“Differential proteomic panning analysis of protein from single stem cells: An alternative to mass spectrometric methods”
Tools and Technologies 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. Emerging evidence indicates that brain tumor stem cells are responsible for recurrence of many of these cancers. It is essential to identify the proteomic differences between normal stem cells and tumor stem cells for targeted destruction of brain tumor stem cells. Existing proteomic technologies still have many constraints and limitations. Our patented Differential Proteomic Panning (DiPP™) technology platform is much more sensitive and effective in identifying the surface markers than any existing methods. This proposal if funded by CIRM will pave the way for promoting DiPP™ to be used for proteomic studies of single-stem-cells.
Statement of Benefit to California:
Malignant brain cancers are a leading cause of cancer death. Three decades of research have resulted in little change to the outcome of 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 mental disabilities. Overall estimates of the incidence of brain cancers in the United States show that about 20,000 will be diagnosed annually with about 2,500 in California. Given these statistics, the costs for the patient and family cannot be overestimated. 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 are projected to cost Californians more than 1.5 billion dollars annually. It is clear that California patients with brain cancers need a new therapeutic approach. One promising approach is to target brain tumor stem cells. It is essential to identify the proteomic differences between normal stem cells and tumor stem cells for targeted destruction of brain tumor stem cells. Existing proteomic technologies still have many constraints and limitations. Our patented Differential Proteomic Panning (DiPP™) technology platform is much more sensitive and effective in identifying the surface markers for targeting brain tumors stem cells, than any existing methods. This proposal if funded by CIRM will pave the way for promoting DiPP™ to be used for proteomic studies of single-stem-cells, which can be used for other brain diseases and injuries as well. Other long-term economic impact is the opportunity to train more student scientists in the field of stem cell technology, facilitating and promoting California's biotechnology industry.
This proposal focuses on the development of methods for single-cell quantitative proteomic analysis of stem cells. The applicants have recently developed a technology, called Differential Proteomic Panning (DiPP), by which protein samples are labeled with different radioactive isotopes, fractionated by a variety of methods and the ratio of isotopes is used as a selection criterion for analysis by mass spectroscopy. The proposal describes validation and further development of the DiPP technology to allow single-cell proteomic analysis of human neural stem cells (hNSCs) and brain tumor stem cells. In terms of impact, reviewers found that this proposal, if successful, could have a considerable impact in the field of stem cell research. Specifically, one reviewer commented that the technology for single-cell quantitative proteomic analysis would facilitate the discovery of new biomarkers for various stages of embryonic, precursor and cancer stem cells; cell-surface proteins unique to these various cell types to permit their purification; and candidate therapeutic molecular targets in cancer stem cells. On the other hand, reviewers had very serious concerns about the feasibility of this proposal. Reviewers also criticized the research plan as vague and disorganized. One reviewer commented that the proposal describes a variety of technologies but lacks details about how they will be applied to specific questions. No information is provided about the source of human neural stem cells (hNSCs), nor how tumor SCs will be identified and purified from resected brain tumors. The proposal also mentions human embryonic and induced pluripotent SCs, but without enough elaboration to determine their role in the overall plan. A reviewer noted that microfluidic device fabrication is discussed conceptually but no design parameters are provided, leaving doubt as to whether the group plans to build it. One reviewer also noted that the proposal will require a new instrument to detect tritium with 95% efficiency in the absence of scintillation fluid. This instrument will require at least a year in development, raising doubt that the technology can be optimized and applied within a 2-year funding period. Finally, the last aim, to perform single-cell proteomics with DiP, was found to lack experimental design details. There was some disagreement among reviewers about the qualifications of the assembled research team. One reviewer found that the principal investigator (PI) did not have a sufficiently strong CV to justify the risk of funding such an uncertain project. Several reviewers raised concerns about the budget, noting that the roles of contractors and consultants are unclear and the budget justification for the large subcontract (~$190K) is incomplete and insufficient. Overall, reviewers found this proposal to be overly ambitious and expressed serious doubts about the project’s feasibility given the enormity of the technical challenges. Reviewers also criticized the proposal’s poorly organized research plan and lack of experimental detail.