Prospective identification of tumorigenic breast cancer cells

Abstract

Despite advances in detection and treatment of metastatic breast cancer, mortality from this disease remains high because current therapies are limited by the emergence of therapy-resistant cancer cells (1, 2). As a result, metastatic breast cancer remains an incurable disease by current treatment strategies. Cancers are believed to arise from a series of sequential mutations that occur as a result of genetic instability and/or environmental factors (3, 4). A better understanding of the consequences of these mutations on the underlying biology of the neoplastic cells may lead to new therapeutic strategies.

In solid tumors, it has been demonstrated that only a small proportion of the tumor cells are able to form colonies in an in vitro clonogenic assay (5–11). Furthermore, large numbers of cells must typically be transplanted to form tumors in xenograft models. One possible explanation for these observations is that every cell within a tumor has the ability to proliferate and form new tumors but that the probability of an individual cell completing the necessary steps in these assays is small. An alternative explanation is that only a rare, phenotypically distinct subset of cells has the capacity to significantly proliferate and form new tumors, but that cells within this subset do so very efficiently (12). To distinguish between these possibilities, it is necessary to identify the clonogenic cells in these tumors with markers that distinguish these cells from other nontumorigenic cells. This identification has been accomplished in acute myelogenous leukemia, where it was demonstrated that a specific subpopulation of leukemia cells (that expressed markers similar to normal hematopoietic stem cells) was consistently enriched for clonogenic activity in nonobese diabetic/severe combined immunodeficient (NOD/SCID) immunocompromised mice, whereas other cancer cells were depleted of clonogenic activity (13–15). Such experiments have not been reported in solid cancers. If this model were also true for solid tumors, and only a small subset of cells within a tumor possess the capacity to proliferate and form new tumors, this finding would have significant implications for understanding the biology of and developing therapeutic strategies for these neoplasms.

To investigate the mechanisms of solid tumor heterogeneity, we developed a modification of the NOD/SCID mouse model in which human breast cancers were efficiently propagated in the mouse mammary fat pad (16). In the present study, we show that solid tumors contain a distinct population of cells with the exclusive ability to form tumors in mice. We refer to these cells as tumorigenic cells, or cancer-initiating cells, because they consistently formed tumors, whereas other cancer cell populations were depleted of cells capable of tumor formation. We identified cell surface markers that can distinguish between these cell populations. Our findings provide a previously uncharacterized model of breast tumor biology in which a defined subset of cells drives tumorigenesis, as well as generating tumor cell heterogeneity. The prospective identification of this tumorigenic population of cancer cells should allow for the identification of molecules expressed in these cells that could serve as targets to eliminate this critical population of cancer cells.

Mice were injected with unsorted T1 and T3 cells and a 2-mm piece of T2. Cells from T4–T9 were isolated by flow cytometry as described in Fig. ​Fig.1.1. All nine tumors tested engrafted in our NOD/SCID mouse model. Except for T2, which was a primary breast tumor, all other tumors were metastases. All of the tumors were passaged serially in mice except for T4.

Cells were isolated by flow cytometry as described in Fig. ​Fig.11 based on expression of the indicated marker and assayed for the ability to form tumors after injection into the mammary fat pads of NOD/SCID mice at 8 × 10^{, 5 × 10, and 2 × 10 cells per injection. For 12 wk, mice were examined weekly for tumors by observation and palpation; then all mice were necropsied to look for growths at injection sites that were too small to palpate. The number of tumors that formed and the number of injections that were performed are indicated for each population. All tumors were readily apparent by visual inspection and palpation except for tumors from the CD24 population, which were detected only upon necropsy.}

Cells were isolated from passage 1 (Mouse passage 1) T1, T2, and T3, passage 2 (Mouse passage 2) T3, and unpassaged (Patients' tumor cells) T1, T4, T5, T6, T8, and T9. CD44^{CD24Lineage populations and CD44CD24Lineage cells were isolated by flow cytometry as described in Fig.} ​Fig.1.1. The indicated number of cells of each phenotype was injected into the breast of NOD/SCID mice. The frequency of tumorigenic cells calculated by the modified maximum likelihood analysis method is ≈5/10 ^{if single tumorigenic cells were capable of forming tumors, and every transplanted tumorigenic cell gave rise to a tumor (}33). Therefore, this calculation may underestimate the frequency of the tumorigenic cells because it does not take into account cell–cell interactions and local environmental factors that may influence engraftment. In addition to the markers that are shown, all sorted cells in all experiments were Lineage^{, and the tumorigenic cells from T1, T2, and T3 were further selected as B38.1. The mice were observed weekly for 4–6.5 mo, or until the mice became sick from the tumors.}

Acknowledgments

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