Isolation of cancer stem like cells from human adenosquamous carcinoma of the lung supports a monoclonal origin from a multipotential tissue stem cell.

There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded in vitro. Primary xenografts and metastatic lesions derived from these cells in NSG mice fully recapitulate both the adenocarcinoma and squamous features of the patient tumor. Interestingly, while the CSLC all co-expressed cytokeratins 5 and 7, most xenograft cells expressed either one, or neither, with <10% remaining double positive. We also demonstrated the potential of the CSLC to differentiate to multi-lineage structures with branching lung morphology expressing bronchial, alveolar and neuroendocrine markers in vitro. Taken together the properties of these ASC-derived CSLC suggests that ASC may arise from a primitive lung stem cell distinct from the bronchial-alveolar or basal stem cells.

Establishment of human ovarian serous carcinomas cell lines in serum free media.

Ovarian cancers are the fifth leading cause of cancer death among US woman. The majority of ovarian cancers belong to a category of serous adenocarcinomas. This type of cancer is often diagnosed at a late stage of the disease. Surgical debulking, followed by chemotherapy is the current treatment. Half of all patients will die within 5 years of diagnosis of the disease. Poor survival may be due to disease progression as a consequence of development of drug resistance, cancer cell heterogeneity within the tumor, or the persistence of cancer stem cells. Cancer stem cells (CSC) are defined as a minority cell type in the tumor, which retains the capacity, through asymmetric division, for self-renewal as well as differentiation into multiple cell types. Through this process, CSC can regenerate the entire tumor phenotype and subsequent metastases. Initial in vitro work in the area of solid tumor CSC biology has focused on the isolation and propagation of cells with CSC-like properties from breast and colon tumors. Breast and colon cell lines with CSC-like properties have been isolated and maintained in vitro for extended periods of time. The in vitro maintenance of these CSC requires growth in hormone-supplemented serum-free media and the use of matrix or growth as tumor spheres (Roberts, Ricci-Vitiani et al., Cammareri et al.). Based on the pioneering work generating breast and colon CSC, our lab has begun to develop methods for the establishment cell lines with CSC-like properties from additional solid tumors. In this article, we describe methods, using defined medium, which allow for the successful establishment of continuous cell cultures from a minority cell type within serous ovarian cancers. The cell lines established using these methods grow in serum-free hormone-supplemented medium either as a monolayer on a matrix, or as tumor spheres in suspension. These cells express markers previously reported for tumor stem cells, including CD44 and CD133, and form tumors that recreate the morphology of the original patient tumor when implanted in immune deficient mice. The introduction of this method will facilitate the expansion of ovarian cancer cells for investigating cancer stem cell biology as well as providing tools to aid in the development of new treatments for this deadly disease.

Isolation and establishment of human tumor stem cells.

Current cancer therapies are based on the ability to inhibit the growth of rapidly dividing cells, the majority of which constitute the tumor. Although for decades, sporadic literature has posited the existence of cancer stem cells (CSCs), only recently has this type of cell been isolated and characterized from solid tumors. Like stem cells from their normal counterpart, CSCs are a rare population that can reconstitute a new tumor with similar composition and phenotype to the tumor of origin. These CSCs represent a small subset of the original tumor, grow indefinitely in vitro, and can form tumors in animals from a very few cells. The cells are slow cycling, capable of self-renewal and give rise to daughter cells that are either self-renewing and pluripotent or transit amplifying, and terminally differentiated. Thus far, CSCs have been isolated from only a small number of tumor types. In most instances, the cells are obtained using selection of, and enrichment for, cells with prospectively identified cell surface markers (Al-Hajj M, et al., 2003). This yields a very limited number of cells, and in many cases these cells cannot be cultured. There is a need for a method for isolation, purification, and expansion of stem cells from a greater spectrum of tumors. There is also evidence for "...a link between normal stem cell regulation and the control of cancer stem cells" (NCI Think Tanks in Cancer Biology, Executive Summary of the Tumor Stem Cell and Self-renewal Genes Think Tank1). We present here a strategy for the isolation and establishment of tumor cell lines that represent a minority of cells in the original tumor. They have the ability to grow indefinitely in vitro, form tumors in mice from less than 100 cells, and share many of the growth requirements and cell surface antigens of normal tissue stem cells from which they may arise.