Early Translational I
Regenerative medicine holds great promise for the treatment of a host of human diseases. The remarkable regenerative potential of stem cells puts them at the forefront in terms of options in regenerative medicine. The use of pluripotent stem cells to generate tissue (sometimes called adult) stem cells is one of the most promising strategies for success in development of novel cell-based therapies and diagnostics. However, the generation of tissue stem cells, from pluripotent stem cells, for potential applications must employ laboratory methods for cell generation, maintenance and expansion that increase the risk of generating cancer stem cells. For example, many pluripotent human embryonic stem cell (hESC) lines are known to be contaminated with cells that have undergone cancer-causing mutations. Furthermore, substantial numbers of mice reconstituted with islet cells derived from induced pluripotent cells (iPSCs) developed tumors. Thus, a critical bottle neck in bringing any pluripotent cell-based therapy to the clinic is overcoming our limited ability to identify malignant cells that contaminate cell cultures intended for regenerative medicine. In addition to applications in regenerative medicine, another aspect of stem cell biology has immense potential for novel therapeutic applications. In order to develop a clearer understanding of breast cancer biology, our group has begun to apply the principles of stem cell biology to breast cancer in humans. Data generated from our laboratory has demonstrated that many common cancer tumors contain populations of tumorigenic, immortal, cancer stem cells (CSCs), as well as and non-tumorigenic cancer cells. Indeed, as few as 100 CSCs were able to form tumors when injected into immunodeficient mice and these resultant lesions contained the full, phenotypically-heterogeneous population of CSCs and non-tumorigenic cancer cells found in the patient’s original malignancy. Since our evidence suggested that stem cells drive tumor development, we hypothesized that resistance of the CSCs would contribute to relapse after cytoxic radiotherapy and chemotherapy. This prediction has now been borne out. Recently, using single cell analyses of stem cell pathways, we have developed a novel way that should make it possible to accurately identify and count cancer stem cells in both pluripotent stem cell cultures and biopsy specimens. This opens the door to a rapid and simple assay that could be used to quickly determine the risk and effectiveness of a treatment regimen for an individual patient.
Statement of Benefit to California:
This research has the potential to significantly benefit the State of California and its citizens. First, this research could solve a potentially serious complication for the use of stem cells in the clinic. For example, transplantation of insulin-producing cells has the potential to cure diabetes. However, there is a risk that the laboratory grown cells used for such treatments could become cancerous. If successful, our studies will reduce this risk and thus help to enable clinical trials to proceed with less risk of this serious complication. Next, this research should lead to a new diagnostic that can be used to optimize the therapy that a patient with cancer receives. This would reduce the side effects that a patient is exposed to and would increase the chances that a particular treatment will be effective. Finally, if these projects are successful, they will generate new instrumentation that must be commercialized. This will lead to more high-paying jobs for the State of California.
The primary goal of this proposal is to fabricate a device that can estimate the frequency of cancer stem cells (CSCs) in complex mixtures of cells derived from patient tumors, and in populations of cells derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) that are intended for transplantation. The applicant proposes three aims. First, the applicant proposes to identify CSCs by determining genes differentially expressed between CSCs and non-tumorigenic tumor cells. Second, the applicant proposes to identify genes that differentiate CSCs that propagate tumorigenesis from undifferentiated hESCs and iPSCs in pluripotent SC populations. Finally, the applicant proposes to create a device that can be used to provide quantitative estimates of tumorigenic cells in complex mixtures of tumor cells or cells derived from hESC or hiPSC. Reviewers recognized the importance of development of a clinical tool for rapid and accurate detection of tumorigenic cells in complex mixtures of tumor cells, or in pluripotent SC populations derived from hESCs or iPSCs that are intended for transplantation. Reviewers stated that the knowledge gained from the project may lead to an understanding of molecular differences that impact prognosis, and judged the potential impact of the project to be high. Reviewers commented that through the better characterization of these tumorigenic cells proposed in the application, it may be possible to reduce the complexity of present cancer stem cell assays toward a more pragmatic test for “real-world” clinical use. Reviewers praised some aspects of the research plan but felt that others were underdeveloped. The proposal was judged to be innovative, and based on a strong foundation of CSC biology from the Principal Investigator’s (PI’s) laboratory. Reviewers praised the applicant for an articulately described plan to develop the single cell analysis devise (SCAD) for the identification and quantification of CSCs in Aim 3. However, reviewers had concerns that its success was dependent on Aims 1 and 2 to develop robust genetic signatures. In several cases, the proposal lacked experimental detail to inspire confidence that the goals of the first 2 aims could be accomplished. Reviewers were concerned that the description of the analysis to identify CSC markers was unclear. They commented that a more rigorous, in vivo characterization would be needed to validate the link between the target genes and cancer initiation. One reviewer commented that the current armamentarium of CSC-specific cell surface proteins and antibodies is not yet sufficient to permit the adequate purification of cell populations needed for discovery of CSC-specific markers, and therefore progress on Aims 1 and 2 would lag. Another serious concern of a reviewer was that the proposal did not address CSC heterogeneity, supported by published evidence that leukemic SCs from individual patients differ; this property of CSCs undermines the implicit assumption in Aims 1 and 2 that all CSCs from particular malignancies or hESCs and iPSC lines are the same, and could significantly impact the experimental plan. Finally, reviewers raised questions as to whether the ambitious goals could be accomplished in the intended time frame. Reviewers were consistent in their praise for the PI and noted his/her prior contribution to the field of CSC biology. Reviewers noted that the research team included highly qualified investigators in bioengineering, stem cell biology, and biostatistics. In addition, the facilities were judged to be outstanding and would fully support the research contained in this proposal. In summary, the proposal was judged to be innovative with potentially high impact to the field. However, the lack of experimental detail did not inspire confidence that the ambitious proposed work could be accomplished in a three-year time frame.