzyme/Prodrug Therapy Mediates Long-term Disease-Free Survival of Mice Bearing Disseminated Neuroblastoma

Neural stem cells and progenitor cells migrate selectively to tumor loci in vivo. We exploited the tumor-tropic properties of HB1.F3.C1 cells, an immortalized cell line derived from human fetal telencephalon, to deliver the cDNA encoding a secreted form of rabbit carboxylesterase (rCE) to disseminated neuroblastoma tumors in mice. This enzyme activates the prodrug CPT-11 more efficiently than do human enzymes. Mice bearing multiple tumors were treated with rCE-expressing HB1.F3.C1 cells and schedules of administration of CPT-11 that produced levels of active drug (SN-38) tolerated by patients. Both HB1.F3.C1 cells and CPT-11 were given i.v. None of the untreated mice and 30% of mice that received only CPT-11 survived long term. In contrast, 90% of mice treated with rCE-expressing HB1.F3.C1 cells and 15 mg/kg CPT-11 survived for 1 year without detectable tumors. Plasma carboxylesterase activity and SN-38 levels in mice receiving both rCE-expressing HB1.F3.C1 cells (HB1.F3.C1/AdCMVrCE) and CPT-11 were comparable with those in mice receiving CPT-11 only. These data support the hypothesis that the antitumor effect of the described neural stem/progenitor cell-directed enzyme prodrug therapy (NDEPT) is mediated by production of high concentrations of active drug selectively at tumor sites, thereby maximizing the antitumor effect of CPT-11. NDEPT approaches merit further investigation as effective, targeted therapy for metastatic tumors. We propose that the described approach may have greatest use for eradicating minimum residual disease.

Olfactory experience modulates apoptosis in the developing olfactory bulb

Early sensory stimulation plays a key role in shaping the structure and function of the developing olfactory system. Here, we provide the first direct evidence for apoptotic cell death in the olfactory bulbs of rat pups during normal development and we also demonstrate that olfactory deprivation by unilateral naris occlusion causes a dramatic increase in apoptotic cell death in the glomerular and granule cell layers of the deprived bulb. The accessory olfactory bulbs displayed a remarkably high basal level of apoptosis but the occluded accessory bulb did not differ in that regard from the control accessory bulb. These results suggest that apoptosis may be an important mechanism by which the olfactory system can adjust its cell numbers in response in sensory stimuli experienced in early life, thereby underlying one form of plasticity in the developing olfactory system.

Neural Stem Cell–Mediated Enzyme/Prodrug Therapy for Glioma: Preclinical Studies

Neural stem cells home to gliomas in mice where they convert a prodrug to 5-fluorouracil, leading to tumor regression. Cellular Assassins Derived from the supporting cells of the brain, gliomas are deadly tumors that can be only temporarily held at bay, but not cured. New ways to treat these cancers are needed. To get regulatory approval to test a new stem cell–based therapy in patients, Aboody et al. performed a series of preclinical experiments in mice with artificially implanted gliomas in their brains. By mimicking closely the treatments that they hoped to perform in humans, these authors were able to show to the satisfaction of the regulatory agency that the treatment was safe and effective enough in the mice to warrant a first-in-human trial in patients. The authors used a neural stem cell line carrying a v-myc gene and a gene for cytosine deaminase. These cells exhibit tropism to human glioma cells. When injected into mice with gliomas, they migrate to the site of the tumor, even when the mice are treated with steroids or radiation, as might be the case for human patients. The cytosine deaminase in the cells provides another anticancer weapon. This enzyme converts the prodrug 5-fluorocytosine (5-FC) to the toxic 5-flurouracil (5-FU), delivering a high concentration of the therapeutic agent directly in and around the tumor and causing it to shrink significantly. Injection of excess numbers of cells or increasing the dose of 5-FU did not result in any abnormalities in the animals; in fact, by 12 weeks after injection, no cells were to be seen in the brain or elsewhere, even when a highly sensitive polymerase chain reaction method was used to look for the v-myc DNA. This targeted cell-based approach to cancer therapy that concentrates the therapeutic agent in the vicinity of the tumor is expected to reduce toxicity to other tissues. Thus, a higher local dose is possible, potentially improving efficacy against the tumor. The phase 1 trial derived from these preclinical results is ongoing; its end will allow evaluation of how well these preclinical in vivo studies set the stage for humans. High-grade gliomas are extremely difficult to treat because they are invasive and therefore not curable by surgical resection; the toxicity of current chemo- and radiation therapies limits the doses that can be used. Neural stem cells (NSCs) have inherent tumor-tropic properties that enable their use as delivery vehicles to target enzyme/prodrug therapy selectively to tumors. We used a cytosine deaminase (CD)–expressing clonal human NSC line, HB1.F3.CD, to home to gliomas in mice and locally convert the prodrug 5-fluorocytosine to the active chemotherapeutic 5-fluorouracil. In vitro studies confirmed that the NSCs have normal karyotype, tumor tropism, and CD expression, and are genetically and functionally stable. In vivo biodistribution studies demonstrated NSC retention of tumor tropism, even in mice pretreated with radiation or dexamethasone to mimic clinically relevant adjuvant therapies. We evaluated safety and toxicity after intracerebral administration of the NSCs in non–tumor-bearing and orthotopic glioma–bearing immunocompetent and immunodeficient mice. We detected no difference in toxicity associated with conversion of 5-fluorocytosine to 5-fluorouracil, no NSCs outside the brain, and no histological evidence of pathology or tumorigenesis attributable to the NSCs. The average tumor volume in mice that received HB1.F3.CD NSCs and 5-fluorocytosine was about one-third that of the average volume in control mice. On the basis of these results, we conclude that combination therapy with HB1.F3.CD NSCs and 5-fluorocytosine is safe, nontoxic, and effective in mice. These data have led to approval of a first-in-human study of an allogeneic NSC-mediated enzyme/prodrug-targeted cancer therapy in patients with recurrent high-grade glioma.

Development of a Tumor-Selective Approach to Treat Metastatic Cancer

Background Patients diagnosed with metastatic cancer have almost uniformly poor prognoses. The treatments available for patients with disseminated disease are usually not curative and have side effects that limit the therapy that can be given. A treatment that is selectively toxic to tumors would maximize the beneficial effects of therapy and minimize side effects, potentially enabling effective treatment to be administered. Methods and Findings We postulated that the tumor-tropic property of stem cells or progenitor cells could be exploited to selectively deliver a therapeutic gene to metastatic solid tumors, and that expression of an appropriate transgene at tumor loci might mediate cures of metastatic disease. To test this hypothesis, we injected HB1.F3.C1 cells transduced to express an enzyme that efficiently activates the anti-cancer prodrug CPT-11 intravenously into mice bearing disseminated neuroblastoma tumors. The HB1.F3.C1 cells migrated selectively to tumor sites regardless of the size or anatomical location of the tumors. Mice were then treated systemically with CPT-11, and the efficacy of treatment was monitored. Mice treated with the combination of HB1.F3.C1 cells expressing the CPT-11-activating enzyme and this prodrug produced tumor-free survival of 100% of the mice for >6 months (P<0.001 compared to control groups). Conclusions The novel and significant finding of this study is that it may be possible to exploit the tumor-tropic property of stem or progenitor cells to mediate effective, tumor-selective therapy for metastatic tumors, for which no tolerated curative treatments are currently available.

Identification of uPAR-positive chemoresistant cells in small cell lung cancer.

Background The urokinase plasminogen activator (uPA) and its receptor (uPAR/CD87) are major regulators of extracellular matrix degradation and are involved in cell migration and invasion under physiological and pathological conditions. The uPA/uPAR system has been of great interest in cancer research because it is involved in the development of most invasive cancer phenotypes and is a strong predictor of poor patient survival. However, little is known about the role of uPA/uPAR in small cell lung cancer (SCLC), the most aggressive type of lung cancer. We therefore determined whether uPA and uPAR are involved in generation of drug resistant SCLC cell phenotype. Methods and Findings We screened six human SCLC cell lines for surface markers for putative stem and cancer cells. We used fluorescence-activated cell sorting (FACS), fluorescence microscopy and clonogenic assays to demonstrate uPAR expression in a subpopulation of cells derived from primary and metastatic SCLC cell lines. Cytotoxic assays were used to determine the sensitivity of uPAR-positive and uPAR-negative cells to chemotherapeutic agents. The uPAR-positive cells in all SCLC lines demonstrated multi-drug resistance, high clonogenic activity and co-expression of CD44 and MDR1, putative cancer stem cell markers. Conclusions These data suggest that uPAR-positive cells may define a functionally important population of cancer cells in SCLC, which are resistant to traditional chemotherapies, and could serve as critical targets for more effective therapeutic interventions in SCLC.

Expression of active caspase-3 in mitotic and postmitotic cells of the rat forebrain

Active caspase‐3 immunoreactivity was detected in the rat forebrain proliferative regions at birth and remained high in these areas for about 2 weeks, during which period labeled cells were present centroperipherally across the olfactory bulb. By the end of the third postnatal week, only a small number of immunolabeled cells remained in these forebrain structures. Active caspase‐3 immunolabeling was localized mostly to cell nuclei and co‐localized partially with TuJ1 and NeuN immunoreactivity, but not with glial fibrially acidic protein, OX‐42, γ‐aminobutyric acid, or terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL)‐positive labeling. Active caspase‐3 and 5‐bromo‐2′‐deoxyuridine (BrdU) double‐labeled nuclei were seen in the proliferative regions after 2 hours and in the periglomerular region of the bulb after 7 days following BrdU injections. Examination of the cells with electron microscopy confirmed that the active caspase‐3‐containing nuclei in the proliferative regions often had infoldings and appeared to be undergoing division. Some of the cells with active caspase‐3‐labeled nuclei in the bulb had synapses on their somata or dendrites. Labeled dendritic spines and a few axon terminals were also observed in the olfactory bulb. Taken together, it appears that a wave of active caspase‐3‐positive cells are dividing in the proliferative zones and then migrating to the bulb as they differentiate into neurons. Therefore, active caspase‐3 may play a role in cellular processes such as neuronal differentiation, migration, and plasticity, in addition to its role in cell death. J. Comp. Neurol. 433:4–22, 2001.