Patent Application
20120295804
The incidence of breast cancer remains relatively high although recent advancements in treatment modalities have improved overall survival. In the United States, breast cancer represents over one-fifth of all cancers (Kakarala, M. and M. S. Wicha. 2008. J. Clin. Oncol. 26:2813-2820). About 10% of hereditary breast cancer has been linked to highly penetrant mutations in BRCA1 and BRCA2 (Simon, M. S, and N. Petrucelli. 2009. Methods Mol. Biol. 471:487-500; Vega, A. et al. 2009. Gynecol. Oncol. 112:210-214). However, 90-95% of breast cancer is sporadic and cannot be attributed to any currently known germline mutations (Simon, M. S, and N. Petrucelli. 2009. Methods Mol. Biol. 471:487-500; Vega, A. et al. 2009. Gynecol. Oncol. 112:210-214). Environmental factors appear to play an important role in sporadic cases (Chia, K. S. 2008. Novartis Found. Symp. 293:143-150). In addition, stress and its associated hormones have been linked to cancer progression (Arranz, A. et al. 2010. Mol. Cancer. 9:261). Breast cancer patients with high body mass indices show poor clinical outcome regardless of hormone receptor/HER2 status of the primary cancer (Phipps, A. I. et al. 2011. Cancer Epidemiol. Biomarkers Prev. 20:454-463).
Breast cancer cells (BCCs) have a predilection to metastasize to the bone marrow, brain, liver and lung (Cocoran, K. E. et al. 2008. PLoS One 3:e2563; Kakarala, M. and M. S. Wicha. 2008. J. Clin. Oncol. 26:2813-2820). In bone marrow, BCCs can form gap junctional intercellular communication (GJIC) with stroma, close to the endosteum (Cocoran, K. E. et al. 2008. PLoS One 3:e2563). These findings are consistent with earlier studies that reported on slower growth rates of BCCs close to the endosteum of mice (Rao, G. et al. 2004. Cancer Res. 64:2874-2881). Loss of connexin 43, a gap junction protein, is linked to malignancy in cancers, breast cancer included (Bodenstine, T. M. et al. 2010. Cancer Res. 70:10002-10011; King, T. J. et al. 2002. Mol. Carcinog. 35:29-41). A key role for GJIC in the quiescence of BCCs within the stromal compartment of bone marrow has been recently reported, with activity attributed to movement of microRNAs from stroma to BCCs for reduced cell cycle activity (Lim, P. K. et al. 2011. Cancer Res. 71:1550-1560). Despite the involvement of GJIC as a facilitator of BCC dormancy, connexins cannot be directly targeted since hematopoietic activity depends on GJIC among bone marrow stromal cells (Milson, M. D. and A. Trump. 2011. Nat. Immunol. 12:377-379).
Octamer-binding transcription factor 3/4 (Oct4) is a member of the POU DNA-binding domain family and a stem cell marker (Liedtke, S. et al. 2008. Biol. Chem. 389:845-850). There are three Oct4 mRNAs: Oct4A, Oct4B and Oct4B1 (Wang, X. and J. Dai. 2010. Stem Cells 28:885-893). The Oct4B transcript can use alternate translational start sites to generate three protein isoforms (Zhang, W. et al. 2010. Biochem. Biophys. Res. Commun. 394:750-754). Although the role of Oct4 in self-renewal of normal adult stem cells remains debatable, Oct4A seems to be relevant for maintaining pluripotency of embryonic stem cells (Wang, X. and J. Dai. 2010. Stem Cells 28:885-893). Oct4 is over-expressed in many tumor types, including breast cancer (Guzman-Ramirez, N. et al. 2009. Prostate 69:1683-1693; Hu, T. et al. 2008. Cancer Res. 68:6533-6540; Suva, M. L. et al. 2009. Cancer Res. 69:1776-1781; Zhang, S. et al. 2008. Cancer Res. 68:4311-4320). Oct4 mediates chemotherapy drug resistance in hepatocellular carcinoma, lung and prostate cancer, and maintains self-renewal of lung cancer stem cells (Wang, X. Q. et al. 2010. Hepatology 52:528-539; Chen, Y. C. et al. 2008. PLoS One 3:e2637). Furthermore, Oct4 expression maintains cell survival through the Oct4/Tcl1/Akt1 pathway in MCF7 BCCs (Hu, T. et al. 2008. Cancer Res. 68:6533-6540).
There have been several reports on the identification of breast cancer stem cells via surface marker expression, including CD44+/CD24−/lin− or ALDH1+, or via efflux of Hoechst dye with the side population (Al-Hajj, M. et al. 2003. PNAS USA 100:3983-3988; Ginestier, C. et al. 2007. Cell Stem Cell 1:555-567; Liu, S, and M. S. Wicha. 2010. J. Clin. Oncol. 28:4006-4012). Researchers have found that certain breast cancer stem cell types, specifically CD44+CD24−/low cells that express Oct4, are potential targets for chemotherapy (Eriksson et al. 2007. Mol. Ther. 15:2088-2093) or drug treatments for breast cancer.
It is evident that not all BCCs are functionally equal and perhaps a particular subset is responsible for the formation of GJIC with bone marrow stroma (Lim, P. K. et al. 2011. Cancer Res. 71:1550-1560). It has now been found that a subset of BCCs with self-renewal and tumor-initiating properties shows preference for GJIC with bone marrow stroma. The identity of BCC subset with preference for dormancy in bone marrow, and perhaps in other organs, provides important information about the mechanism by which BCCs adapt dormancy and also provides an understanding of how the process could potentially be reversed to prevent tertiary metastasis (Cocoran, K. E. et al. 2008. PLoS One 3:e2563; Lim, P. K. et al. 2011. Cancer Res. 71:1550-1560).
The present invention is a breast cancer biomarker which comprises a breast cancer cell with a phenotype OCt4hi/CD44hi/med/CD24−/+. In the present invention it has been shown that cells with the OCt4hi/CD44hi/med/CD24−/+ phenotype represent dormant, metastatic breast cancer cells that can exist in bone marrow. As a result, the breast cancer cells of the present invention establish gap junctional intercellular communication with bone marrow stroma.
The present invention is also a method for developing a breast cancer prognosis for a patient which comprises detecting the presence of the biomarker cells of the present invention in a patient sample wherein the presence of said biomarker is indicative of a the presence of dormant metastatic breast cancer cells in the patient and a poor prognosis for the patient. Additionally, the present invention is a method of diagnosing metastatic breast cancer which comprises detecting the presence of the biomarker of the present invention in a patient sample, wherein the presence of the biomarker indicates that the patient has metastatic breast cancer.
Yet another object of the present invention is a method for identifying chemotherapeutic agents that can kill dormant metastatic breast cancer cells in bone marrow which comprises that steps of contacting a breast cancer cell with a phenotype Oct4hi/CD44hi/med/CD24−/+ in vitro with an agent; and determining whether the agent is capable of killing said breast cancer cell, wherein death of said breast cancer cell is indicative of the ability of the agent to kill dormant metastatic breast cancer cells in bone marrow.
The identity of tumor-initiating cells in breast cancer has been examined, however, there is no clear consensus on the exact phenotype of this population. The expression of stem cell markers such as Oct4, Sox2, and Nanog has been linked to breast cancer (Bourguignon, L. Y. et al. 2008. J. Biol. Chem. 283:17635-17651; Hu, T. et al. 2008. Cancer Res. 68:6533-6540; Lengerke, C. et al. 2011. BMC Cancer 11:42). The present invention is focused on Oct4-expressing cells, based on the role of this gene as a regulator of other stem cell genes (Yang, J. et al. 2010. PLoS One 5:e10766), as well as its detection in adult and embryonic stem cells (Greco, D. J. et al. 2007. Stem Cells 25:3143-3154; Johansson, H. and S. Simonsson. 2010. Aging 2:815-822). Oct4 exists as multiple isoforms, e.g., Oct4A, Oct4B, and Oct4B1 (Asadi, M. H. et al. 2010. Int. J. Cancer 128:2645-2652). In the present invention, the status of cells as being either Oct4-expressing (Oct4+) or lacking expression of Oct4 (Oct4−) was not the only basis for identifying cells. Instead, Oct4+ or Oct4− cells have been subdivided in the instant invention, depending on the level of Oct4 gene expression. To identify subsets of BCCs, BCCs were stably transfected with pEGFP1-Oct3/4 and different BCC subsets were then isolated based on the intensity of GFP emission, since GFP emission was shown to correlate with Oct4 protein levels. Studies with the stably transfected BCCs indicated that the subset with the highest Oct4 expression consisted of breast cancer-initiating cells that expressed certain stem cell genes.
Thus, the present invention is a population of BCCs with preference for establishing dormancy in bone marrow. This subset of BSCs shows self-renewal, divides asymmetrically, has a long doubling time, exhibits tumor-initiating properties, and expresses high levels of Oct4 and other stem cell-associated genes. Moreover, the breast cancer stem cells identified as being high Oct4 expressing cells, designated Oct4hi, have been shown to be independent of CD44/CD24 status, which has been reported by others to be a critical feature of breast cancer stem cells. Also of importance to the instant invention is the finding that the breast cancer stem cells expressing high levels of Oct4 exhibit functional gap junction intercellular communication (GJIC) with bone marrow stroma, indicating that these cells can establish dormancy and remain resistant to chemotherapy. Thus, targeting these particular BCCs provides a new tool for therapy of breast cancer. Finally, a hierarchy of BCC subsets has also been identified, which can be used as a basis to identify combinations of markers to demarcate cancer cell subsets for diagnosis and prognosis, as well as to develop novel treatments and further the understanding of breast cancer dormancy and resurgence. These discoveries have led to the compositions and methods of the present invention. Hence, the present invention provides a novel BCC subset that can be exploited in the diagnosis and treatment of breast cancer.
A role of miRNA and GJIC in BCC quiescence within the stromal compartment of bone marrow was recently reported (Lim, P. K. et al. 2011. Cancer Res. 71:1550-1560). Based on this finding, experiments were performed to identify of BCC subset or subsets that are responsible for cancer cell dormancy. Initial experiments focused on Oct4-expressing BCCs because of its involvement in pluripotency and tumorigenesis. Relevant Oct4 isoforms expressed in breast cancer were identified by screening a panel of eight BCC lines, by western blots with an antibody that detects all isoforms of Oct4. The eight BCC lines studied included MDA-MB-231, MDA-MB-468, MDA-MB-453, T47D, MCF-7, HCC1954, HCC1143, and BT549. The results showed light bands at the predicted size for Oct4A (45 kDa) and undetectable bands at the regions expected for Oct4B proteins in all cell lines. Positive controls, using extracts from mesenchymal stem cells, showed predictable bands for Oct4B at 18, 21 and 29 kDa. These data provided evidence of the role of Oct4A as a mediator of pluripotency and the inability of Oct4B to sustain self-renewal (Wang, X. and J. Dai. 2010. Stem Cells 28:885-893).
BBC lines MDA-MB-231 and T47D were selected for subsequent studies due to their established differences in hormone receptor expression. RT-PCR with primers specific for Oct4A and Oct4B showed bands at the predicted size (350 bp) for both transcripts, although detectable bands for Oct4B required 40 cycles. The primers used are shown below in Table 1. Real-time RT-PCR confirmed lower Ct values for Oct4A indicating higher levels of its mRNA.
Due to the relatively low expression of Oct4, experiments were performed to determine if the low expression could be explained by low frequency of Oct4+ BCCs in the cell lines. Immunocytochemistry was performed in MDA-MB-231 and T47D cells in culture with the same anti-Oct4 antibody used in the western blots. Cells that stained bright green were designated as Oct4+/hi BCCs and were found in the following frequencies: 1.0%±0.37 for MDA-MB-231 and 0.84±0.17 for T47D (mean±SD, n=5). Thus, the expression of Oct4 was low in these cell lines, a finding that is consistent with low frequency of other stem cells. Although the levels of expression were low in the cell lines, the Oct4+ cells did cluster.
Oct4 expression was next studied in primary human breast cancer tissue samples and compared with normal tissues surrounding the malignant regions. The demographics of patients that served as sources for tissue are described in Table 2. Western blot analyses indicated low levels of Oct4 protein in the patient tumor samples and undetectable expression in the control, non-malignant tissue. Hematoxylin-eosin staining showed no evidence of tissue necrosis. Parallel staining for Oct4 protein indicated intense labeling for the highly malignant tissue with moderately to poorly differentiated areas. Similar staining with the surrounding normal tissue showed light to negative staining.
Experiments were then performed to determine if Oct4+ BCCs can be identified in the blood of patients presenting with different stages of breast cancer, including and excluding treatment (Table 3). Results of flow cytometry on blood samples from three representative patients are shown in
The low frequencies of Oct4+ BCCs within the eight cell lines examined (<1%), as well as their resistance to chemotherapy in patients indicated that the level of Oct4 expression may be correlated with the maturity of the BCC. Therefore, different BCC subsets were selected based on Oct4 expression. To do this, MDA-MB-231 and T47D were stably transfected with an Oct4 reporter vector, pEGFP1-Oct3/4 (Gerrard, L. et al. 2005. Stem Cells 23:124-133). The transfected cells were sorted into three subsets based on GFP expression levels. The top 5% of GFP expressers were designated Oct4hi and the lowest 5%, Oct4−. The population between the extremes were designated Oct4med. GFP intensities were validated for correlation with Oct4 protein by intracellular flow cytometry of pEGFP1-Oct3/4 transfectants. The Oct4− population was similar to isotype control. In contrast, the mean fluorescence intensity (MFI) of cells labeled with APC-anti-Oct4 correlated with GFP intensities. The MFI of Oct4hi was approximately 8 fold higher than Oct4med, indicating that GFP intensity was proportional to Oct4 protein levels. The Oct4− population was not due to the loss of pEGFP1-Oct3/4 since treatment of the stable transfectants with a G9a histone methyltransferase inhibitor, BIX01294 (Shi, Y. et al. 2008. Cell Stem Cell 3:568-574), induced GFP expression in which 92.5%±4.4% of the Oct4− BCCs reverted to high GFP expression.
Experiments were then performed to quantify the frequency of tumorsphere formation in BCC subsets. In order to accurately quantify the frequency of tumorsphere formation in serial passages, the stability of Oct4hi cells in culture was first examined. After two weeks in culture, the sorted Oct4hi BCCs emerged as two distinct populations with regard to GFP intensities. The percentage of Oct4hi cells was 36.5±5.9 (mean±SD, n=15), suggesting that the Oct4hi cells differentiated rapidly in culture. This could not be explained by contaminants from Oct4− cells during sorting since all cells were Oct4hi immediately after sorting. Furthermore, only singlet was sorted, indicating that the Oct4hi cells could not be explained by doublets of two low GFP cells. Based on the instability of cultures to maintain Oct4hi BCCs, subsequent experiments were performed with freshly sorted cells.
Three sorted BCC subsets were then compared for tumorsphere formation. Oct4hi cells, when plated at 1 cell/well, formed a tumorsphere in low attachment 96-well plates, whereas Oct4med and Oct4− cells did not form tumorspheres. Oct4med cells showed small clusters of less than 20 cells, which were then designated as negative tumorsphere. Single-cell derived tumorspheres from Oct4hi BCCs (MDA-MB-231 and T47D) were tested for serial passaging in low attachment plates four times. The frequencies of tumorsphere formation from the parental unsorted cells, Oct4med, and Oct4hi BCCs, showed similar results for both MDA-MB-231 and T47D. Oct4hi BCCs showed greater than 96% efficiency in tumorsphere formation at each passage. The tumorsphere sizes in each passage were comparable, suggesting similar cellular maturity. The small tumorspheres from Oct4med BCCs could not be serially passed. Of note, cells at the periphery of the tumorspheres showed a decrease in GFP signal, suggesting differentiation within the tumorsphere. It was concluded that since PO Oct4− BCCs did not form tumorspheres, these were differentiated as compared to the primitive status of Oct4hi cells.
Evidence of self-renewal in noble agar was investigated with the three BCC subsets. The cultures in noble agar were followed and the images captured at the time when one Oct4hi BCC formed two cells. Comparable GFP intensities were observed for the two cells, suggesting self-renewal. The half-life of GFP is about 20 hours. Since the GFP intensities of the parent and daughter cells were similar, the results indicated that continued expression of Oct4 must be replacing the degraded GFP. The progeny of Oct4med showed reduced GFP intensity. The Oct4− BCCs divided into two cells, with undetectable GFP.
The two-cells from the Oct4hi cultures were expanded for two weeks in suspension cultures. The cells in the suspension cultures were re-plated in noble agar and the results showed colonies of cells with varied GFP intensities and different sizes, indicating that the original cloned cell generated heterogeneous populations of BCCs. These data indicated that the original cell was tumor-initiating with self-renewal properties. Oct4med and Oct4− cells did not expand into cells that can form colonies in noble agar.
The cell cycle status of BCC subsets was then examined; specifically, the cell cycle phase of Oct4hi, Oct4med and Oct4− BCCs was compared. Doubling times in proliferation assays were determined. Oct4hi BCCs showed approximately 3-fold greater doubling time than unsorted, Oct4med and Oct4− BCCs (
Relative proliferation rates of BCC subsets were determined, specifically rates of proliferation for Oct4hi and Oct4−. Bright-field and fluorescence images of cell divisions of representative Oct4+ and Oct4− cells were obtained by video time-lapse microscopy at different time points. The first cell division for Oct4+ cells was 7 hours after the start of time-lapse imaging, and the cell cycle times of the daughter cells were markedly different. One daughter divided at 38 hours, whereas the other daughter did not divide until 64 hours. The cell division at 38 hours resulted in three cells, and one of the cells died. In contrast, for the Oct4− cell, the cell cycle rates of the daughters and granddaughters were similar. The first cell division occurred 2 hours after the start of time-lapse imaging, and the second and third divisions of all the progeny occurred by 30 hours and 59 hours, respectively.
The production of mixed colonies from single Oct4hi cells, and the images from the time lapse studies, indicated that Oct4hi cells may be asymmetrically dividing. Recent evidence has suggested that when stem cells asymmetrically divide, they give rise to daughters with differing proliferative rates (Cicalese, A. et al. 2009. Cell 138:1083-1095; Costa, M. R. et al. 2011. Development 138:1057-1068). To test whether BCC subsets gave rise to daughters with differing proliferative rates, MDA-MB-231 cell divisions were monitored and the daughters tracked by time-lapse video microscopy. Due to the qualitative nature of visualizing GFP by immunofluorescence, cells were classified as Oct4− and Oct4+, rather than the Oct4−, Oct4med and Oct4hi. Cell cycle times of individual Oct4− and Oct4+ cells were monitored. Unlike the population doubling times calculated from cell proliferation (
Experiments were then performed to determine if drug resistance genes played any role in the differentiation of BCC subsets. Since Oct4hi BCCs showed an immature phenotype, this subset was tested for its ability to exclude Hoechst dye, a characteristic commonly reported for stem cells. Oct4hi cells were incubated in the presence or absence of verapamil, which inhibits multi-drug resistance transporters, and then dye exclusion was tested in four different experiments. Oct4hi, Oct4med and Oct4− cells were tested. The Oct4hi population showed a 3-fold difference in dye retention with verapamil (
It was then determined if different expressions of ABCG2 and MDR1 drug resistance genes could explain the results of the Hoechst dye exclusion experiments (
Next, stem cell gene expression was studied in the BCC subsets. Freshly sorted Oct4hi cells were cultured for one week to obtain Oct4hi and Oct4med cells and then compared to the parental post-sorted Oct4− cells. The analyses of stem cell PCR arrays are presented as a fold change of Oct4hi/med vs. Oct4− cells (
The qPCR array data was validated by western blots for specific stem cell genes with nuclear extracts from Oct4hi, Oct4med and Oct4− MDA-MB-231 and T47D. The stem cell markers employed included Sox2, Musashi-1, Nanog, Notch and REST (Rezza, A. et al. 1010, J. Cell Sci. 123:3256-3265; Singh, S. K. et al. 2008. Nature 453:223-227). For extract purity and normalization purposes, acetylated histone H3 validated nuclear proteins and ribosomal protein L28 identified cytoplasmic proteins. As expected, the intensities of Oct4 bands were denser for Oct4hi than Oct4med and undetectable in the Oct4− subsets (
Next, it was determined if there were differences in the expression of hormone receptors (estrogen receptor or ER and progesterone receptor or PR) in Oct4hi, Oct4med and Oct4− BCCs. Intracellular flow cytometry indicated no change in the subsets from triple negative MDA-MB-231. Although the parental T47D were ER+/PR+, the spread in fluorescence intensities for the three subsets indicated varied receptor levels. The results also indicated a small population that was ER within the Oct4hi subset.
Oct4hi BCCs, therefore, have been shown to have functions consistent with stem cells. Further experiments were performed to determine if Oct4hi BCCs could be serially passed in female nude BALB/c mice. Oct4hi, Oct4med and Oct4− BCCs (200 cells) were injected subcutaneously in the dorsal flanks of mice (P1). In ten mice studied, nine mice formed palpable tumors. At a tumor size of approximately 0.5 cm3, similar cells were acquired from dissociated tumors and the injections were repeated as for P1, until passage 5 (P5). The time for tumor formation in each passage was similar to P1. The tumors in 10 mice injected with Oct4− and Oct4med BCCs regressed after 2 weeks, providing further support for a stem cell role for Oct4hi-expressing cells.
It was then determined if tumors could be initiated with less than 200 Oct4hi BCCs and, if so, whether the formation of palpable tumors was time-dependent. Mice were injected with 10, 100, 200, or 1000 Oct4hi BCCs in the dorsal flank (n=5 per dose group). The time required to attain a tumor size of 0.5 cm3 was plotted against the number of cells injected (
Both triple-negative MDA-MB-231 and triple-positive T47D are expected to respond to carboplatin treatment, a drug which acts through a mechanism independent of hormone receptor status. Thus, the responsiveness to carboplatin was investigated in Oct4hi cells from both cells lines. Tumors were injected into the dorsal flanks of mice as before. When the tumors were 0.5 cm3 the mice were injected with carboplatin intra-peritoneally (i.p.). After the second injection of carboplatin, a significant (p<0.05) reduction was observed in the tumors of unsorted BCCs as compared to Oct4hi and also to unsorted BCC not treated with carboplatin. Eight days after treatment with carboplatin, tumors were undetectable in mice injected with unsorted BCCs (
It was then determined if Oct4hi BCCs, at sites of distant metastasis, such as bone marrow, could resist carboplatin treatment. Bone marrow was selected for study because it is well-established that BCCs can attain dormancy in this organ, close to the endosteum (Lim, P. K. et al. 2011. Cancer Res. 71:1550-1560; Rao, G. et al. 2004. Cancer Res. 64:2874-2881). Mice were injected intravenously with 103 Oct4hi MDA-MB-231. After 1 day, carboplatin was injected i.p. at 3-day intervals. One week after the second injection, femurs were decalcified, processed, and examined for GFP+ cells. The results indicated there was drug resistance in GFP+ BCCs close to the endosteum of bone marrow. Similar cells were not detected in the cellular regions of the marrow, which were flushed from femurs prior to the analyses of the endosteal regions.
It has been reported that BCC quiescence could be partly explained by GJIC between BCCs and bone marrow stroma, which is located close to the endosteum (Lim, P. K. et al. 2011. Cancer Res. 71:1550-1560). As already discussed, Oct4hi BCCs could not regress completely despite treatment with carboplatin. Thus, it was determined if Oct4hi BCCs show preference for GJIC with stroma.
Connexin (Cx) expression was studied using plasma membrane extracts from unsorted, Oct4hi, Oct4med and Oct4− BCCs. Western blots indicated that Cx26, Cx32 and Cx43 were expressed at higher levels in Oct4hi cells, and that Cx43 and Cx26 were expressed at higher levels than Cx32 within the Oct4hi fraction (
Dormancy of the BCC subsets was then studied by first assessing the in vitro invasion abilities of different subsets from MDA-MB-231 and T47D cell lines. Cells, labeled with CFDA-SE, were seeded onto matrigel inserts and then allowed to migrate for 48 hours. Cell migration was based on combining the fluorescence intensities within the lower chambers and underside of the inserts. A significant (p<0.05) increase in invasion by Oct4hi cells as compared to the other subsets was observed.
Since the in vitro studies indicated preference for GJIC with bone marrow stroma (
Experiments were performed to determine whether GJIC occurred in vivo by repeating the injections with CFDA-SE-labeled Oct4hi and Oct4− BCCs. The goal was to determine if the BCCs entering bone marrow at the endosteal area can form GJIC, which would be indicated by dye transfer to neighboring cells. Dye transfer was assessed by labeling the scraped cells with PE-anti-cytokeratin. Oct4− cells were mostly yellow due to merging of CFDA-SE dye (green) and PE-anti-cytokeratin (red). In the case of Oct4hi cells, the CFDA-SE dye was observed away from the yellow cells, indicating that CFDA-SE is moving to other cells.
CD44+/CD24−/low/lin− have previously been identified as markers of breast cancer stem cells (Al-Hajj, M. et al. 2003. PNAS USA 100:3983-3988). In the present invention, Oct4hi BCCs exhibit tumor-initiating properties. As a result, it was determined if Oct4hi BCCs showed a phenotype consistent with the role of breast cancer stem cells. Two week cultured Oct4hi cells were selected in order to compare the progenies, based on GFP intensities. The cells were studied by flow cytometry for CD44 and CD24 expression. The results indicated that the population with brightest GFP emission included a subset that expressed CD24. The bright GFP cells were dissected by analyzing the upper 5% and it was observed that this subset comprised the CD24+ cells. Cells with reduced GFP intensity were mostly negative for CD24, indicating that CD24 expression correlated with Oct4.
Experiments were then performed to determine whether there were differences in CD44 expression. CD24+ cells were depleted by negative selection and the remaining cells were analyzed for CD44 by flow cytometry. The results indicated variations in CD44 expression within the Oct4hi subset. Similarly, Oct4med BCCs showed varied CD44, including a subset with dim CD44 expression.
A subset of GFPhi BCCs expressed CD24. Thus, it was determined if this could be related to cell size. Flow cytometry was repeated by gating on the Oct4med/hi population and the results indicated approximately 4% of the cells were CD24+. The same subset was analyzed based on forward scatter. The data indicated CD24+ cells within the larger subset. A hierarchy of BCCs was constructed, based on phenotype, and is shown in
The results considered together indicate that a subset of Oct4hi BCCs represents a population of cells that can act as cancer stem cells in distant tissues, such as bone marrow. The results that support this conclusion include the identification of these cells in the blood of patients that had undergone chemotherapy or that had negative lymph nodes in the presence of a tumor, and the fact that only high-expressing Oct4 BCCs could sustain tumors in vivo in mice. The finding of Oct4hi cells in a patient that had undergone six cycles of chemotherapy and radiation indicates that Oct4hi BCCs are resistant to treatment, consistent with the resistance to carboplatin demonstrated in mice. Moreover, the result showing negative lymph nodes with the presence of Oct4hi BCCs in blood lends support to the debate concerning the lack of utility of lymph node dissection in some breast cancer treatment (Guiliano, A. E. et al. 2011. JAMA 305:569-575; Kawada, K. and M. M. Taketo. 2011. J. Clin. Oncol. 26:2813-2820). Thus, the BCC subset identified in the present invention may be a useful marker for breast cancer diagnosis and prognosis that has greater utility than lymph node dissection alone.
Another important finding of the present invention was the fact that the BCC subset of Oct4hi cells may be a more primitive cell group than stem cells. This conclusion was reached based on the finding that stem-like subpopulations within tumors cannot be clonally expanded sue to spontaneous differentiation, yet, while maintaining the Oct4hi BCCs in culture, the subset was able to be expanded since the cells differentiated into Oct4med and Oct4low cells. Thus, the Oct4hi BCC subset, exhibits stem cell properties but also adapts dormancy in distant sites, leading to evasion of treatment. Given that a large percentage of cancer resurgence has been purported to occur in cells from bone marrow (Pantel, K. and M. Otto. 2001. Sem. Cancer Biol. 11:327-337), the finding of a marker for a population of such BCCs will provide for new insights into cancer therapy, cancer diagnosis and cancer prognosis.
The ability of Oct4hi cells to enter bone marrow and resist chemotherapy is an important finding for investigation into the effects of the microenvironment on cancer cell survival. It is possible that immune mediators in the microenvironment serve to protect Oct4hi cells and opens new areas for investigation. The finding that Oct4hi cells were dependent on stem cell networks while Oct4low cells exhibited proliferation-associated genes may explain a mechanism where the Oct4hi cells maintain dormancy in bone marrow.
Finally, although CD44/CD24 expression has been a accepted method for isolating BCC stem cells, the results of the present investigations indicate that the cellular properties of Oct4 expression are independent of CD44/CD24 status. In fact, Oct4 has now been identified as a unique marker for identifying stem-like subsets of cells in breast tumors. Moreover, the results of the present invention indicate that establishing a single phenotype for tumor-initiating cells is not likely. Instead, a hierarchy of such cells has now been identified, a hierarchy based on function and phenotype. The hierarchy now discovered combines the Oct4, CD44 and CD24 status of different breast cancer cell subsets, with Oct4hi cells identified as the most primitive cell type. Thus, the present invention is a population of BCCs that have stem cell characteristics but also are identified as exhibiting functional GJIC with bone marrow stroma, indicating the cells establish dormancy and would remain resistant to chemotherapy. Thus, these cells provide a useful target for testing new cancer therapies for their ability to affect cancer dormancy and reverse drug resistance.
Contemplated by the present invention is a biomarker for breast cancer based on the detection of Oct4hi cells within samples taken from a patient, such as tissues or biological fluids of patients. Commonly used patient samples would include but not be limited to tumor samples, blood samples, or bone marrow samples. These patient samples can be screened for the presence of Oct4 cells of various types, i.e., Oct4hi, Oct4med, Oct4low. The cells identified may also be screened for CD44 and CD24 status as a further method for phenotyping cells as breast cancer stem cells. In a preferred embodiment, the breast cancer biomarker of the present invention comprises a breast cancer cell with a phenotype Oct4hi/CD44hi/med/CD24−/+. In the present invention it has been shown that cells with the Oct4hi/CD44hi/med/CD24−/+ phenotype represent dormant, metastatic breast cancer cells that can exist in bone marrow.
The Oct4 expression profile and Oct4 subset analysis also provides for a method of cancer prognosis and diagnosis. Cancer prognosis would be aided by identifying whether the patient has Oct4− cells that are likely dormant cells which are drug resistant. The presence of the Oct4hi cells would indicate that a patient's cancer would, therefore, be identified as one that is likely to reoccur, indicating a poor prognosis. As a result, the present invention is also a method for developing a breast cancer prognosis for a patient which comprises detecting the presence of the biomarker cells of the present invention in a patient sample wherein the presence of said biomarker is indicative of a the presence of dormant metastatic breast cancer cells in the patient and a poor prognosis for the patient. In the context of the present invention a “poor” prognosis is one where cancer reoccurrence is expected to occur. Additionally, the present invention is a method of diagnosing metastatic breast cancer which comprises detecting the presence of the biomarker of the present invention in a patient sample, wherein the presence of the biomarker indicates that the patient has metastatic breast cancer. Again, the patient samples would include but not be limited to tumor samples, blood, or bone marrow samples.
Finally, the Oct4hi cells themselves provide for a useful system for testing new therapies for their ability to affect dormant cancer cells, which could lead to new treatments for cancer. Thus, another object of the present invention is a method for identifying chemotherapeutic agents that can kill dormant metastatic breast cancer cells in bone marrow which comprises that steps of contacting a breast cancer cell with a phenotype Oct4hi/CD44hi/med/CD24−/+ in vitro with an agent; and determining whether the agent is capable of killing said breast cancer cell, wherein death of said breast cancer cell is indicative of the ability of the agent to kill dormant metastatic breast cancer cells in bone marrow.
Although specific embodiments of the present invention have been described, it should be understood that such embodiments are by way of example only and merely illustrative of but a small number of the many possible specific embodiments that can represent applications of the principles of the present invention. Various changes and modifications obvious to one skilled in the art to which the present invention pertains are deemed to be within the spirit, scope and contemplation of the present invention as further defined in the appended claims.
The following examples are provided to further illustrate the present invention.
Liquid and 10× powdered DMEM were purchased from Gibco (Grand Island, N.Y.). Noble agar, propidium iodide, fetal bovine sera, RPMI 1640, Hoechst 33342 dye, verapamil, and mouse monoclonal IgG to β-actin were purchased from Sigma (St. Louis, Mo.). Biocoat Matrigel Matrix, anti-human CD44-APC, and anti-human CD24-PE were purchased from BD Biosciences (Franklin Lakes, N.J.). Vybrant CFDA-SE Cell Tracer, CyQUANT Cell Proliferation Assay, Platinum SYBR Green qPCR SuperMix-UDG Kit, SuperScript III reverse transcriptase, RNase A, Platinum Taq polymerase, Dynabeads pan mouse-IgG, Connexin Antibody Sampler Pack, and Geneticin G418 were purchased from Invitrogen (Carlsbad, Calif.). HyGLO HRP Chemiluminescent Detection Kit was purchased from Denville Scientific (Metuchen, N.J.). Restore Western Blot Stripping Buffer and NE-PER Nuclear and Cytoplasmic Extraction Kit were purchased from Thermo Scientific (Waltham, Mass.).
The following antibodies were purchased from Abcam (Cambridge, Mass.): rabbit polyclonal anti-Oct4, mouse anti-progesterone receptor (PR) mAb, rabbit polyclonal anti-estrogen receptor (ER) a, rabbit polyclonal anti-Sox2, rabbit polyclonal anti-Nanog, rabbit polyclonal anti-Musashi, rabbit polyclonal anti-ABCG2, rabbit polyclonal anti-REST and FITC-polyclonal goat anti-rabbit IgG. APC-anti-rabbit IgG and polyclonal goat anti-ribosomal protein L28 were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Rabbit polyclonal IgG to acetyl-histone H3 was purchased from Upstate Cell Signaling Solutions (Lake Placid, N.Y.). Antibodies to p15, p16, Cdk4, and cyclin D1 were purchased from Cell Signaling Technology.
The use of all human tissues was approved by the Institutional Review Board of the University of Medicine and Dentistry of New Jersey-Newark Campus. All subjects signed the approved consent forms. Stromal cells were cultured from bone marrow aspirates. Left over surgical tissues from malignant (invasive ductal carcinoma) and normal areas of the mammary gland was obtained from Brookdale University Hospital, Brooklyn, N.Y. The studies were approved by the Institutional Review Board of UMDNJ and Brookdale University Hospital. Peripheral blood was obtained from patients with breast cancer.
MDA-MB-231 and T47D were stably transfected with pEGFP1-Oct3/4. This vector expresses green fluorescent protein (GFP) under the control of Oct4 regulatory region (Gerrard, L. et al. 2005. Stem Cells 23:124-133). Dose response toxicity curves indicated that 600 μg/ml G418 as optimum for MDA-MB-231 and 400 μg/mL for T47D. The stable transfectants were maintained in the same concentration of G418. Immediately before all assays, the cells were sorted with the FACSDiva (BD Biosciences). Selection of subsets was based on the intensity of GFP of singlets. The top 5% was designated Oct4hi and the lower 5%, Oct4−. Those between the two extremes were designated Oct4med. GFP intensity correlated with Oct4 protein, as indicated by immunofluorescence for intracellular Oct4.
GJIC was assessed by CFDA-SE dye exchange from BCCs to stroma in co-cultures, as described previously (Ramkisson, S. H. et al. 2007. Cancer Res. 67:1653-1659). Briefly, BCCs and stroma were co-cultured at equal ratios in α-MEM with 10% FCS, in the presence or absence of 300 μM 1-octanol. CFDA-SE dye transfer was assessed on an EVOS fl fluorescence imager (AMG Micro, Bothell, Wash.).
RNA extraction was performed via RNeasy Mini Kit from (Qiagen, Valencia, Calif.). Total RNA (1 μg) were immediately reverse transcribed using dNTPs (0.2 mM), random hexamers (50 μM), and SuperScript III reverse transcriptase (200 U). Incubation conditions were 25° C. for 5 min, 50° C. for 60 min, and 70° C. for 15 min. Real-time PCR was performed with 200 ng cDNA using Platinum SYBR Green qPCR SuperMix-UDG Kit (Invitrogen) and then analyzed on the 7300 Real-Time PCR System (Applied Biosystems, Foster City, Calif.). The analyses were performed with an initial incubation of 50° C. for 2 min followed by 95° C. for 2 min. After this, the cycling conditions were as follows: 94° C. for 15 sec and 60° C. for 45 sec, for 40 cycles. Primer sequences are described in Table S3.
Gene expression analyses were performed with Taqman Stem Cell Pluripotency Array (Applied Biosystem), by quantitative RT-PCR, using ABI 7900. The fold change between subsets were calculated using the ΔΔCt method as follows: (CtOct4(hi)−CtGene of Reference)/(CtOct4(−)−CtGene of Reference).
The fold changes were entered into Ingenuity Pathway Analysis (Ingenuity® Systems, www.ingenuity.com) for pathway networks, as described previously (Lim, P. K. et al. 2011. Cancer Res. 71:1550-1560). The analyses allowed for the identification of complex biological interactions based on at least one published reference in the database. Biological predictions were made based on protein-protein interactions, and the insights into molecular pathways were gathered.
Mice were injected intravenously with 103 BCCs, stably transfected with pEGFP1-Oct3/4. After 24 hours, mice were injected i.p. with carboplatin (50 mg/kg), followed by a second dose after 3 days. One week after the final injection of carboplatin, mice were euthanized, and the femurs were removed. Femurs were rinsed and the cells flushed using a 27 gauge needle attached to a syringe with PBS to remove the cells within the central region of the cavity. After this, the femurs were fixed overnight in 4% formaldehyde at 4° C. After this, the femurs were transferred to decalcification solution (Cal-Ex Decalcifier, Fisher Scientific, Pittsburgh, Pa.) overnight at 4° C. After this, femurs were rinsed in running distilled water for 4 hours and then embedded in Optimal Cutting Temperature (O.C.T.) compound (Tissue-Tek, Redding, Calif.). After this, tissues were section in 10 μm with a cryostat-microtome HM550 (Walldorf, Germany). Slides were examined with an EVOS fl fluorescence imager.
BCCs were added to sterile coverslips placed within 6-well plates. The next day, after adherence, cells were washed with 1×PBS, fixed with 3.7% formaldehyde for min, permeabilized with 0.1% Triton X-100 in PBS and blocked in 1% BSA in PBS for 1 h. The blocking buffer was washed with PBS and the cells were incubated with anti-Oct4 (1:500 dilution). The antibody was diluted in 0.1% BSA/0.1% Triton X-100 in PBS. After 30 min, the cells were washed in PBS and then incubated with goat anti-rabbit IgG-FITC (1:1000 dilution) for 2 h in the dark. Nuclei were stained with 300 nM DAPI, and F-actin was stained with Texas Rex-X phalloidin. Green emission was observed using the 518 nm filter.
Oct4 staining in primary breast tissues was performed by fixing in 4% paraformaldehyde overnight followed by incubation in 20% sucrose overnight. The tissues were embedded in O.C.T. compound and then sectioned into 5 μm slices as described above. Sections were placed on slides and then de-paraffinized in xylene. After this, the sections were rehydrated with consecutive washes in decreasing concentrations of ethanol: 100%, 90%; 80% 70%. Slides were washed twice in PBS and then incubated in 0.25% Triton X-100 for 5 min. This followed by blocking in 1% BSA for 1 h. Slides were incubated overnight at 4° C. with anti-Oct4 (1:500 dilution). The antibody was diluted in PBS containing 0.1% Triton X-100 and 0.1% BSA. Diaminobenzidine (DAB) detection for Oct4 was performed using the DAKO Envision+System-HRP according to manufacturer's protocol.
Western blots were performed as previously described (Trzaska, K. A. et al. 2007. Stem Cells 25:2797-2808). Cell extracts from surgical tissues were obtained by homogenizing in the NP-40 cell lysis buffer containing protease inhibitors (Invitrogen). For intracellular proteins with cell lines, whole cell extracts were prepared with the NP-40 buffer and also nuclear/cytoplasmic extracts with NE-PER Nuclear and Cytoplasmic Extraction kit. For membrane proteins, extracts were prepared with Qproteome Plasma Membrane Protein kit (Qiagen).
BCC extracts (20 μg) were subjected to electrophoresis on 4-20% SDS-PAGE (Bio-Rad; Hercules, Calif.). Proteins were transferred to PVDF membranes, and membranes were incubated overnight in the respective primary antibodies. This was followed by 2 h incubation with HRP-conjugated secondary antibodies at 1:2000 final dilutions. The latter was detected with chemiluminescence. Membranes were stripped with Restore Western Blot Stripping Buffer and then re-probed for other proteins, including β-actin mAb (1:4000 dilution). All bands were normalized to (3-actin.
BCC subsets were seeded at one cell per well in serum-free media in 96-well low-adhesion plates (Costar, Corning, N.Y.). At day 10, wells with spheres containing greater than 20 cells were designated as tumorsphere-positive. One tumorsphere was dissociated, first enzymatically with trypsin and then mechanically with a syringe attached to a 27-gauge needle. After this, the cell suspension was passed through a 40 μm mesh (BD cell strainer cap tube). One cell, with similar phenotype, was re-assessed for green fluorescence and then re-seeded at 1 cell/well. This method was continued serially more than 5 times.
The assay was established with two layers of noble agar in 60 mm Petri dishes. The bottom layer contained 4 mL of 0.6% agar, and the top layer contained the cells in 4 mL of 0.3% agar. The agar was prepared with a stock of 1.8% diluted in deionized water. Agar was autoclaved and then diluted to the working concentration with sterile deionized water and 2×DMEM. The bottom agar was allowed to solidify at 37° C. for 10 min. After this, the top agar was added with BCCs at concentrations between 101 and 105 at log10 dilutions. Plates were incubated and examined with EVOS fl fluorescence imager.
Intracellular flow cytometry for Oct4 was performed by the following consecutive treatments: fixed in 4% formaldehyde for 15 min at 4° C., permeabilized in 0.1% Triton X-100 for 30 min, incubated with anti-Oct4 for 30 min at 4° C., washed once with cold PBS and then incubated with goat anti-rabbit IgG-APC for 30 min in the dark at 4° C. After this, cells were washed with PBS and then immediately analyzed on the FACSCalibur (BD Biosciences).
Cell surface labeling for CD44/CD24 with pEGFP1-Oct3/4 stable transfectants were performed by first washing with PBS, fixing in 4% formaldehyde as for intracellular labeling, incubating with anti-CD44-APC for 30 min followed by a second labeling with anti-CD24-PE. All incubations occurred for 30 min at 4° C. in 2% FBS/PBS. The cells were immediately analyzed by gating cells, based on green (GFP) emission, with the FACSCalibur. The data were analyzed with CellQuest software (BD Biosciences).
Side population analysis of the pEGFP1-Oct4 stable transfectants was performed using the LSR II (BD Biosciences). Stable transfectants (106) were washed in PBS, resuspended in phenol-free, Ca2+/Mg2+-free 1× Hank's Balanced Salt Solution containing 2% FBS and then incubated in titrations of Hoechst 33342 and verapamil. Optimal titrations and conditions were determined to be 5 μg/ml Hoechst 33342 (90 min incubation at 37° C.) and 400 μM verapamil (10 min pre-incubation). Cells were then washed, maintained on ice, and incubated in propidium iodide (5 μg/ml) to gate for viability. The analyses were done by gating on the top and lower 5% of GFP-expressing cells, designated Oct4hi and Oct4−, respectively. The cells between the two extremes were also analyzed (Oct4med).
Doubling time was performed by seeding BCCs at 5×103/well into 96-well plates. After four days cell numbers were determined by CyQUANT Cell Proliferation Assay Kit (Invitrogen). Cell numbers were calculated on a standard curve of fluorescence intensity vs. known cell densities. Calculations for doubling times were based on the following: A=A0*2n, where A=final cell number, A0=initial seeding density and n=number of divisions. Doubling time was taken by dividing the incubation time by the number of divisions. The fluorescence method was validated by manual cell count.
Cell cycle analyses were performed with BCCs (106). Cells were washed in PBS and then resuspended in 0.1% hypotonic sodium citrate solution containing 5 μg/ml propidium iodide and 200 μg/ml DNase-free RNase A. Cells were incubated for 30 min at room temperature and then immediately analyzed on FACSCalibur (BD, San Jose, Calif.).
BD BioCoat™ Matrigel™ Matrix (0.2 ml) was added into 8 μm FluoroBlok cell culture inserts. These inserts prevent the plate reader from detecting emission in the upper chamber. After solidification of the matrigel at 37° C. for 1 h, the inserts were placed in 24-well culture plates containing 0.5 mL DMEM with 10% FCS. BCCs (2×104) in sera-free media were added to the inner wells. The cells were allowed to migrate for 2 h at 37° C. After this, the inserts were removed, and the cells within the inner chambers were gently removed with a Q-tip. The wells under the membranes were then transferred to another well for labeling with 10 μM CDFA-SE, diluted in PBS for 1 h. After this, the wells were gently washed with PBS to remove excess CFDA-SE and then transferred to another well containing PBS for analyses on Victor 3V Multi-well plate reader (Perkin Elmer, Waltham, Mass.) at 485 nm/535 nm. Controls included MCF12A non-tumorigenic breast epithelial cells.
Time-lapse microscopy of MDA-MB-231 cells was performed with Axiovert 200M fluorescence microscope (Carl Zeiss, Inc.) at constant conditions of 37° C. and 5% CO2. Brightfield and fluorescence images were acquired every 10 min for up to 68 h using a 10× objective (Zeiss) and an AxioCam MRm camera with Axiovision software v4.6 (Zeiss). Individual images were adjusted for brightness using the Axiovision software and exported to ImageJ (National Institutes of Health, Bethesda, Md.), where the movies were assembled. Individual cells were tracked manually.
Female athymic BALB/c mice (4 weeks) were obtained from Harlan Laboratories (Somerville, N.J.) and housed in a laminar flow hood at an AAALAC-accredited facility. The use of mice was approved by the Institutional Animal Care and Use Committee, New Jersey Medical School (Newark, N.J.).
Serial passage of different BCC subsets was performed by injecting BCCs in dorsal flanks of mice. The cells were resuspended in PBS and then mixed with matrigel at 1:1 ratio in 0.2 mL total volume. The cells were injected at different numbers, but in a constant volume to study dose effects on the time for tumor growth. Parallel analyses were performed with unsorted BCCs. Tumors were monitored daily for 30 days and measured in two dimensions with a caliper and volume was calculated using the following: formula V=πr2h, where r=radius and h=height. Subsequent passages occurred by sorting the same cell subset (top 5% highest GFP) and then repeating the injection as above.
Migration of BCC subsets to bone marrow of nude mice was studied by intravenous injection of 103 pEGFP1-Oct3/4 stable transfectants. After 72 h, the femur was flushed to eliminate the cells within the central region. After this, femurs were transected longitudinally and the endosteal cells were acquired by scraping with a blunt spatula. Cells were then labeled by immunocytochemistry for cytokeratin, as described above.
GJIC was assessed by dye transfer from BCCs to stroma in nude BALB/c was investigated by injecting CFDA-SE-labeled Oct4hi BCCs intravenously. After 72 h, the endosteal region cells were collected as above and then labeled for cytokeratin.
Carboplatin responses to tumor growth were assessed by subcutaneous injection of 200 Oct4hi BCCs in matrigel in the dorsal flank. For unsorted BCCs, 106 cells were injected. At −0.5 cm3 tumors, mice were injected with carboplatin (50 mg/kg), twice, at 3-day intervals. Tumor sizes were recorded every 2 days with a caliper, as described for serial passage.
Extracts from MSCs served as positive control in the western blots for Oct4B. MSCs were cultured from bone marrow (BM) aspirates as described (Greco, S. J. et al. 2007. Stem Cells 25:3143-3154). The use of human bone marrow aspirates followed a protocol approved by the Institutional Review Board of The University of Medicine and Dentistry of New Jersey-Newark campus. Unfractionated bone marrow aspirates were cultured in DMEM with 10% FCS in Falcon 3003 dishes. After 3 days, red blood cells and granulocytes were removed with Ficoll Hypaque. After four cell passages, the adherent cells were asymmetric, CD14−, CD29+, CD44+, CD34−, CD45−, SH2+, prolyl-4-hydroxylase− (Potian et al., 2003).
At 40-50% confluence stably transfected BCCs were treated with G9a histone methyltransferase inhibitor, [2-(Hexahydro-4-methyl-1H-1,4-diazepin-1-yl)-6,7-dimethoxy-N-[1-(phenylmethyl)-4-piperidinyl]4-quinazolinamine] (BIX01294, Enzo Life Sciences, Farmingdale, N.Y.). BIX01294 and similar chemicals can induce Oct4 in negative cells, in reprogramming (Huangfu, D. et al. 2008. Nat. Biotech. 26:795-797; Shi, Y. et al. 2008. Cell Stem Cell 3:568-574). Cells were treated with 2.7 μM BIX01294. After 24 h, images were taken by fluorescence microscopy. Control cells were untreated or treated with vehicle (dimethyl sulphoxide, DMSO).
This application claims the benefit of U.S. Provisional Application No. 61/487,378, filed May 18, 2011, which is herein incorporated by reference in its entirety.
This invention was made with government support under Grant No. W81XWH-10-1-0413 awarded by the U.S. Department of Defense. The government has certain rights in the present invention.
| Number | Date | Country | |
|---|---|---|---|
| 61487378 | May 2011 | US |
