CD44+/CD242 ovarian cancer cells demonstrate ... - Springer Link

Clin Exp Metastasis (2012) 29:939–948 DOI 10.1007/s10585-012-9482-4

RESEARCH PAPER

CD44+/CD242 ovarian cancer cells demonstrate cancer stem cell properties and correlate to survival Erhong Meng • Beverely Long • Paula Sullivan • Steve McClellan • Michael A. Finan • Eddie Reed • Lalita Shevde • Rodney P. Rocconi

Received: 30 November 2011 / Accepted: 30 April 2012 / Published online: 18 May 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Cancer cells with the surface marker profile CD44?/CD24- have previously been described to possess cancer stem cell-like properties. This manuscript evaluates those properties in ovarian cancer cell lines. The proportion of CD44?/CD24- cells corresponded to the clinical aggressiveness of each ovarian cancer cell line histologic subtype. CD44?/CD24- cells demonstrated enhanced progressive differentiation as well as showing a 60-fold increase in Matrigel invasion in both SKOV3 and OV90 cell lines (p \ 0.001 each) compared to other phenotypes. CD44?/ CD24- demonstrated significant resistance to all chemotherapy agents used in all cell lines, with a 71–93 % increase in resistance compared with baseline. Using a threshold of 25 % CD44?/CD24– ovarian cancer cells found in ascites, patients with [25 % CD44?/CD24- were significantly more likely to recur (83 vs. 14 %, p = 0.003) and had shorter median progression-free survival (6 vs. 18 months, p = 0.01). In conclusion, the CD44?/CD24- phenotype in ovarian cancer cells demonstrate cancer stem cell-like properties of enhanced differentiation, invasion, and resistance to chemotherapy. This CD44?/CD24- phenotype correlates to clinical endpoints with increased risk of recurrence and shorter progression-free survival in patients with ovarian cancer. Keywords CD44?/CD24- Ovarian cancer Cancer stem-like cells Chemoresistance Survival

E. Meng B. Long P. Sullivan S. McClellan M. A. Finan E. Reed L. Shevde R. P. Rocconi (&) University of South Alabama, Mitchell Cancer Institute, 1660 Springhill Avenue, Mobile, AL 36604, USA e-mail: [email protected]

Abbreviations bFGF Basic fibroblast growth factor BSA Bovine serum albumin CK7 Cytokeratin 7 EGF Epidermal growth factor FACS Fluorescence-activated cell sorting FCM Flow cytometry FITC Fluorescein isothiocyanate PFS Progression free survival

Introduction In 2011, it is estimated that ovarian cancer will account for 21,990 new diagnoses and 15,460 deaths in the United States alone, making it the most lethal of all gynecologic malignancies [1]. Although the majority of ovarian cancer patients present with advanced stage III/IV disease, over 75 % will achieve a complete clinical response to cytoreductive surgery followed by combination chemotherapy. Despite this effective initial response, the majority of patients will experience a recurrence and unfortunately succumb to progressive disease. One hypothesis associated with this complete response followed by recurrence revolves around cancer stem cells. The initial ‘‘complete’’ clinical response of ovarian cancer would represent the majority of cancer cells that are sensitive to cytotoxic therapy; however, the small percentage of clinically undetectable cancer stem cells would ultimately survive due to their innate chemoresistance. Combined with their ability to proliferate and form new tumors these cancer stem cells would lend themselves to an eventual recurrence [2–4]. As such, irrespective of initial response rates, if chemotherapy fails to eradicate cancer stem cells then cancer may regenerate and a recurrence or progression of disease can occur [5]. By this hypothesis, the

123

940

likelihood of recurrence and death would thereby correlate to the inherent proportion of cancer stem cells within a given tumor. Although a consensus in defining a ‘‘cancer stem cell’’ has eluded us, the conceptual basis for a stem cell origin of cancer is supported by observations that certain sub-populations of cancer cells appear to acquire stem cell-like properties such as the capacity of self-renewal, and the ability to differentiate [4, 6–9]. Other enhanced cancer properties described within cancer stem cells include enhanced aggressiveness [10], invasion, migration [11–13], tumorigenesis [14, 15], and chemoresistance [14, 16]. Effective curative therapy for ovarian cancer has yet to be developed. Describing the innate differences between ovarian cancer stem cells and ‘‘normal’’ differentiated nonstem cell ovarian cancer cells and identifying their distinct phenotypic attributes is of vital importance and holds promise in future discoveries in targeted therapy. One important cancer stem cell marker phenotype CD44?/CD24- was originally described in breast cancer stem cells [17] with properties of serial passaged tumorigenicity with low numbers of CD44?/CD24- cells. This phenotype has been associated with breast cancer lymph node metastasis [18], brain metastasis [19], chemoresistance [20], and as a poor prognostic factor [21]. Recently this phenotype has been implicated to be an ovarian cancer stem cell marker as well with properties of self-renewal and chemoresistance [22, 23]. Our objective was to evaluate a sub-population of CD44?/CD24- cells for cancer stem cell-like properties in ovarian cancer.

Methods Cell culture Ovarian cancer cell lines ES2, SKOV3, OV90 and TOV112D (purchased from American Type Culture Collection within 6 months of initiation of project) were cultured in monolayer. ES2 and SKOV3 cell lines were maintained in DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 1 % sodium pyruvate (Invitrogen), 0.2 % non-essential amino acids (Invitrogen), and 5 % FBS in a humidified atmosphere containing 5 % CO2 at 37 °C. OV90 and TOV112D cell lines were maintained in a 1:1 mixture of medium 199 and MCDB 105 plus 10 % FBS. Monolayer cancer cell lines were then cultured under conditions to enrich for cells with stem-like properties by suspending in serum-free corresponding medium supplemented with 5 lg/mL insulin (Sigma, St. Louis, MO), 20 ng/mL human recombinant epidermal growth factor (EGF, Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF; Invitrogen), and 0.4 % bovine serum albumin

123

Clin Exp Metastasis (2012) 29:939–948

(BSA; Sigma) in 6-well Ultra-Low Attachment plates (Corning, Corning, NY) and subsequent organization into spheres. Spheroid forming cells were kept for 5 days in same conditions. Fluorescence-activated cell sorting (FACS) FACS was used to sort ovarian cancer cell lines (TOV112D, OV90, SKOV3, and ES2) into two phenotypically distinct populations: CD44?/CD24- and all other phenotypes. Cells were suspended in PBS and labeled with anti-human CD24-fluorescein isothiocyanate (FITC) antibody: 20 lL antibody per one million cells in a final volume of 100 lL. TM (BD Pharmingen ), and anti-human CD44-PerCP-Cy5.5 antibody: 0.25 lg antibody per one million cells in a final volume of 100 lL (eBioscience). A total of about 1.0 9 106 cells were incubated with these two antibodies for 1 h at 4 °C in the dark. Unbound antibody was washed off and cells were analyzed or sorted on a BD FACSCalibur within 1 h after staining. Gating was established using the isotype controls TM FITC-labeled mouse IgG2a (BD Pharmingen ) and PerCPCy5.5-labeled rat IgG2b (eBioscience). Serial differentiation experiment After sorting, the purity of each phenotype was checked and 2 9 104 per well of either CD44?/CD24- or other phenotypes were re-seeded. Sorted cells were cultured in DMEM/F12 containing 1 % sodium pyruvate (Invitrogen), 0.2 % non-essential amino acids (Invitrogen) and 5 % FBS in 6-well plate; sorted OV90 were re-seeded in a 1:1 mixture of Medium 199 (Invitrogen) and MCDB 105 (Cell Applications Inc, 140 San Diego, CA) with 15 % FBS. Seven days later, they were re-collected and re-analyzed through flow cytometry (FCM). Matrigel invasion assay After FACS sorting was performed, each ovarian cancer phenotype was evaluated for invasion properties using the Matrigel invasion assay for the SKOV3 and OV90 cell TM lines. Matrigel -coated chamber (BD Pharmingen) were rehydrated for 2 h in humidified tissue culture incubator at 37 °C, 5 % CO2. After rehydration, carefully remove the medium without disturbing the layer of Matrigel Matrix on the membrane. Cell suspension was prepared in serum-free medium with cell concentration adjusted to 105 cells/mL. The lower chambers of the 24-well plate were filled with 750 lL serum-free medium containing 10 lg/mL fibronectin as a chemotactic factor. 500 lL serum-free medium containing 5 9 104 cells were added to the upper chambers. The plate was incubated in a humidified tissue culture incubator at 37 °C, 5 % CO2 atmosphere for 24 h. Then


cells were fixed, stained and counted under the microscope. Numbers of invaded cells at least ten consecutive fields were enumerated and their average was calculated. Data are expressed as mean number of migrating cells ± standard error of the mean per field. Transwell migration assay Sterile 6.5 mm insert with 8 lm Polycarbonate membrane were hydrated with 500 lL serum free medium for 90 min at 37 °C. Sorted cells were collected and washed extensively with medium containing 0.1 % FBS and re-suspended in the same medium. 100 lL containing 1 9 105 cells were added onto upper chambers and 600 lL medium containing 0.1 % FBS were added in the lower chambers. The cells were allowed to adhere onto transwells for 1 h at 37 °C. Then, the medium in the lower chambers were removed, and 600 lL fresh migration medium (containing 10 % FBS) were added to the lower chamber. Migration was allowed to proceed for 6–8 h at 37 °C. Then cells were fixed and stained as described above. The cells attached to the upper surface of the filters were removed carefully with cotton swabs. Migrated cells were counted under the microscope as described in invasion assay. Cell proliferation assay Ovarian cancer cell lines SKOV3, TOV112D, and ES2 cell lines were plated at density of 4,000 cells per well in 96 well tissue culture plates and treated with IC50 concentrations of carboplatin, paclitaxel, and carboplatin plus paclitaxel in monolayer as well as in spheroid forming cells enriched for CD44?/CD24- cells. The following day, fresh medium was added to each well and treated with IC50 concentrations for each cell line and kept at 37 °C in a humidified 5 % CO2 atmosphere. For spheroids, cells were seeded at a density of 4,000 cells per well fed with 0.4 % BSA (Sigma), 5 lg/mL insulin (Sigma), 20 ng/mL recombinant human EGF (Invitrogen) and 10 ng/mL recombinant fibroblast growth factor-basic (bFGF, Invitrogen) in ultra-low attachment 96-well plates (Corning, Corning, NY). After 3 days, spheroid-forming cells were treated with IC50 concentrations. The mixture of 20 lL PMS/MTS was added to each well every 24 h followed by incubation at 37 °C incubator for 3 h. Absorbance was read at 490 nm. The assays were carried out up to 72 h. Soft agar colony formation assay Soft agar colony formation assay was performed as Shevde et al. [24] described. Cells (5,000 cells/well) were suspended in 0.35 % Bactoagar (BD Bioscience) DMEM/F12 medium containing 5 % FBS and seeded in 6-well tissue culture plate

941

(Corning, NY, USA). The agar containing cells was allowed to solidify overnight in a humidified tissue culture incubator at 37 °C, 5 % CO2 atmosphere. Additional DMEM/F12 medium containing 5 % FBS was overlaid on the agar and the cells were allowed to grow for about 28 days. Visible colonies ([50 cells) were counted on five randomly selected 409 microscopic fields in each well. To evaluate the effect of chemotherapy on colony formation, SKOV3 cells were sorted into CD44?/CD24- and other phenotypes. Sorted cells were seeded onto soft agar in 6-well plate. The next morning, cells were treated with 20 lM carboplatin, and 300 lL fresh media with 20 lM carboplatin. Fresh media with 20 lM carboplatin was added every 4 days. Visible colonies ([50 cells) were counted on five randomly selected 409 microscopic fields in each well. Clinical correlation After IRB approval (USA #03-092) for collection and storage for human derived specimens, ascites was obtained on 19 consecutive patients undergoing primary ovarian cancer debulking surgery (i.e., prior to chemotherapy) for advanced stage IIIC/IV papillary serous ovarian cancer. Ascites was immediately processed and centrifuged. After washing with PBS, the cells were incubated in ACK lysing buffer (Lonza, Walkersville, MD, USA) to remove the erythrocytes. Cells from each sample were incubated with FITC labeled anti-human Cytokeratin 7 (CK7) antibody (Millipore) in order to confirm gating properties that contained highest purity of ovarian cancer cells. Anti-human TM CD24-FITC antibody (BD Pharmingen ) and anti-human CD44-PerCP-Cy5.5 antibody (eBioscience) was used to determine the percentage of ovarian cancer cells that possessed the CD44?/CD24- phenotype. Gating was established using the isotype controls as described above for CD44 and CD24 and FITC-conjugated IgG2b for CK7. Clinicopathologic data was abstracted and correlated to percentage of CD44?/CD24- phenotype found.

Results CD44?/CD24- correlates with histological subtype and aggressiveness First, we analyzed the correlation of CD44?/CD24- cells to the clinical aggressiveness of ovarian cancer. The proportion of CD44?/CD24- phenotype correlated to the clinical aggressiveness typically seen with each histologic subtype derived from ovarian cancer cell lines. In order of increasing aggressiveness of histologic subtypes with respective CD44?/CD24- percentages: endometrioid = TOV112D at 0.5 %; papillary serous = SKOV3 at 66 %

123

942


Table 1 The proportion of CD44?/CD24- cells in ovarian cancer cell lines correlates to innate clinical aggressiveness seen with histologic subtypes Cell line

Histologic subtype

Aggressiveness

CD44?/ CD24- (%)

TOV112D

Endometroid

??

0.5

SKOV3

Papillary serous

???

66

OV90

Papillary serous

???

77

ES2

Clear cell

?????

99

and OV90 at 77 %; and clear cell = ES2 at 99 % (Table 1; Fig. 1). CD44?/CD24- demonstrate enhanced differentiation The ability to differentiate into both stem cells and non-stem cells is an innate property of cancer stem cells. As such, FACS sorted CD44?/CD24- phenotype showed enhanced progressive differentiation into multiple phenotypes compared to non-CD44?/CD24- phenotype. An immediate post-sort calculation of CD44?/CD24- revealed a purity of 92.4 % for OV90, which differentiated to 62.4 % CD44?/ CD24- cells 7 days later. Similarly, over the same time frame, the CD44?/CD24- phenotype in SKOV3 cells differentiated from 98.4 to 6 %. Fig. 1 The proportion of CD44?/CD24- cells in ovarian cancer cell lines correlates to innate clinical aggressiveness seen with histologic subtypes. FCM was utilized to calculate ovarian cancer cell lines (TOV112D, SKOV3, OV90, and ES2) for the proportion of various phenotypes containing CD44 and CD24. The percentage of CD44?/ CD24- cells (GATE Q1 in FCM) found at baseline within these cell lines corresponded to the clinical aggressiveness typically seen with histologic subtypes. In order of increasing aggressiveness, a TOV112D (endometrioid) = 0.5 %; b SKOV3 (papillary serous) = 66 %; c OV90 (papillary serous) = 77 %; and d ES2 (clear cell) = 99 %

123

Conversely, other phenotypes maintained a more consistent proportion over a 7-day observation period. The percentage of other phenotypes remained stable from 99.6 to 91.3 % for OV90 and 94.9 to 97.2 % for SKOV3 from post-sort to day seven calculations. Of note, the other phenotype in OV90 cells also showed some multipotentiality albeit at much lower rates. Additionally, CD44?/ CD24- cells demonstrated a slower growth potential over a 72 h period compared to other phenotypes as measured by absorbance at 490 nm (Fig. 2). CD44?/CD24- cells demonstrate enhanced cancer invasion, migration, and colony formation Aggressive properties of cancer include the ability to invade tissues locally as well as migrate and metastasize. Therefore, we evaluated the ability of CD44?/CD24phenotype to invade via Matrigel invasion assay. OV90 and SKOV3 cell lines were sorted via FACS into two groups (CD44?/CD24- and other phenotypes) and plated for invasion. CD44?/CD24- demonstrated a 60-fold increase in invasive properties for both cell lines (p \ 0.001 for each). Transwell migration assay also confirmed a significantly higher rate of migrated cells in CD44?/CD24- phenotype compared to other phenotypes (OV90 = 104.3 vs. 20.4; p \ 0.0001 and SKOV3 = 181.3 vs. 53.0; p \ 0.0001) (Fig. 3).


943

Fig. 2 CD44?/CD24- cells are more likely to demonstrate enhanced differentiation as well as slower growth potential. FACS was used to sort ovarian cancer cell lines SKOV3 and OV90 into two phenotypically distinct populations: [1] CD44?/CD24- and [2] all other phenotypes. CD44?/ CD24- demonstrated enhanced progressive differentiation into multiple phenotypes from an immediate post-sort to 7 day later calculation. a CD44?/ CD24- varied from 92.4 to 62.4 % for OV90 and other phenotypes maintained consistency from 99.6 to 91.3 %; b CD44?/CD24varied from 98.4 to 6 % for SKOV3 and other phenotypes maintained consistency from 94.9 to 97.2 % for SKOV3; and c using cell proliferation assay, CD44?/CD24- demonstrated slower growth potential than other phenotypes

123

944


Fig. 3 CD44?/CD24- cells demonstrated enhanced invasion and migration. a Matrigel invasion assay was performed in both OV90 and SKOV3 cell lines. Each cell line was sorted via FACS into CD44?/CD24- and other phenotypes and plated for invasion. Compared to other phenotypes, CD44?/CD24showed a 60-fold increase in invasion in both OV90 and SKOV3 cell lines (p \ 0.001 each). b Transwell migration assay was performed in both OV90 and SKOV3 cell lines. Each cell line was sorted via FACS into CD44?/CD24and other phenotypes cells for migration assay. Compared to other phenotypes, CD44?/ CD24- showed a significant increase in migration ability in both OV90 (5.1-fold) and SKOV3 (3.4-fold) cell lines. (p \ 0.001 for each)

In soft agar colonization experiment utilizing SKOV3 cells, CD44?/CD24- demonstrated a 2.5-fold increase in colony formation as defined as the number of colonies greater than 50 cells per 409 high power field (47.6 vs.

123

18.3; p \ 0.0001). In the presence of carboplatin chemotherapy, the number of colonies was significantly increased in CD44?/CD24- compared to other phenotypes (13.8 vs. 1.6, respectively; p \ 0.0001) (Fig. 4).


945

consecutive advanced stage ovarian cancer patients were evaluated for the proportion of CD44?/CD24– cells (range 3.7–97.7 %). Using a threshold of 25 % CD44?/CD24– cells, patients with [25 % CD44?/CD24- cells were significantly more likely to recur (83 vs. 14 %, p = 0.003) and had shorter median progression-free survival (6 vs. 18 months, p = 0.01) (Fig. 6).

Discussion

Fig. 4 CD44?/CD24- cells demonstrated enhanced colony formation. SKOV3 was sorted into CD44?/CD24- and other phenotypes for soft agar colony formation assay. a CD44?/CD24- cells demonstrated a significant increase in colony formation compared to other phenotypes (47.6 vs. 18.3 colonies, respectively; p \ 0.0001). b With carboplatin chemotherapy treatment, the number of colonies was significantly increased in CD44?/CD24- compared to other phenotypes (13.8 vs. 1.6, respectively; p \ 0.0001)

Spheroids enriched for CD44?/CD24- cells demonstrated chemoresistant properties in vitro Ovarian cancer cells were grown in spheroid forming conditions to enrich percentage of CD44?/CD24- cells. These stem-cell enriched spheroid cells were treated with various chemotherapies typically used for ovarian cancer (carboplatin, paclitaxel, and carboplatin ? paclitaxel) and compared to monolayer cells. In SKOV3, TOV112D and ES2 cell lines, spheroid enriched cells demonstrated significant resistance to all chemotherapy agents used, with a 71–93 % increase in resistance compared with monolayer (Fig. 5). Proportion of CD44?/CD24- cells correlate to recurrence and survival in ovarian cancer patients ascites After confirmation by CK7 immunofluorescence staining, ovarian cancer cells obtained from the ascites of 19

By a far margin, ovarian cancer represents the most lethal of all gynecologic malignancies. The majority of patients are diagnosed with bulky advanced stages due to a lack of both symptoms as well as effective screening. Despite these disadvantages, ovarian cancer is unique in its relative initial chemosensitivity compared to other solid malignancies of the abdomen and pelvis. In fact, nearly 75 % of all patients with stage III or IV disease will have a complete clinical remission with front line combination platinum/taxane based chemotherapy. Unfortunately, all but 10–15 % of advanced stage patients will ultimately recur and die of their disease. There is no doubt that better strategies are needed for ovarian cancer treatment. However, many questions remain. What causes this dramatic response followed by a recurrence? Could the cancer stem cell hypothesis explain this phenomenon? If so, could we apply this knowledge to the development of targeted therapies? As such, the identification and characterization of ovarian cancer stem cells could provide vital information to more effective targeted therapy. This report describes a sub-population of CD44?/CD24- ovarian cancer cells that demonstrate many of the characteristics of cancer stem cells. Historically, this cell phenotype was one of the first cancer stem cell to be described as a breast cancer stem cell, possessing stemness traits such as expression of Oct4 [25]/Sox/Nanog [26], serial transplantability [17] and differentiation into diverse phenotypes [27]. This study demonstrated that the CD44?/CD24- ovarian cancer cell phenotype correlates with in vitro aggressiveness as well as containing the properties of enhanced differentiation, invasion, migration, tumorigenicity, and resistance to chemotherapy. CD44?/CD24- ovarian cancer cell phenotype demonstrated an incremental aggressiveness that coincided with the clinical aggressiveness typically seen in each histologic subtype. Endometrioid ovarian cancer is comparatively an indolent and slow growing histologic subtype clinically, and the TOV112D cell line showed a baseline rate of 0.5 % CD44?/CD24- cells. Clear cell cancer of the ovary is an extremely aggressive histologic subset clinically and the ES2 cell line showed 99 % CD44?/CD24- cells. Papillary serous, the most common subtype demonstrated an

123

946

Clin Exp Metastasis (2012) 29:939–948 b Fig. 5 Cells enriched for CD44?/CD24- cells via spheroid forma-

tion demonstrated significant resistance to chemotherapy compared to monolayer controls. MTS cell survival assay was performed on: a TOV112D, b SKOV3, and c ES2 cell lines. CD44?/CD24- cells were enriched within each cell line by growing cells into spheroids by using non-adherent growth conditions and compared to monolayer cell lines in a chemotherapy panel. In all cell lines evaluated, spheroid cells enriched for CD44?/CD24- cells were significantly more resistant to carboplatin, paclitaxel, and carboplatin ? paclitaxel than its monolayer control

Fig. 6 The proportion of CD44?/CD24- cells found within primary ovarian cancer ascites correlated to the risk of recurrence and progression free survival (PFS). Ascites from 19 consecutive patients with stage IIIC/IV primary ovarian cancer was obtained. FACS gating was determined by identifying ovarian cancer cells by staining for CK7. Next, ovarian cancer cells were analyzed for percentage of CD44?/CD24- cells. An arbitrary threshold of 25 % CD44?/ CD24- was set. Patients with [25 % CD44?/CD24- cells were significantly more likely to experience recurrence than those with \25 % cells, 83 vs. 14 % respectively (p = 0.003). Compared to \25 % CD44?/CD24- cells, median PFS was significantly less for patients with [25 % cells

intermediate 66–77 % CD44?/CD24- rate in the SKOV3 & OV90 cell lines. Although variability exists clinically amongst these histologic subtypes, it is generally accepted that endometrioid, papillary serous, and clear cell histologies have increasing clinical aggressiveness. The CD44?/ CD24- percentage in vitro correlates to this clinical aggressiveness. However, considering the vast heterogeneity of cancer, the CD44?/CD24- phenotype is unlikely to be the sole reason for this clinical phenomenon. Frequently, in vitro cancer cell line explorations can differ from clinical correlations. However, in our evaluation of ascites from 19 consecutive patients with advanced stage III or IV papillary serous ovarian cancer, the percentage of CD44?/CD24- cells found ([25 %) had a direct correlation to both risk of recurrence (83 vs. 14 %, p = 0.003) as well as shorter median progression-free survival (6 vs. 18 months, p = 0.01). This robust clinical data coincides with the hypothesis that the proportion of

123


cancer stem cells and their inherent slower growth and chemoresistance predisposes to ‘‘nests’’ of clinically undetectable cancer stem cells [28]. These nests possess the necessary molecular machinery for further tumorigenesis and self-renewal which portends earlier clinical recurrence despite this initial ‘‘response’’ to chemotherapy. As such, irrespective of response rates, if chemotherapy fails to eradicate cancer stem cells then cancer may regenerate and a recurrence or progression of disease can occur. Combined with in vitro data that demonstrates cancer stem cell properties, this clinical correlation mandates further evaluation. In fact, this cell surface marker could hold promise as either a clinical biomarker for prognosis as well as targeted therapy in future research endeavors. In conclusion, effective durable treatment of advanced stage ovarian cancer eludes us. The ovarian cancer cell phenotype CD44?/CD24- exhibits many properties of cancer stem cells such as in vitro aggressiveness, enhanced differentiation, invasion, migration, colony formation, quiescence, and chemoresistance. Paramount to these findings in ovarian cancer cell lines is the clinical correlation to advanced stage ovarian cancer patients. Our data provides evidence to the rationale that the proportion of ovarian cancer stem cells could provide important information allowing us to predict which patients are likely to recur. As such, CD44?/CD24- could hold promise as either a clinical biomarker for prognosis as well as an opportunity to develop targeted therapy in future research endeavors. Acknowledgments This work was supported by the Gynecologic Cancer Foundation Ovarian Cancer Research Award: Sherri’s From a Whisper to a Roar, Women’s Motorcycle Foundation Award. Conflict of interest The authors declare that they have no conflict of interests to disclose.

References 1. Siegel R, Ward E, Brawley O, Jemal A (2011) Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin 61(4):212–236 2. Dean M, Fojo T, Bates S (2005) Tumour stem cells and drug resistance. Nat Rev Cancer 5(4):275–284 3. Frank NY, Margaryan A, Huang Y, Schatton T, Waaga-Gasser AM, Gasser M, Sayegh MH, Sadee W, Frank MH (2005) ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma. Cancer Res 65(10):4320–4333 4. Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414(6859):105–111 5. Rocconi RP, Matthews KS, Kemper MK, Hoskins KE, Barnes MN (2008) Chemotherapy-related myelosuppression as a marker of survival in epithelial ovarian cancer patients. Gynecol Oncol 108(2):336–341 6. Al-Hajj M, Becker MW, Wicha M, Weissman I, Clarke MF (2004) Therapeutic implications of cancer stem cells. Curr Opin Genet Dev 14(1):43–47

947 7. Al-Hajj M, Clarke MF (2004) Self-renewal and solid tumor stem cells. Oncogene 23(43):7274–7282 8. Jordan CT, Guzman ML, Noble M (2006) Cancer stem cells. N Engl J Med 355(12):1253–1261 9. Sunayama J, Sato A, Matsuda K, Tachibana K, Watanabe E, Seino S, Suzuki K, Narita Y, Shibui S, Sakurada K, Kayama T, Tomiyama A, Kitanaka C (2011) FoxO3a functions as a key integrator of cellular signals that control glioblastoma stem-like cell differentiation and tumorigenicity. Stem Cells 29(9):1327–1337 10. Joshua B, Kaplan MJ, Doweck I, Pai R, Weissman IL, Prince ME, Ailles LE (2012) Frequency of cells expressing CD44, a head and neck cancer stem cell marker: correlation with tumor aggressiveness. Head Neck 34(1):42–49 11. Wu Q, Guo R, Lin M, Zhou B, Wang Y (2011) MicroRNA-200a inhibits CD133/1? ovarian cancer stem cells migration and invasion by targeting E-cadherin repressor ZEB2. Gynecol Oncol 122(1):149–154 12. Zhao BC, Zhao B, Han JG, Ma HC, Wang ZJ (2010) Adiposederived stem cells promote gastric cancer cell growth, migration and invasion through SDF-1/CXCR4 axis. Hepatogastroenterology 57(104):1382–1389 13. Cheng L, Wu Q, Guryanova OA, Huang Z, Huang Q, Rich JN, Bao S (2011) Elevated invasive potential of glioblastoma stem cells. Biochem Biophys Res Commun 406(4):643–648 14. Luo L, Zeng J, Liang B, Zhao Z, Sun L, Cao D, Yang J, Shen K (2011) Ovarian cancer cells with the CD117 phenotype are highly tumorigenic and are related to chemotherapy outcome. Exp Mol Pathol 91(2):596–602 15. Ray A, Meng E, Reed E, Shevde LA, Rocconi RP (2011) Hedgehog signaling pathway regulates the growth of ovarian cancer spheroid forming cells. Int J Oncol 39(4):797–804 16. Van Phuc P, Nhan PL, Nhung TH, Tam NT, Hoang NM, Tue VG, Thuy DT, Ngoc PK (2011) Downregulation of CD44 reduces doxorubicin resistance of CD44CD24 breast cancer cells. Onco Targets Ther 4:71–78 17. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100(7):3983–3988 18. Tiezzi DG, Valejo FA, Marana HR, Carrara HH, Benevides L, Antonio HM, Sicchieri RD, Milanezi CM, Silva JS, de Andrade JM (2011) CD44(?)/CD24(-) cells and lymph node metastasis in stage I and II invasive ductal carcinoma of the breast. Med Oncol. doi:10.1007/s12032-011-0014-x 19. McGowan PM, Simedrea C, Ribot EJ, Foster PJ, Palmieri D, Steeg PS, Allan AL, Chambers AF (2011) Notch1 inhibition alters the CD44hi/CD24lo population and reduces the formation of brain metastases from breast cancer. Mol Cancer Res 9(7):834–844 20. Nicolini A, Ferrari P, Fini M, Borsari V, Fallahi P, Antonelli A, Berti P, Carpi A, Miccoli P (2011) Stem cells: their role in breast cancer development and resistance to treatment. Curr Pharm Biotechnol 12(2):196–205 21. Lee HE, Kim JH, Kim YJ, Choi SY, Kim SW, Kang E, Chung IY, Kim IA, Kim EJ, Choi Y, Ryu HS, Park SY (2011) An increase in cancer stem cell population after primary systemic therapy is a poor prognostic factor in breast cancer. Br J Cancer 104(11):1730–1738 22. Alvero AB, Chen R, Fu HH, Montagna M, Schwartz PE, Rutherford T, Silasi DA, Steffensen KD, Waldstrom M, Visintin I, Mor G (2009) Molecular phenotyping of human ovarian cancer stem cells unravels the mechanisms for repair and chemoresistance. Cell Cycle 8(1):158–166 23. Shi MF, Jiao J, Lu WG, Ye F, Ma D, Dong QG, Xie X (2010) Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line. Cell Mol Life Sci 67(22):3915–3925 24. Shevde LA, Samant RS, Paik JC, Metge BJ, Chambers AF, Casey G, Frost AR, Welch DR (2006) Osteopontin knockdown

123

948 suppresses tumorigenicity of human metastatic breast carcinoma, MDA-MB-435. Clin Exp Metastasis 23(2):123–133 25. Liu CG, Lu Y, Wang BB, Zhang YJ, Zhang RS, Chen B, Xu H, Jin F, Lu P (2011) Clinical implications of stem cell gene Oct-4 expression in breast cancer. Ann Surg 253(6):1165–1171 26. Wu K, Jiao X, Li Z, Katiyar S, Casimiro MC, Yang W, Zhang Q, Willmarth NE, Chepelev I, Crosariol M, Wei Z, Hu J, Zhao K, Pestell RG (2011) Cell fate determination factor Dachshund

123

Clin Exp Metastasis (2012) 29:939–948 reprograms breast cancer stem cell function. J Biol Chem 286(3):2132–2142 27. Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S, Pierotti MA, Daidone MG (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/ progenitor cell properties. Cancer Res 65(13):5506–5511 28. Li L, Neaves WB (2006) Normal stem cells and cancer stem cells: the niche matters. Cancer Res 66(9):4553–4557