A novel and generalizable organotypic slice model to evaluate stem cell potential for targeting pediatric brain tumors
New Faculty I
In children, cancers are the deadliest of diseases and second only to accidents as the leading cause of death. Cancers of the brain are the worst. Our current forms of therapy for these diseases can best be described as brutal: brain surgery followed by administration of very high doses of very toxic drugs and exposure to high doses of radiation. The deadliest of the brain cancers are the malignant gliomas. All children with this type of cancer die and in all cases the course of the disease and its treatment are horrific. About two-thirds of children can survive the rest of the types of brain cancers but two-thirds of these survivors go on to have a recurrence of their cancer. Even more heartbreaking is the fact that those that do survive are usually left with lifelong disabilities. It is clear that a new medical approach to brain cancers is needed. Importantly, stem cells appear to be able to provide the basis for a new approach. That is, stem cells, because of their seemingly natural ability to seek out diseased tissue, could be used to deliver therapy directly to the cancer, lessening the reliance on the harsh treatment methods that we currently use, and increasing the chance of eliminating the cancer once and for all. It is obvious that medical research on stem cells for the treatment of brain cancers is an important and necessary endeavor. The problem is that it is likely that, by doing this research in the usual fashion, it is going to take a very long time to come up with new therapies, perhaps decades. The usual fashion includes using animal models to test each therapeutic variation, each possible way of using different stem cells to treat different brain cancers. An additional and very troublesome problem is that once stem cells are introduced into the animal, they become very difficult to track as they blend in with the animal tissue, since they are not an organ and they are very small. So it becomes difficult to say, exactly, where the cells are going and what the cells are doing. What we suggest in this grant proposal is that there is a new and much more efficient way to do this. By using living slices of rat brain that we can keep alive in the laboratory for many weeks, we can introduce stem cells into to these living “brains in a dish” and then watch the cells’ every move. In this way, we can try many variations on a theme in a relatively short period of time before actually moving into experiments in the whole animal. This allows us to much more quickly identify and abandon techniques that aren’t working and arrive at techniques that do. This also allows us to use far fewer animals for this type of research. Importantly, we believe that this approach to medical research lends itself to applications for a wide variety of other brain diseases and diseases of other organs of the body as well.
Statement of Benefit to California:
The malignant brain cancers comprise the leading cause of cancer death. Three decades of research have afforded little to allow us to change the outcome in these lethal brain cancers. For example, virtually all patients die after being diagnosed a diffuse brainstem glioma. Of the two-thirds of patients who survive at least 5 years after being diagnosed with any brain cancer, more than two-thirds go on to have a recurrence of their disease. Moreover, the treatments that these patients suffer can only be described as brutal and most of those that do survive are left with life-long consequences of the disease and its treatment, including disturbances in mental abilities, movement, and hormonal balance, which prevent them from re-entering the work force or, in the case of children, from ever entering the work force. Overall estimates of the incidence of brain cancers in the United States show that about 20,000 will be diagnosed annually with about 2500 in California. The costs for the patient and family cannot be overestimated and should be clear, given the statistics above. The economic costs are also grim: repeated use of physician, inpatient, outpatient and laboratory services as well as lost future earnings and occurrence of secondary diseases incur costs of more than 1.5 billion dollars annually in California alone. It is clear that California patients with brain cancers need a new therapeutic approach. One promising approach is to use stem cell’s natural ability to seek out diseased cells, such as cancer cells. Since part of our current therapeutic approach involves administering very large amounts of very toxic drugs and radiation, we may be able to use stem cells to deliver these drugs directly, and only, to the cancer, thus sparing the rest of the body and brain of the damaging effects of these drugs. This not only would spare the patient from the ravaging effects of the treatment but also allow much higher effective drug concentrations at the cancer itself, resulting in a much more efficient destruction of the cancer cells. Unfortunately, there is not, at present, a comprehensive and efficient way of assessing whether or not the stem cell can adequately perform this particular function in the living animal, which types of cancers might be best suited to this approach, when, in the cancer progression, it is best to institute this type of treatment, and whether or not repeated treatments might be appropriate. The work described in this grant proposal is based on using a novel “brain-in-a-dish” method to answer these very questions. Given the applicant’s extensive training, the participation of established stem cell and clinical collaborators, and the demonstration of competence by way of work that has already been done, it is expected that the work will promote new clinical therapies for the brain cancers and will pave the way for using the techniques described for devising therapies for other brain diseases and injuries as well.
SYNOPSIS: The clinical vehicle for this proposal is malignant glioma. Conventional therapeutic strategies are unsuccessful for treating this cancer, and new tactics are required. One emerging strategy is to use the tumor tracking capacity inherent in many stem cell populations to deliver therapeutic agents to the brain cancer cells. The objective of this applicant's proposal is to define activation states for optimizing tumor targeting behaviors. The study plan has three specific aims: Aim one is simply to carry on with current activities in the applicant's laboratory aimed at isolating and characterizing stem cells from pediatric brain tumors. Aim two is to program these stem cell populations to target brain tumors. The applicant will focus on the therapeutic activation state, which he defines as the combinatorial presence of molecular markers (chemotactic receptors, metallo proteases) and biological functions, (migration and homing capacity). The applicant proposes to culture stem cells in a three-dimensional extracellular matrix, or in rat brain slices. He will challenge these cells with a wide range of growth factors and cytokines to identify agents that induce matrix remodeling activities. The applicant also proposes to manipulate components of the three dimensional extracellular matrix in his stem cell cultures to identify proteins necessary for optimal therapeutic activation. The third specific aim is to standardize and optimize a potentially high throughput platform for stem cell programming. Towards this end, the applicant touts a micro fabricated fluidic chamber system that overcomes the technical limits of other chemotaxis assays. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The strength of this study plan is that it addresses an important clinical problem. Maligant gliomas are, for all practical purposes, incurable and new therapeutic approaches are desperately needed. That said, the work proposed here does not inspire confidence that this project will significantly advance the field. The study plan is descriptive in flavor and unrealistically broad in scope. The level of innovation is low. Specific concerns with the feasibility of the scientific plan are detailed. The studies described for aim one involve 5 extracellular matrix components and 9 different growth factor/chemokines used in combinatorial fashion. In other studies for aim one the applicant will test for activation of signal transduction pathways that mediate the response to the growth factors/chemokines/matrix components outlined above. The scope of this work is similarly broad. Similarly, the work in the third Aim seems descriptive in tone and unrealistically broad in scope. The clinical translational potential seems minimal. The research plan presented here lacks insight into the long term goal (eg. the therapeutic value of the research) and is mired with vague experiments. The stem cell populations to be studied are treated as a single entity despite their diversity (banked neural and mesenchymal stem cells, human ES cells) and the readouts on the ex vivo experiments are addressed very superficially. The time line is unrealistic, as is the personnel/effort planned. Overall, even if such a research plan were executed, its impact and benefit remain unclear. The applicant needs to address the relevance, pros and cons of implanting patients with the activated stem cells in a more precise manner. What is the fate of the stem cells? Will they proliferate once in vivo? Will they lose their "activation" status? Can they eliminate tumor cells or do they simply "home" in on the tumor cells? Where are his controls? Is the described therapeutic activation truly superior to the cells' innate ability to track tumors? While the applicant is not expected to address all of these issues in his 5 year plan, their absence from the discussion and merit evaluations he presents are indicative of poor insight into the planned work. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant received his PhD from Mount Sinai School of Medicine in 1994. He conducted postdoctoral research at MIT and Harvard from 1994 through 1998. This formal scientific training was followed by a seven-year hiatus in industry. In 2005 he joined the Children's Hospital of Orange County Research Institute as a senior scientist. Curiously, his CV also lists current faculty positions at Thomas Jefferson University, and Cal State Fullerton. A position as an assistant adjunct professor is pending at UC Irvine. The applicant has studied caveolin since his graduate student days at Mount Sinai and lists a number of JBC publications in that area. A career development plan for the applicant has been provided, but is vague. To guard against intellectual isolation, a career development committee is in place with consultants and coinvestigators. His collaborators/consultants are dispersed between California and Philadelphia (where the most relevant mentors are present). One concern with this career development plan is that the applicant's scientific life will be rather disjointed. For example, he will be teaching stem cell biology at Cal State Fullerton, auditing courses at UC Irvine, and serving on stem cell oversight committees at CHOC. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The chief medical hospital of CH OC provided a letter describing core facilities available to Dr. Lee and the history of the hospital's commitment to stem cell research. The track record of CH OC in promoting career development puts young faculty is unknown to this reviewer. DISCUSSION: In general, the reviewers found that the application was not strong. The proposal was diffuse, experiments were descriptive, aims were not well discussed, and the expected results are inconclusive. One reviewer questioned whether the institution could provide adequate support for the proposal.