Intrinsic Resistance of Tumorigenic Breast Cancer Cells to Chemotherapy

Xiaoxian Li; Michael T. Lewis; Jian Huang; Carolina Gutierrez; C. Kent Osborne; Meng-Fen Wu; Susan G. Hilsenbeck; Anne Pavlick; Xiaomei Zhang; Gary C. Chamness; Helen Wong; Jeffrey Rosen; Jenny C. Chang


J Natl Cancer Inst. 2008;100(9):672-679. 

In This Article

Abstract and Introduction

Background: Tumorigenic breast cancer cells that express high levels of CD44 and low or undetectable levels of CD24 (CD44+/CD24-/low) may be resistant to chemotherapy and therefore responsible for cancer relapse. These tumorigenic cancer cells can be isolated from breast cancer biopsies and propagated as mammospheres in vitro. In this study, we aimed to test directly in human breast cancers the effect of conventional chemotherapy or lapatinib (an epidermal growth factor receptor [EGFR]/HER2 pathway inhibitor) on this tumorigenic CD44+ and CD24-/low cell population.
Methods: Paired breast cancer core biopsies were obtained from patients with primary breast cancer before and after 12 weeks of treatment with neoadjuvant chemotherapy (n = 31) or, for patients with HER2-positive tumors, before and after 6 weeks of treatment with the EGFR/HER2 inhibitor lapatinib (n = 21). Single-cell suspensions established from these biopsies were stained with antibodies against CD24, CD44, and lineage markers and analyzed by flow cytometry. The potential of cells from biopsy samples taken before and after treatment to form mammospheres in culture was compared. All statistical tests were two-sided.
Results: Chemotherapy treatment increased the percentage of CD44+/CD24-/low cells (mean at baseline vs 12 weeks, 4.7%, 95% confidence interval [CI] = 3.5% to 5.9%, vs 13.6%, 95% CI = 10.9% to 16.3%; P < .001) and increased mammosphere formation efficiency (MSFE) (mean at baseline vs 12 weeks, 13.3%, 95% CI = 6.0% to 20.6%, vs 53.2%, 95% CI = 42.4% to 64.0%; P < .001). Conversely, lapatinib treatment of patients with HER2-positive tumors led to a non–statistically significant decrease in the percentage of CD44+/CD24-/low cells (mean at baseline vs 6 weeks, 10.0%, 95% CI = 7.2% to 12.8%, vs 7.5%, 95% CI = 4.1% to 10.9%) and a non–statistically significant decrease in MSFE (mean at baseline vs 6 weeks, 16.1%, 95% CI = 8.7% to 23.5%, vs 10.8%, 95% CI = 4.0% to 17.6%).
Conclusion: These studies provide clinical evidence for a subpopulation of chemotherapy-resistant breast cancer–initiating cells. Lapatinib did not lead to an increase in these tumorigenic cells, and, in combination with conventional therapy, specific pathway inhibitors may provide a therapeutic strategy for eliminating these cells to decrease recurrence and improve long-term survival.

Conventional chemotherapies are initially effective in controlling tumor growth,[1] yet many patients relapse over time. At least two major explanations exist for these observations. The first is that all cancer cells acquire resistance, resulting in decreased overall sensitivity to therapy over time. In this case, the relative proportion of cells in residual tumors with tumorigenic properties would be expected to be similar before and after treatment. The second explanation is that a rare subpopulation of cells with tumorigenic potential is intrinsically resistant to therapy. In this case, the relative proportion of cells in residual tumors with tumorigenic properties would be expected to increase after treatment. Analogous to the propensity of dandelion roots to regenerate weeds, regrowth of tumors from an intrinsically chemotherapy-resistant subpopulation has been termed the "dandelion hypothesis".[2] Consistent with this hypothesis, we have previously shown[3] that the gene expression pattern of residual tumor cells surviving after treatment is different from that of cells in the initial tumor, with differential expression of genes involved in cell cycle arrest and survival pathways in particular. This hypothesis provides a unified explanation for the successes and failures of cytotoxic chemotherapy—namely, that although the majority of cells in the original tumor may be killed by chemotherapy, the most important target, a small population of therapy-resistant cancer cells that possess tumorigenic capacity, is spared, thereby allowing tumor regrowth. A combination of treatments that target both subpopulations would therefore be essential to prevent tumor regrowth and relapse.

A highly tumorigenic subpopulation of breast cancer cells was recently described.[4] Using cells from metastatic pleural effusions, this subpopulation of epithelial cells was identified by flow cytometry as expressing CD44 (CD44+) but low or no CD24 (CD24-/low) or a panel of nonepithelial lineage markers (Lin-). This subpopulation can form mammospheres in vitro and was shown to be enriched for tumorigenic cells by its ability to form xenograft tumors in immunocompromised mice.[4] The tumorigenicity of CD44+/CD24-/low cells has been confirmed in subsequent studies.[5,6] In addition, recently published data show that epidermal growth factor receptor (EGFR) signaling may be required for cancer self-renewal and that HER2-positive cancers may have increased self-renewal properties.[7]

In this study, we tested the dandelion hypothesis directly in human breast cancer patients in the neoadjuvant setting by evaluating whether residual tumors after chemotherapy were enriched for the tumorigenic CD44+/CD24-/low cell population and whether these cells had enhanced mammosphere-forming efficiency (MSFE). We also analyzed residual tumors from patients undergoing treatment with lapatinib, a dual-specificity inhibitor of EGFR and HER2, for the proportion of tumorigenic cells and MSFE before and after this therapy. We also assessed whether—consistent with the dandelion hypothesis, in which both dividing daughter cells and tumorigenic cancer cells must be targeted to prevent relapse—lapatinib together with conventional therapy would lead to a high pathological complete response rate.


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