The goal of this proposal is to develop a prospective brain tumor model originating in PDGF over-expressing hES cells, and to utilize this model for physiological investigations of tumors and their interactions with non-tumor neighbors. We will study this model using the same techniques used on neural stem and progenitor cells. Gliomas often present clinically as seizure, and epileptic foci are found adjacent to experimental gliomas. Tumor cells, even if non-excitable, may interact reciprocally with electrically active neighbors. These questions are almost completely unexplored. Because we are exploring a novel concept, federal funding seems unlikely at this stage. This project is composed of three parts: Part I. Construct hES cell lines over expressing platelet-derived growth factor (PDGF) under control of the nestin promoter, and matched control lines. Over expression of platelet-derived growth factor (PDGF) can initiate brain tumor formation, and CNS gliomas often express PDGF. Part II. Transplant PDGF-over expressing hES cells into developing mouse brain, and follow emergence tumors. Other groups have reported that undifferentiated hES cells, transplanted into the ventricles of embryonic mice, will migrate from the ventricles into the brain parenchyma, differentiate, and integrate into the host environment. No evidence of teratoma or immune rejection was seen. In this model only a small number of cells relative to the total population of the brain will be of human origin. Part III. Study physiological reactions to the tumor and mechanisms by which host brain cells may be recruited by PDGF-overexpressing hES cells. Our hypothesis is that the tumor and surrounding cells are recapitulating in part the interactions between neuroprogenitor cells and their more differentiated surround. We will determine the extent to which the physiological characteristics and cell-cell signaling mechanisms of PDGF-secreting hES cells, neighboring naïve hES cells, and adjacent mouse neurons and non-neuronal cells reflect those seen during migration and differentiation of neural progenitors. Our ultimate goal is to look at recruitment and possible transformation of cell neighbors into the tumor, and ask if disruption of synchronous activity and/or blockade of transmitter systems disrupt tumor or surrounding cell migration, proliferation or recruitment.
Statement of Benefit to California:
Glioma is among the most intractable cancers, in part because invasion of the normal brain by tumor cells makes surgery only palliative. Tumor progenitor cell birth and migration is similar to normal brain development. We propose to create and study a model in which a small number in human ES cell-derived neuroprogenitor cells resident in mouse brain will express genes that will induce them to become tumors. We will then investigate them as we do normally developing cells, essentially considering the tumor as a developing tissue. Our hope is that this novel approach will yield unique insights leading to potential therapies. Any progress towards reducing the virulence of glioma will be of immense benefit to the State of California and its citizens.
SYNOPSIS: This proposal aims to model brain tumorigenesis in the mouse using hES cells engineered to overexpress PDGF. This applicant will engineer hES cells to produce PDGF driven by the nestin promoter as a way to mimic the effect of PDGF-producing CNS gliomas. In addition to establishing the cell lines, transplant studies will examine their growth and differentiation in the forebrain of young mice to evaluate the effects of PDGF production on the differentiation and tumorogenicity of the hES cells, and molecular and physiological studies will be performed to determine how these cells integrate and interact with the host cells. SIGNIFICANCE AND INNOVATION: This work is innovative and significant in that there are few if any studies looking at the physiological interactions of glioma cells and host neurons; this is significant toward the development of countermeasures for seizure disorders that often accompany gliomagenesis in humans. Also, the potential recruitment of host stem and progenitor cells to a growing tumor in the brain has been looked at in only a few studies, and these do support a notion of chemotactic events that beckon host cells from the SVZ and perhaps other areas to the tumor. The work is innovative because there is no such hESC tumor generating model in mice that reviewers are aware of, and the work is sure to uncover interesting transformed cell-host brain interactions that recapitulate some aspects of normal neurogenesis but in a tumorigenic setting that contributes to abnormal cell differentiation and abnormal host cell-tumor cell interactions. Perhaps these could be thwarted using transmitter and biophysical sequelae to both affect tumor growth and abnormal circuitry actions that can contribute to seizures. STRENGTHS: The PI has a strong track record in applying state-of-the-art neurophysiological approaches toward understanding neurodevelopmental issues, and substantial experience with the techniques that will be used here to evaluate the physiological effects of cell transplantation and tumor formation. Viewing tumors as a “tissue” and the hypothesis that host-tumor cell interactions might be important in understanding how progenitor cell interactions shape CNS development is truly “almost completely unexplored”. This is a novel and potentially interesting brain tumor-host cell interaction model and new insights are to be gleaned from the tumor cell-host brain interactions. The notion of looking at “recruitment and possible transformation of cell neighbors into the tumor” is very interesting and of interest to the field because the effects of tumors on the physiology of surrounding host tissue, and the possible recruitment of host cells by growth factors emanating from a tumor, are both subjects of great importance about which little is known. This is an understudied area of cancer and stem cell biology, and the idea that altering synchronous activity and possibly blocking transmitter systems might disrupt tumor cell migration, proliferation, recruitment and integration is truly novel and worthy of study. WEAKNESSES: The main weakness of the proposal is that there is no reason to assume that PDGF-overexpressing hES cells will make tumors in this model. This work could attract federal funding one day, but the PI will have to show tumorigenicity of the PDGF-ES cell lines. The PI has also exhibited limited recent funding from these agencies, and this does not help the cause. There is a glaring hint of using ES cells only to respond to this RFA, since the Holland mouse models are already available and perfect for studying many of these physiological issues. The rationale that "it uses human ES cells to develop a disease model that circumvents using mouse cells to model human disease by using the mouse as a host for a limited number of human brain cells” is not completely unreasonable, but also not the greatest reason for doing all of this difficult model development. At several points, the applicant raises the possibility that transplanted cells may exhibit de-differentiation or induce de-differentiation in surrounding host cells; however, it is not clear how this de-differentiation would be distinguished from a failure of the cells to mature in the first place. Reversion to a less mature state would be interesting, but it is not clear how this could be demonstrated in the complex environment surrounding a mass of transplanted cells, where following the fate of individual cells over time would be impossible. Because the expression of PDGF is controlled by the nestin promoter, it will be expected to persist so long as nestin expression does. Use of an inducible promoter that could be turned on and also turned off by delivery and cessation of an exogenous compound would allow for more clear-cut interpretations of the necessity for PDGF in the ongoing interaction between the transplanted and host cells. With the experiments as presently envisioned, the onset of nestin and PDGF expression should begin as the hES cells differentiate, but the continued maintenance of PDGF production will depend on whether nestin expression is maintained. RECOMMENDED CHANGES: The PI is strongly encouraged to test his approaches on the RCAS mouse model already developed and used by the Holland group, and also possibly recruit Dr. Holland as a collaborator to help generate these new hESC lines. The PI’s collaborator Dr. Barberi is certainly skilled in ES cell studies, but this PDGF model could benefit from the expertise of Dr. Holland (including getting his seal of approval that this hES cell approach is in fact worth the effort). The PI should also consider use of an inducible promoter, rather than or in addition to the proposed nestin promoter constructs, and explain how de-differentiation could be distinguished from failure to differentiate initially. DISCUSSION: The investigator is regarded as having a good track record, and the goals of the proposal were deemed appropriate in discussion. However, PDGF over-expression as a cause of tumorigenicity in hESC is an assumption not supported by evidence. It was taken on faith that PDGF over-expression would cause tumors in hESC, but perhaps the failure might also be of interest since it would be new information. One panel member indicated that while PDGF may be overexpressed in human brain tumors, studies on Akt and Ras in adult cell models also have been published, so why focus solely on PDGF in hESCs? Hyper-excitability at the tumor periphery is an under-studied area worthy of more investigation, and physiological studies here are useful. The question is whether this neurophysiological model is useful to the field. One reviewer indicated that the applicant's approach in the context of tumors inducing seizures was potentially more targeted to treating epilepsy, rather than to curing cancer. In this case, there are probably better models for seizure.