Tools and Technologies II
The regulation of the development of humans and cancer are closely related. Small differences in the rate of self renewal, expansion and differentiation of tissues can provide premalignant cells a selective advantage resulting in tumors. As cancers progress they may accumulate additional mutations that result in increasingly divergent behavior and opportunities of evading host defenses and therapeutic intervention. Differential killing of the early stages of cancer is difficult because of collateral damage. However, an opportunity of significantly impacting breast cancer mortality may reside in instructing early stage disease to adopt a normal tissue fate instead of progressing to invasive and metastatic stages of the disease. The demonstration that only some, and perhaps very few, of the cells of some breast cancers are responsible for both perpetuating the tumor and generating the diversity of cell types within the tumor, has defined a breast cancer stem cell as one that has the capacity to self renew but also retains the potential to differentiate to a more specialized state. These cancer stem cells present the opportunity of differentiation therapy as an alternative or adjuvant to cytoxic therapies. One proven way to identify new compounds that might direct the differentiation of cancer stem cells requires testing thousands of chemicals. To use automated methods of screening candidate chemicals that makes this strategy feasible, suitable cell lines are needed. Nearly all of the cancer cell lines currently used were isolated under conditions selective for growth but not necessarily differentiation potential. By starting with conditions that inhibit the differentiation of mouse mammary cancer stem cells and promote their self renewal in cell culture, we have improved the culture conditions necessary to isolate breast cancer stem cells that retain the potential to differentiate. We have successfully propagated mouse cancer stem cells and similar cells from human tumor surgical. We propose to extend our efforts to isolate a larger number human cancer stem cell lines from representative types of early and late stage disease for eventual use in identifying new therapeutic candidates for differentiation therapy. We will characterize these cell lines with regard to their tumorigenicity, behavior in culture, gene expression profile and sensitivity to a panel of chemical compounds. The selective propagation, amplification and characterization of human cancer stem cell lines from representative types of early and late stage disease will provide the necessary tools to use current screening methods to identify new drugs that may significantly decrease the incidence of late stage breast cancer.
Statement of Benefit to California:
Breast cancer afflicts nearly one in ten women during their lifetime. While progress is being made in certain targeted therapies, major advances in treating very early stage disease could have a great impact on preventing the progression of the disease to stages that are difficult to cure. In some cancers like colorectal cancer, stem cells are clearly the target of genetic changes that accumulate and cause the progressive loss of cellular control. In breast cancer there is increasing evidence for cancer stem cells that are responsible for the propagation of the disease but also retain the potential to differentiate to a benign fate. It is the stem cell characteristic of breast cancer stem cells that is the opportunity for the development of an alternative to cytotoxic cancer therapies. The differential killing of cancer cells with minimum impact on normal cells is the difficult requirement that leads to the well know adverse side effects of radiation and cytotoxic chemotherapy. Differentiation therapy uses the alternative strategy of inducing cancer stem cells to adopt a benign fate. However the search for chemicals and biologics that might efficiently instruct breast cancer stem cells to adopt a benign fate has been restricted by the availability of representative breast cancer stem cell cultures. Our goal is to amplify the breast cancer stem cells of representative human tumors for use in identifying compounds and gene targets that regulate their differentiation to benign cell fates. Periodic treatment of women at very early stages of disease who are at increased risk of the disease with therapeutics that instruct breast cancer stem cells to differentiate to a benign fate could greatly diminish invasive breast cancer incidence and progression. Our proposal would provide new tools to discover therapeutics for differentiation therapy. In part this proposal is facilitated by the Athena Project that coordinates clinical research activities across the multiple campuses of the University of California and by CIRM support of a Stem Cell Laboratory that provides equipment for expanding the culture of cells in low oxygen environments. Citizens of the California will benefit from this research by accelerating progress in substantially decreasing the incidence of invasive breast cancer.
Breast cancer stem cells (CSCs) have been identified in both human and mouse tumors, but propagating pure populations of such cells in vitro has been difficult due to spontaneous differentiation. Previously, the Principal Investigator (PI) discovered improvements that allow the proliferation of mouse mammary CSCs in a highly enriched state with less spontaneous differentiation. In the proposed research, this technology will be extended to human breast tumors in order to isolate and characterize breast CSC lines, which retain the potential for differentiation. The applicant will first obtain and characterize surgical samples from breast cancer patients with representative types of early and late stage disease. Next, the applicant will establish and amplify approximately 24 new human breast CSC lines. Finally, the candidate CSC lines will be characterized for their tumorigenicity, histological diagnosis, tumorsphere formation, gene expression profile, differentiation potential, cell surface markers, intermediate filament protein content, and hormone receptor expression. Reviewers recognized that the proposal focuses on a clear, unmet need to improve breast cancer therapeutic strategies, and they acknowledged that the project addresses a translational bottleneck: the isolation, expansion, and maintenance of breast CSCs. Although the project could result in an increased number of CSC lines available for testing new compounds to treat cancer, reviewers questioned the proposal’s novelty as similar efforts are already underway at a large number of labs that are exploring the use of selective targeting to attack tumor-initiating cells or CSCs. Additionally, proposed approaches appear to differ little from those being pursued by multiple labs, and reviewers were unconvinced that the project would yield a novel approach or represented an innovative solution to a critical bottleneck for translational stem cell research. Reviewers raised several serious concerns about the proposal’s experimental design and feasibility. Although the PI provided significant preliminary data from a mouse model, only very limited data from human tumors were included. It was unclear whether human breast carcinoma samples could be propagated routinely or if the phenotype of the cultured cells would accurately reflect in vivo CSC biology. Reviewers expressed additional concerns about the cellular heterogeneity of specimens, the rationale for the number and types of CSC lines to be generated, or which specific genotypes and phenotypes would be informative. Another weakness was the lack of a thorough comparison of generated CSC lines with cell lines derived from the original tumor. Moreover, reviewers found the study primarily descriptive, and it was unclear how the abundant data produced by the many proposed analyses would inform the selection of human CSC lines. The reviewers unanimously praised the PI’s qualifications and his/her long track record in the fields of cancer biology and cell biology. Although the PI has expertise in studying mammary carcinoma, there was concern that he/she had only limited experience with human CSCs or CSC line derivation. Overall, the research team appeared appropriate and well qualified; however, the role and commitment to the project of one Co-Investigator was unclear. In summary, this proposal describes the development of breast CSC lines as tools for advancing cancer therapeutic strategies. Despite the proposal’s potential impact and focus on a key translational bottleneck, reviewers’ enthusiasm was severely diminished by a lack of innovation, unclear rationale, and an unfocused experimental approach. Thus, the reviewers did not recommend this application for funding.