While invasion and metastasis is the major cause of breast cancer treatment failure, we do not know when the invasive cancer cell emerges during the early stages of breast cancer. The answer to this question has profound implications for breast cancer screening, therapy to prevent breast cancer, and the treatment of advanced metastatic disease. The objective of this proposal is to study the stage of human breast cancer designated as ductal carcinom in situ (DCIS) in an attempt to understand how early invasive and metastatic cancer cells can arise. For this research, we will develop and employ a wholly new technology to microdissect three-dimensional structures of living human DCIS lesions. The technology will provide the means to conduct studies of the nature of DCIS that was never before possible. Our team has pioneered the development of laser capture microdissection (LCM) as a widely used method for procuring subpopulations of cells under direct microscopic visualization. Currently, LCM is restricted to stained histologic sections of nonviable tissue. We will extend this technology into the three-dimensional realm to create a new technology capable of microdissecting living tissue structures. This technology is required to attain the goal of isolating, cultivating, and studying living DCIS native lesions within an intact duct segment. DCIS is a pathologic entity that can only be diagnosed under the microscope. Consequently, in the past DCIS lesions could only be studied by first identifying them as nonviable stained tissue sections. Thus, fundamental questions about the neoplastic nature of DCIS in vivo and the existence of DCIS cancer stem cells could not previously be answered. At the microscopic level, human breast cancer is a heterogenous mixture of invading carcinoma cells, adipose cells, stroma, fibrocystic lesions, normal appearing ducts and lobules, and accompanying premaligant DCIS lesions. Moreover, following dissaggregation and culture of human breast cancer fragments (in the manner done in all prior studies), the cells that grow out cannot be distinguished as being derived from cancer cells or from DCIS lesions. Live tissue laser microdissection (LTM) now offers a means to isolate pure DCIS lesions for organ culture propagation and analysis using animal model transplantation.

DCIS lesions are defined pathologically as breast ducts filled with growing neoplastic cells. The difference between DCIS and invasive breast cancer is that in the case of DCIS the neoplastic cells have not yet invaded through the wall of the duct. We will test the hypothesis that DCIS neoplastic cells already possess the capability to invade and metastasize and that this malignant potential is delayed or held in check because the DCIS cells are confined inside the duct and do not have access to the blood and lymph vascular system outside the duct. We will microdissect living DCIS structures consisting of a truncated segment of the duct containing the DCIS. We will then transplant the structures into the mammary fat pad of immunodeficient animal models to test if the contained human DCIS cells can invade and grow because the duct wall is breached and the cells gain access to the outside breast stroma.

Our hypothesis that DCIS cells possess full malignant potential has far-reaching implications for the exploding field of cancer stem cells. Cancer stem cells are hypothesized to be a subpopulation of precursor cancer cells that are immortal, resistant to therapy, and that retain all the properties of invasion and metastasis. Clinicians are performing tests to determine if cancer stem cells should constitute a major therapeutic target for future cancer therapies. The logic is that conventional treatments, which don't affect the cancer stem cells, will only delay the re-emergence of the cancer, since a small portion of cancer stem cells can re-capitulate the entire cancer. This proposal will address a basic question in the cancer stem cell field, particularly for breast cancer. The question is, How early does the cancer stem cell arise? Does it exist at the level of the so-called premalignant lesions? If it does, then what keeps it in check until it invades and metastasizes? To approach these questions, a major objective of the proposal will be to propagate living human DCIS organoids and to attempt to isolate DCIS cancer stem cells as continuous lines. This is a high-risk proposal because (a) living DCIS has never been microdissected and isolated, (b) living human DCIS has never been fully tested for its malignant properties in vivo, and (c) no attempts have been made to isolate strains of DCIS cancer stem cells.

The demonstration that DCIS cells already possess the capacity to invade, and perhaps metastasize, can profoundly alter future strategies for cancer screening and treatment. It will be important to identify the molecular and tissue structural factors that keep the DCIS cancer stem cells in check, since these will be the main determinant of when human breast cancer first becomes malignant. If DCIS stem cells can be isolated, it will then be possible in the future to test if these cells are actually derived from a non-neoplastic normal breast stem cell. Finally, the availability of continuous lines of human DCIS stem cells (adult nonembryonic) can become the foundation for a field of mechanistic studies addressing the regulation of these cells in the tissue microenvironment, as well as the application of such cells to testing new drugs.