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RN1-00578-1: Tumor Neovascularization by a Non-angiogenic Mechanism: Plasticity-based Contributions by Brain Stem Cells.

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Every year, 2,040 people in California are afflicted by brain cancer. Glioblastoma multiforme, the most prevalent and aggressive form of brain cancer in adult, is believed to stimulate the formation of new blood vessels which are required for this cancer to progress to malignancy and metastasize to other tissues. New blood vessels are believed to be generated by angiogenesis, the process whereby pre-existing blood vessels form new vascular branches that supply oxygen- and nutrient-deprived tissue. Angiogenesis has classically been proposed to be the predominant mechanism of vascular remodeling in adults, putatively linking angiogenesis to the development of solid tumors, ischemia, autoimmune disorders and Alzheimer disease. Our research focuses on neural stem cells that were originally proposed to differentiate to only neuronal cell types. Unexpectedly, we have found that these stem cells can be diverted away from neural lineages and instead be induced to become blood vessels. Thus, neural stem cells are more flexible or “plastic” than previously believed, expanding the importance of the adult neural stem cell in maintaining the cellular composition and function of brain. Our goal is to test the potential of brain stem cells to promote tumor growth of by forming blood vessels. We will also develop methods to arrest the generation of blood vessels by these stem cells so as to block the tumor growth and expansion. The discovery and continued study of neural stem cell-mediated blood vessel formation could define the neural stem cell as a relevant target of vascular therapy.

Statement of Benefit to California (provided by applicant)

Gliomas, the most prevalent brain cancer in adult 4, are dependent upon the circulatory system to acquire growth-promoting nutrients and to metastasize to breast, lung, colon and skin. Despite the latest therapies directed against the vasculature of grade IV gliomas, an 85% recurrence rate persists 1, as does the dismal median survival of <1.5 years 4 (2). The latest therapies failed, eliciting significant side effects because they target factors common to normal and cancer blood vessel formation. We have obtained evidence that a distinct, brain stem cell-mediated pathway supplies gliomas, but not healthy brain tissue, with blood vessels. The ultimate goal of our research effort is to develop novel therapies which acutely target this stem cell to specifically block the formation and maintenance of brain tumor capillaries.

Review

SYNOPSIS: The ultimate goal of this research effort is to develop novel therapies targeted against formation of aberrant capillaries that sustain brain tumors to understand the cellular and molecular mechanisms by which vascular networks are maintained and remodeled in normal versus cancerous brain. Evidence from the applicant indicates that glioma progenitor cells can convert to endothelial cell (EC)-marker expressing cells in a hypoxic microenvironment typical of the tumor. The research proposed here will test the cancer stem cell hypothesis for high grade malignant gliomas in humans, which are highly vascularized tumors. The applicant wishes to explore the notion that the endothelial cells within malignant gliomas are actually derived from glioma stem cells. The applicant also wishes to explore the idea that the pathway from neural stem cell (NSC) to endothelial cell can be traveled in both directions.

In the first aim, the applicant will characterize the stem cell niche that triggers the ‘conversion’ of neural progenitor cells to vascular cells. The main goal of the experiments is to show that the conversion is not a cell fusion event. The applicant would also like to define signaling pathways and “molecular switches” that modulate the conversion event. For the “molecular switch” line of work the applicant will take both a directed and an unbiased approach. The directed approach will focus on the role of two specific molecular factors. An unbiased approach will use expression profiling of cells cultured with normal oxygen and low oxygen to detect potential candidate genes.

In the second aim, the Principal Investigator (PI) will determine if glioma-derived cancer stem cells contribute to pathologic vascularization of gliomas. Based on prior work, the applicant speculates that glioma stem cells are the sources of tumor neovascularization. Thus, the applicant will define and characterize the subfraction(s) of mouse and human glioma cancer stem cells (CSC) that bring about the cell types and invasive properties of gliomas and determine if these CSCs maintain vasculogenic potential. Mouse glioma models and human glioma tissue will be isolated in collaboration with physicians. Those clones that exhibit CSC characteristics will be screened for vascular (and neural) differentiation potential under hypoxic and normoxic conditions. The conversion of CSCs to ECs and pericytes that incorporate into the vasculature of the tumor mass will be quantified at different time points.

In Specific Aim 3, the applicant asks if the relationship between neural stem cells and endothelial cells is symmetrical. The PI will determine whether ECs can generate NSC under the right environment and whether transforming mutations of the EC-to-NSC pathway can lead instead to cancer stem cell formation. The PI proposes a broad-based attack to address a number of questions that are raised by preliminary results. Fate mapping experiments, expression profiling and other tactics will be used to determine whether endothelial cells can be differentiated into stem cells in vivo and whether they can be targeted by oncogenic transformation to differentiate into glioma stem cells. Other experiments to identify effectors of endothelial cell dedifferentiation are also proposed.

STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: Vascularization is a determinant of the growth and metastasis of solid tumors. Induction of vascular network remodeling by angiogenesis is thought to be the major mechanism for tumor vascularization. The studies seem to be more directly relevant to cancer biology than regenerative medicine, although plasticity of stem cells is of general interest to stem cell science. The strength of this research plan is the interesting scientific premise that the vasculature of malignant gliomas may actually be derived from glioma stem cells rather than from conventional angiogenic mechansims. This notion – if validated by experimental test – would have significant impact on anti-angiogenic strategies for the treatment of malignant glioma. That said, there are some significant flaws in the experimental design that diminish enthusiasm.

Oxygen signaling is coming to be recognized as important for regulation of stem cell fate, but there are still considerable misunderstandings in the biology community about the normal oxygen concentrations at the tissue level in the body. This proposal suffers from some of these misconceptions, that may make the in vitro work difficult to interpret. In Aim 1 the PI states that ‘normoxia’ or atmospheric oxygen conditions are the stimulus for generation of EC and other cells from NSC. Atmospheric oxygen is 21% and most incubators (because of the 5% CO2 taken up for buffering) are 20% oxygen. Arterial blood is 12% oxygen, therefore atmospheric oxygen is physiologically hyperoxic and produces measurable oxidant stress. The oxidant stress signaling is likely the operative issue here and this is not acknowledged. For reference, mean venous oxygen levels are about 5.3%, mean brain tissue oxygen levels across mammalian species normally are about 1.5%. In many parts of the brain 1% oxygen at the tissue level is not hypoxic. This misconception affects numerous areas of the study plan. For example, in the gene expression studies in Aim 1, the truly normoxic condition for gene expression is being missed. Instead the investigators are comparing oxidatively stressed cells (oxygen toxic) to oxygen deprived cells. Similarly, in Aim 3 the PI states that certain oxygen conditions did not induce ECs to dedifferentiate, but nothing like a physiologically normoxic condition is mentioned.

Regarding the design and feasibility of the research plan, the scope of the studies proposed is unrealistically broad, and throughout the application it is difficult to separate what has already been done from things that are proposed for the years ahead. A considerable amount of information that should have been presented in the preliminary results section is instead embedded within the study plan, which has little discussion of controls, potential pitfalls and alternative approaches. The preliminary results did not have figure legends, and thus the significance of results is difficult to judge. Another concern is the reliance on undergraduate researchers for critical procedures to be executed without a clear plan by the PI for supervision.

Much of the work proposed in Aim 1 involves a rat glioma cell line. This line is not well accepted within the neuro-oncology community as a model of human glioma. There may be considerable heterogeneity in human glioma stem cells as there are in virtually all other cancer stem cells. The research would be more compelling if the aims were reversed, and various human stem cell clones isolated and tested for the potential to generate vascular cells. Similarly, the rat glioma cells may be responsive to oxygen conditions in a way that is different from human cells depending on the nature of the mutations that led to transformation.

As tissue sources for Aim 2, the applicant will use commercially available mouse glioma models and human glioma tissue obtained from collaborators. The human studies should be emphasized if the PI has access to tumor samples, and the mouse work minimized, because the heterogeneity of human glioma CSC are the interest. Although the tissue sources for the study seem well thought out, the rest of Aim 2 requires more detail. How will the applicant identify glioma stem cell subtypes that drive vascularization? The strategy for isolating the CSC clones is also unclear. While single cell sorting experiments are described, the markers used for cell sorting are not identified. It is also unlikely that the products of FACS sorting experiments will retain viability and be tumorigenic in in vivo studies. Practical experience from a number of laboratories suggests that these studies will be very difficult.

In Aim 3, the applicant proposes that vascular ECs may represent a cellular precursor for progenitor cells of the CNS. The PI has made the most interesting observation that certain culture conditions reproducibly convert ECs to an NSC-like state. Assuming that this system can be robustly repeated, the PI proposes both gene expression analysis of this differentiation process as well as a focused look at signaling. Is it possible that other characteristics of the culture technique are the cause of the signaling? What else is known that would inform the design of the studies? It is hard to justify such an expensive foray into the differentiation mechanism without more scholarship in support of the research design.

Additional experiments in Aim 3 are presented as a bit of a laundry list and the feasibility and likelihood of success of each experiment is not clear. NSC-like cells will be tested to ascertain contributions to neuronal, astrocyte, ECs and pericyte lineages. The PI will also test whether EC-targeted malignant transformation induces EC dedifferentiation to glioma CSCs. These EC transformation studies would benefit from a control using NSC exposed to the same activators of transformation. The PI will also attempt to identify factors critical to the dedifferentiation of ECs, but does not seem to take into account differences in protein profiles that are relevant to these studies. In addition, s/he will try to define mediators of EC dedifferentiation by gene profiling, and trace the dedifferentiation of endogenous ECs. The idea is a good one but the proposed experiments are not ideal based on the molecular tools that will be used.

QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The PI received a Bachelor’s degree in 1991. After a three-year hiatus as a research technician, the PI enrolled in a graduate program and received a PhD in 2001. The PI conducted postdoctoral research from 2001-2005 where s/he discovered that adult neural stem cells possess a capacity for plasticity. In 2006 the applicant joined the faculty of the applicant institution where s/he now serves as Assistant Professor of Molecular and Cell Biology. The PI’s dissertation research culminated in multiple first-author papers, which appeared in well-regarded journals, and his/her postdoctoral studies culminated in first author publications in top tier journals.

The applicant has given some thought to his career development. A key problem that s/he recognizes at the applicant institution is translating basic science into clinical applications – an important goal of stem cell research. To address this problem, the PI has established a key collaboration at a local hospital. Ultimately the PI envisions a collaborative effort with the hospital to take the form of developing therapeutic approaches which through the deliberate up- or down- regulation of brain stem cell vascular potential will improve patient diagnosis. The applicant states that research support from CIRM would constitute a critical first step in forging a meaningful interaction of research clinicians at the hospital and the applicant institution, bolstering stem cell programs at both institutions. The PI might also consider contacting neurosurgeons at other local institutions for the possibility of obtaining more tumor explants for CSC isolation, and as potential collaborators. Also, other area hospitals are likely to provide collaborators. The PI attends a bimonthly neural stem cell research discussion and plays an integral part in converting a graduate level developmental biology course into a stem cell-focused journal club forum. The PI has been appointed a faculty mentor, and has forged another important relationship with a professor in the applicant institution’s bioengineering department.

INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant institution has a strong stem cell and biology environment. The Chairman of Molecular and Cell Biology has written a letter documenting strong institutional commitment to the candidate. The applicant has received a generous startup package to retrofit his/her research laboratory and has the appropriate environment, equipment, and interactions for conducting the research. The applicant has been given a year’s grace from teaching duties and assigned a faculty mentor to monitor his/her career development. A group of scientists at the applicant institution has informally organized a neural stem cell interest group which is a good addition to the strong intellectual environment. The institutional track record in fostering the career development of its young faculty is exemplary. Recruitments in the area of stem cell biology are continuing with the latest recruit being housed in a new science building.

DISCUSSION: The PI tackles the difficult and important clinical problem of gliomas by attempting to characterize the niche that supports the transition to malignant cells. One reviewer feels that the proposal has some charm. This is a strong investigator from a top-tier university who has done solid work, and there is some validity to the claim that this PI’s earlier work on plasticity came along when the concept was still in dispute. The hypothesis is interesting, but unfortunately it suffers from a fatal flaw. In the studies as proposed, the PI will never attain a truly normoxic state. The stroke-in-a-dish studies have never yielded therapies because investigators used the hyperoxic control, and they never examined the dynamics of oxygen concentrations in the intermediate ranges which are comparable to physiologic oxygen states. Aims 1 and 3 are dependent on oxygen environment, and cancer stem cells often reside in the hypoxic core of a tumor. If the PI wants to isolate glioma cancer stem cells, s/he will not get them unless s/he creates physiologically relevant normoxic conditions. There was some concern that the PI was obtaining gliomas from a pediatric source, which might not yield sufficient number of cases to conduct the study. The PI should look at adult tumors for more samples. Also, the heterogeneity issues will be an enormous problem in assessing samples.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

  • None