Journal of the National Cancer Institute Advance Access originally published online on April 29, 2008
JNCI Journal of the National Cancer Institute 2008 100(9):672-679; doi:10.1093/jnci/djn123
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ARTICLES |
Intrinsic Resistance of Tumorigenic Breast Cancer Cells to Chemotherapy
Affiliations of authors: Breast Center, Dan L. Duncan Cancer Center, Department of Molecular and Cellular Biology, Division of Biostatistics and Department of Surgery, Baylor College of Medicine, Houston, TX 77030
Correspondence to: Jenny C. Chang, MD, Breast Center at Baylor College of Medicine, 1 Baylor Plaza BCM 600, TX 77030 (e-mail: jcchang{at}bcm.edu).
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ABSTRACT |
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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.
Prior knowledge Breast cancer cells that express high levels of CD44 and low levels of CD24 (CD44>/CD24>/low) may be responsible for tumor recurrence. HER2-positive cancers may have increased self-renewal properties. Study design Breast cancer patients were treated with neoadjuvant chemotherapy (HER2-negative patients) or the epidermal growth factor receptor/HER2 inhibitor lapatinib (HER2-positive patients). Cells that were isolated from biopsy samples taken before and after therapy were assayed for the percentage of CD44>/CD24>/low cells and the ability to form mammospheres in vivo as an indication of self-renewal. Contributions Treatment with chemotherapy increased the percentage of CD44>/CD24>/low cells and the formation of mammospheres, whereas treatment with lapatinib non–statistically significantly reduced the percentage of CD44>/CD24>/low cells and the formation of mammospheres. Implications Tumorigenic CD44>/CD24>/low cells may be chemotherapy resistant. Lapatinib treatment did not increase the proportion of these tumorigenic cells in HER2-positive breast cancers. Limitations Breast cancers and samples taken from them contain several different cell types; thus, it is difficult to distinguish the roles of each cell type and those of cell–cell interactions in the effects observed. CD44>/CD24>/low cells have been shown to have high tumorigenic potential and to be resistant to chemotherapy and radiation therapy, but their role in metastasis is still unclear.
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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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Subjects and Methods |
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Patients and Clinical Samples
Patients with locally advanced breast cancer (>5 cm in diameter or with clinically palpable axillary adenopathy) were enrolled in phase 2 clinical trials from January 18, 2000, to December 21, 2006. These phase 2 studies were designed to identify biological correlative markers of response to therapy. In a prospectively planned protocol, a subset of these patients were enrolled in an institutional review board–approved "stem cell" protocol, in which the biopsies collected were examined for tumorigenic markers. All patients provided written informed consent. Patients with HER2-negative tumors (n = 31) were randomly assigned to receive preoperative docetaxel (T) or doxorubicin and cyclophosphamide (AC) for 12 weeks at standard doses. Patients who received conventional chemotherapy had biopsies taken at baseline and at weeks 3 and 12. Patients with HER2-positive tumors (n = 21) received lapatinib (an inhibitor of the EGFR/HER2 tyrosine kinase) for 6 weeks at 1500 mg daily, followed by T and trastuzumab for 12 weeks at standard doses. These patients had biopsies taken at baseline and at weeks 2, 4, and 6 of lapatinib therapy. For both studies, clinical response according to standard Response Evaluation Criteria in Solid Tumors criteria (8) was assessed in all patients by physical examination before and after therapy by two experienced breast specialists (JCC, CKO). Pathological complete response (pCR) after preoperative therapy was defined as complete disappearance of all invasive cancer or only residual minute foci (<1 cm in diameter) (8).
Immunohistochemistry
Standard methods for immunohistochemical analysis for estrogen receptor (ER), progesterone receptor (PgR), and HER2 have been described in detail elsewhere (9). Subjective estimation of the proportion of stained cells on the entire slide (0, none; 1, <1%; 2, 1%–10%; 3, >10%–33%; 4, >33%–67%; and 5, >67%) and the intensity of positive signal (0, none; 1, weak; 2, intermediate; and 3, strong) of all slides were semiquantitatively evaluated by light microscopy as described (9). The overall score was expressed as the sum of the proportion and intensity scores. Tumors were regarded as expressing ER and PgR if the overall score was greater than 3 and HER2 positive if the overall score was 5 or greater or if HER2 gene amplification, as detected by fluorescence in situ hybridization, was present.
Analytic Flow Cytometry and Mammosphere-Forming Efficiency
A longitudinal section of each core biopsy was examined by standard hematoxylin–eosin (H & E) staining and for the expression of the epithelial marker cytokeratin 8 (CK8). Only samples that contained at least 50% epithelial cancer cells were analyzed. The remaining portion of the same core biopsy was placed immediately in cold RPMI-1640 supplemented with 10% heat-inactivated newborn calf serum (HINCS, Invitrogen, Carlsbad, CA). Within an hour, the samples were minced and digested in 10–15 mL of mammary epithelial growth medium (MEGM) with 250–300 U/mL of collagenase at 37°C. The samples were filtered, washed with Hank’s Balanced Salt Solution (HBSS, Invitrogen), and subjected to hypotonic shock (9 mL of sterile H2O for 10 seconds followed with 1 mL 10x HBSS) to lyse red blood cells. Approximately 106 single cells from each biopsy sample were resuspended in HBSS supplemented with 2% HINCS, incubated for 15 minutes at 4°C with mouse monoclonal anti-CD44, anti-CD24, and anti–Lin antibodies (phycoerythrin-conjugated anti-CD2, anti-CD3, anti-CD10, anti-CD16, anti-CD18, anti-CD31, and anti-CD140B; Pharmingen, San Diego, CA) according to the manufacturer's instructions. The cells were then washed twice with 10 mL of HBSS, resuspended with HBSS supplemented with HINCS and propidium iodide (10 µg/mL), and analyzed using Dako MoFlo flow cytometry (Dako, Carpinteria, CA). Side and forward scatter were used to eliminate debris and cell doublets. Lin> cells were further analyzed for CD44 and CD24 staining. The percentage of CD44>/CD24>/Lin> cells was assessed in samples from biopsy cores taken before, during, and after therapy. Cells stained only with 10 µg/mL propidium iodide without antibody staining were used as negative controls.
Portions of the collagenase-digested core biopsies were used in MSFE assays. Isolated single-cell suspensions were plated on nonadherent (polyhema-coated) plastic and counted with a hematocytometer, and 20 000 cells were then seeded into a six-well ultralow attachment plate supplemented with 2 mL MEGM. Medium was replaced every 3–4 days. The primary mammospheres were allowed to grow for 3 weeks. Mammospheres were counted by week 3. MSFE was calculated by dividing the number of mammospheres by the number of seeded cells. In addition, established mammospheres were serially passaged by dissociation, and single cells were replated on fresh nonadherent plastic to form secondary mammospheres, which were counted using a Leica MZ16 F microscope after 3 weeks.
Xenograft Transplantation Assays of Tumorigenicity
Tumorigenicity of human breast cancer samples before and after chemotherapy was assayed by xenograft transplantation into immunocompromised severe combined immunodeficiency (SCID)/Beige mice. As stated earlier, biopsy samples were examined after H & E and CK8 staining and were used only if they were found to contain 50% or more epithelial cancer cells. The biopsies were minced with a blade to yield approximately 1-mm3 fragments. For each clinical sample, two 3-week-old female SCID/Beige mice (Charles River Laboratories, Wilmington, MA) were used as xenograft transplant hosts. Briefly, host mice were anesthetized with 1.5 µL/g body weight Rodent Combination III (ketamine at 37.6 mg/mL, xylazine at 1.92 mg/mL, and acepromazine at 0.38 mg/mL), and both #4 mammary fat pads from each mouse were cleared of endogenous mammary epithelium by the method of DeOme (10). Each of the four cleared fat pads was then implanted with a single tumor fragment. The mice were killed 10–20 weeks after transplantation by anesthesia overdose with subsequent cervical dislocation. Tumor outgrowths, identified either because they were palpable or detected at histological evaluation, were counted. Growth of transplanted fragments was confirmed by bromodeoxyuridine incorporation (0.01 cm3/g body weight of a 25 mg/mL solution in phosphate-buffered saline injected 2 hours before mice were killed). All mice were maintained and treated in accordance with the National Institutes of Health Guide for the Care and Use of Experimental Animals with approval from the University of Texas at M. D. Anderson Institutional Animal Care and Use Committee.
Statistical Analysis
Spearman rank correlation analysis was used to investigate associations between the baseline percentage of CD44>/CD24>/low cells and MSFE, for all patients and for patients stratified by HER2 status (positive vs negative). Baseline percentages of CD44>/CD24>/low cells were compared between patients with HER2-negative vs HER2-positive and ER-negative vs ER-positive tumors using the Wilcoxon rank sum test. Linear mixed-effects models were used to model the changes in the relative proportions of CD44>/CD24>/low cells and of MSFE over time. Specifically, the intercept of an individual patient was considered as a random effect and gave an estimate of the initial value of CD44>/CD24>/low or MSFE. Time was included as a continuous covariate with both linear and quadratic terms. Data from chemotherapy-treated subjects (HER2 negative) and lapatinib-treated subjects (HER2 positive) were modeled separately. The resulting models provided estimates of linear and/or quadratic time trends while taking into account correlations among a given patient's responses over time. Mean of observed values and associated 95% confidence intervals (CIs) were reported as twice the SEM. P values were computed by model-based contrasts. All statistical tests were two-sided, and P values less than .05 were considered statistically significant.
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Results |
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Clinical Outcomes
Patients with HER2-negative tumors (n = 31) were randomly assigned in a phase 2 study to receive 12 weeks of preoperative T or AC treatment. Their mean age was 47 years (range = 31–69 years), and mean tumor size was 6.7 cm (range = 3–13 cm) at baseline. Pathological complete response/near pathological complete response (pCR/npCR) was observed in 7 of the 31 (23%) patients (Table 1). Not surprisingly, most patients (six of seven [86%]) who achieved pCR had hormone receptor–negative tumors.
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Patients with HER2-positive tumors (n = 21) were randomly assigned to 6 weeks of preoperative lapatinib treatment. Their mean age was 54 years (range = 28–84 years), and mean tumor size was 10 cm (range = 2.5–30 cm) at baseline. The clinical response after treatment was 63%. Following lapatinib treatment, all patients then received 12 weeks of preoperative trastuzumab and T treatment. The pathological complete response rate (pCR/npCR) on completion of all preoperative therapy was 62%, which was much higher than expected (Table 1).
Percentage of Epithelial Cells in Biopsy Samples Taken Before and After Treatment
To evaluate the effect of chemotherapy on the tumorigenic subpopulation of cells compared with other cells present in the primary tumor, we examined tumor specimens taken before, during, and after treatment for changes in the relative proportion of tumorigenic cell types. All biopsies were examined after H & E and CK8 staining and determined to contain 50% or more epithelial cancer cells before analysis. The mean percentage of epithelial cancer cells (ie, cells staining for CK8) was unchanged with chemotherapy, from baseline (63%, 95% CI = 51% to 75%) to week 3 (53%, 95% CI = 33% to 73%) and week 12 (68%, 95% CI = 44% to 92%) (Figure 1, A). Similarly, the mean epithelial cancer cell content was unchanged at baseline (73%, 95% CI = 65% to 81%) and weeks 2 (65%, 95% CI = 55% to 75%), 4 (60%, 95% CI = 46% to 74%), and 6 (65%, 95% CI = 51% to 79%) of lapatinib treatment (Figure 1, B). Hence, the proportion of cancer epithelial to stroma cells was unchanged after therapy.
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Effect of Chemotherapy on Tumorigenic Cells in Patients With HER2-Negative Tumors
No statistically significant difference was observed in the percentage of CD44>/CD24>/low cells at baseline in ER-positive vs ER-negative tumors before treatment (5.6% vs 3.9%, P = .312). In matched human breast cancer biopsies (n = 31 pairs), the percentage of CD44>/CD24>/low cells increased with chemotherapy, from a mean of 4.7% (95% CI = 3.5% to 5.9%) at baseline to 13.6% (95% CI = 10.9% to 16.3%) after 12 weeks of chemotherapy (P < .001) (Figure 2, A), indicating an enrichment of tumorigenic breast cancer cells. This statistically significant increase was observed regardless of molecular subtype—triple negative (ER, PgR, and HER2 negative) or ER positive (Figure 2, B and C). No difference in enrichment of tumorigenic cells was observed with AC vs T (data not shown).
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We next performed biological studies to further characterize the cell population remaining after chemotherapy. Consistent with the increase in the relative proportion of tumorigenic cells, the ability of these cells to form mammospheres, as determined by MSFE assays, was statistically significantly increased after chemotherapy in matched pre- and postchemotherapy samples (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) (Figure 3, A). This increase in MSFE was observed regardless of molecular subtype, that is, in both triple negative (ER-, PgR-, and HER2-negative) and ER-positive samples (Figure 3, B and C). In addition, samples with a higher percentage of CD44>/CD24>/low cells had increased MSFE, with a positive correlation coefficient of r = 0.45 (P = .027) (Figure 4, A). Primary mammospheres could be serially passaged to yield secondary mammospheres, showing enrichment for mammosphere-initiating cells, with a statistically significant threefold increase in secondary mammosphere formation (median difference = 10.0 mammospheres/1000 cells, 95% CI = 4.5 to 19.4; P = .016) (Figure 4, B). These results suggest enhanced symmetric self-renewal in cancers compared with asymmetric self-renewal, as observed in normal mammary epithelium (11–13).
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We established human breast cancer xenografts in immunocompromised SCID/Beige mice using human breast cancer biopsy samples from patients who were treated with conventional chemotherapy. The number of xenografts that grew out from the postchemotherapy biopsy samples was almost twice that of pretherapy specimens, increasing from 4 of 14 (29%) to 7 of 14 (50%) patient samples transplanted. These data are consistent with the possibility that cells surviving T and AC chemotherapy were enriched for a tumorigenic CD44>/CD24>/low subpopulation and for cells with increased potential to form mammospheres in culture and tumors in immunocompromised mice.
Effect of EGFR/HER2 Inhibition on Tumorigenic Cells in Patients With HER2-Positive Tumors
Recently published data suggest that EGFR signaling is required for cancer cell self-renewal in mammospheres (7), suggesting that these tumorigenic cells may be susceptible to therapeutic agents targeting this signaling pathway in vivo. If so, then the relative percentage of CD44>/CD24>/low cells should not change as a function of treatment. Consistent with this prediction, and in contrast with chemotherapy treatment, lapatinib treatment did not increase the percentage of tumorigenic cells. Instead, it led to a non–statistically significant decrease in the percentage of tumorigenic cells in matched biopsies from a mean of 10.0% (95% CI = 7.2% to 12.8%) at baseline to 7.5% (95% CI = 4.1% to 10.9%) after 6 weeks of lapatinib (Figure 5, A). Interestingly, the baseline CD44>/CD24>/low values were higher in the HER2-positive breast cancers than in the HER2-negative cancers (10.0%, 95% CI = 7.2% to 12.8%, vs 4.7%, 95% CI = 3.5% to 5.9%).
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Consistent with its effect on tumorigenic cells, lapatinib treatment again led not to an increase, as with chemotherapy, but to a non–statistically significant decrease in self-renewal capacity as measured by 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%; Figure 5, B). Targeting both tumorigenic cells and dividing daughter cells would therefore be essential in preventing cancer relapse. Consistent with its effect on tumorigenic cells, use of lapatinib to augment conventional therapy increased the pathological complete response rate by three- to fourfold (Table 1), compared with the published rate with conventional therapy alone (14–16).
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Discussion |
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We have shown for the first time, to our knowledge, that tumorigenic cancer cells are intrinsically resistant to conventional AC or T chemotherapy. The tumorigenic cancer cells that remained after chemotherapy had unique properties of enhanced self-renewal, as demonstrated by the formation of mammospheres, and may display increased propensity for tumor formation, as demonstrated by increased formation of xenograft outgrowths. Inhibition of EGFR/HER2 signaling through lapatinib treatment led to a decrease, albeit non–statistically significant, in the percentage of tumorigenic cancer cells and in self-renewal (ie, MSFE) in HER2-positive cancers. This observation may explain, in part, the large survival benefit conferred by targeting this signaling pathway together with chemotherapy when compared with chemotherapy alone (17).
Lapatinib has been shown to statistically significantly prolong survival in patients with metastatic HER2-positive breast cancer (18). Recent published data show that EGFR signaling is required for mammosphere formation and that HER2-positive cancers have increased self-renewal properties (7). These data may, in part, explain the observation of a higher percentage of CD44>/CD24>/low cells in HER2-positive than HER2-negative cancers. We here and others (16, 19) have reported pathological complete response rates, a validated surrogate marker of long-term survival, of approximately 10%–20% with 12 weeks of conventional chemotherapy in patients with locally advanced breast cancer. Results of this study are encouraging and suggest that inhibition of key regulatory pathways responsible for self-renewal could augment the effects of conventional therapy and improve clinical outcome.
The mammosphere culture technique was first used by Reynolds and Rietze (20) for the cultivation of neural cells in conditions that promoted neural stem cell enrichment, although some data have suggested that these neurospheres may not be clonal in origin (21). However, using similar cultivation techniques, Wicha et al. (22–24) showed that normal mammary stem cells undergo primarily asymmetric self-renewal to form mammospheres, whereas the differentiated cells undergo apoptosis. Malignant cells behave similarly, with a higher propensity for symmetric self-renewal. Recent publications (7,25) further support increased MSFE as an indicator of enhanced self-renewal. In this study, we reported a statistically significant correlation (r = 0.45) between MSFE and the percentage of CD44>/CD24>/low tumorigenic cells. Many commonly associated biological relationships, like ER with PgR, in clinical samples show similar correlation coefficients (26). We have also demonstrated self-renewal capacity with a statistically significant increase in secondary mammosphere formation, suggesting enhanced symmetric self-renewal.
This study has several potential limitations. Several potential pitfalls, such as cellular heterogeneity, exist with the use of human breast cancer biopsy samples. In addition, the functional significance of CD44>/CD24>/low cells is still unclear. These cells have tumorigenic potential relative to other cell populations in the tumor (4), are resistant to radiation therapy (25,27), have decreased MSFE, and. based on our findings. may also play a role in intrinsic chemoresistance. However, their importance in metastasis, and thus in disease recurrence and breast cancer mortality, is not yet well understood. In addition, our results with xenograft transplantation are suggestive and not conclusive of increased tumorigenicity after chemotherapy.
In summary, we have provided evidence supporting the intrinsic chemoresistance of tumorigenic breast cancer cells. Targeting residual cells with stem cell self-renewal properties in combination with conventional chemotherapy may provide a specific approach to prevent cancer recurrence and improve long-term survival.
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Funding |
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Breast Cancer Research Foundation (to J.C.C.); Emma Jacobs Clinical Breast Cancer Fund (to J.C.C.); Helis Foundation (to J.R., J.C.C., M.T.L.); Breast Cancer SPORE (P50 CA50183 to C.K.O., J.C.C., M.T.L.); National Cancer Institute (R01 CA112305-01 to J.C.C.); Glaxo Smith Kline (grant-in-aid to J.C.C.); US Army Medical Research and Materiel Command (DAMD17-01-0132 and W81XWH-04-1-0468 to J.C.C.).
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NOTES |
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The two first authors (X. Li and M. T. Lewis) and two senior authors (J. Rosen and J. C. Chang) contributed equally to this study. The sponsors had no role in the study design, data collection and analysis, interpretation of the results, the preparation of the manuscript, or the decision to submit the manuscript for publication.
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Manuscript received November 2, 2007; revised February 18, 2008; accepted March 18, 2008.
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