Infinite Expansion of Breast Tumor Samples in Culture

Institution: California Pacific Medical Center Research Institute
Investigator(s): Shanaz Dairkee, Ph.D. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8WB-0032 Award: $311,400
Award Type: STEP Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2002)
Experimental models of breast cancer encompass a very small number of cell lines, which were developed several decades ago. These eight, common cell lines are mostly derived from late stage metastatic tumors. This reliance on non-representative forms of breast cancer compromises the current effort to analyze the key genes and proteins involved in earlier, progressive stages of the disease. In addition, screening new drugs against these cells might give erroneous information on their potential clinical translation. It is very difficult to develop new breast cancer-derived cell lines for certain underlying technical reasons. Attempts to culture primary breast tumor cells usually fail, because the cells die out after a few cell divisions.

We propose a novel approach towards inducing continuous growth or 'immortalization' in breast tumor cells, which should be applicable to a wide spectrum of breast tumors. Our specific aims are to: 1) Employ tumor cultures developed from an unselected series of breast cancer patients while introducing the telomerase gene, which is known to confer continued proliferation potential by maintaining the ends of human chromosomes. 2) Evaluate similarities between the original tumor tissue from patients and the immortalized cultures developed from this tissue. We will examine cellular characteristics that are important from the current standpoint of treatment, such as estrogen receptors, HER-2 overexpression, and p53 mutations. This analysis will ascertain that such immortalized cell lines can serve as reliable model systems for breast cancer. Finally, 3) Determine why the continued growth potential of tumor cells is not evident in culture. These studies will improve basic understanding of the immortalization phenotype, which is a hallmark of malignancy, and provide additional insights towards its control.

At the present time, surgically excised tumor tissues are routinely archived as fixed or frozen samples. This approach is valuable for molecular profiling, but is not amenable to tests that require living cells. We anticipate that completion of these studies will lead to a new infrastructure for procuring an unlimited supply of live tumor cells from the majority of breast cancer cases. This will advance knowledge in tumor cell biology and rapidly lead to the identification of new molecular targets for the treatment of patients at all stages of disease. Finally, our process might lead to "individualized" therapy, where potential therapeutic agents are tested against a woman's tumor cells before deciding on the drug used for treatment.

Final Report (2004)
An important prerequisite towards the approval of novel cancer therapeutics for clinical use is their ability to inhibit the survival and growth of a panel of 60 immortal cell lines, which comprise what is commonly referred to as the “In Vitro Screen.” In fact, with regard to breast cancer, this panel consists of only 6 independent tumors, maintained in culture for 2-3 decades. Moreover, because these cell lines were originally derived from aggressively proliferating tumors, only a subset of breast cancer clinical cases are represented by such model systems. Consequently therapeutic targets, which are lacking, or eliminated over time from these cellular tumor models, but displayed by many clinical cancer samples, remain understudied. Thus, new basic science and clinical approaches will require the development of new breast cancer cell lines that better represent the breadth of tumor heterogeneity.

A major obstacle for creating new cancer cell lines is the limited lifespan of tumor cells derived from patient samples in the laboratory. Using CBCRP support we first studied the molecular properties of cell cultures derived from clinical tumor samples, which prevent their continuous proliferation in vitro. A complete understanding of how culture conditions impose growth arrest in tumor cells could provide novel insights regarding disease prevention and management. To develop clinically representative model systems, we determined the extent of phenotypic variation before and after cell culture in clinical samples of tumor tissue. Next, to induce continuous proliferation in the desired cellular breast cancer models, we introduced the telomerase gene, hTERT, into cell cultures encompassing a variety of histological types, grades, and stages of disease presentation. We subsequently evaluated changes in gene expression introduced by hTERT manipulation. Finally, to determine whether hTERT-transduced primary tumor cultures yield xenografts, we inoculated authenticated cells into immune compromised (nude) mice.

We found that a proportion of primary tumor samples placed in culture displayed elevated expression of negative growth regulators, such as p21, TGFP, TPRI and TPRII. Introduction of hTERT into such primary tumor cultures has resulted in 16 novel immortal cell lines. Prior to immortalization, the tumor cultures were compared to the original tumor tissue by global gene expression profiling (i.e., DNA arrays) in order to determine the degree of similarity before and after cell culture. This analysis has consistently demonstrated greater concordance between these two groups than with immortal cell lines, such as those used in the NCI’s “In Vitro Screen.” Our gene array studies were done in collaboration with Dr. Stefanie Jeffrey at Stanford University. To study the ability of our hTERT immortalized cells to form tumors, they were inoculated into one of two sites: the mammary fat pad, or the renal capsule of 7-8 week old immune deficient female mice. The inoculated cells developed palpable lesions, approximately 0.5 to 0.9 cm in diameter, within 4 weeks at the mammary fat pad injection site in three cases. A histologic comparison (i.e, microscopic appearance) of the tumor in the cancer patient with the tumor in the mouse model, displayed close similarities. Thus, we were able to (1) produce permanent breast cancer cell lines directly from a variety of human tumors, (2) demonstrate the retention of the key genetic profile compared to the original patient tumor, (3) show that these new cell lines were different from existing tumor lines, and (4) show that the new cell lines were capable of forming tumors in mice.

In addition to the goal of making these lines widely available to other investigators in the field as novel tools for drug development, these tumor cell cultures are currently facilitating long-term research goals in our laboratory. For example, in conjunction with fibroblasts isolated from the same clinical samples that were used in the development of the primary tumor cell lines, we have initiated studies (1) to link the gene expression profiles of epithelial and stromal cell cultures to various histological phenotypes, and (2) to determine independent, and interactive effects of these cell types in functional assays, and how they relate to clinical characteristics, as well as tumor behavior and patient prognosis. As a result of CBCRP innovative funding, we have been able to secure NIH R01 funding to pursue these goals.

A molecular 'signature' of primary breast cancer cultures; patterns resembling tumor tissue.
Periodical:BMC Genomics
Index Medicus: BMC Genomics
Authors: Dairkee SH, Ji Y, Ben Y, Moore DH, Meng Z, Jeffrey SS.
Yr: 2004 Vol: 5 Nbr: 1 Abs: Pg:47-56

Oxidative stress pathways highlighted in tumor cell immortalization: association with breast cancer outcome.
Index Medicus: Oncogene
Authors: Dairkee SH, Nicolau M, Sayeed A, Champion S, Ji Y, Moore DH, Yong B, Meng Z, Jeffrey SS.
Yr: 2007 Vol: 26 Nbr: 43 Abs: Pg:6269-79.