Cancer Letters

Cancer Letters

Volume 339, Issue 2, 10 October 2013, Pages 237-246
Cancer Letters

Ovarian tumor initiating cell populations persist following paclitaxel and carboplatin chemotherapy treatment in vivo

https://doi.org/10.1016/j.canlet.2013.06.014Get rights and content

Highlights

  • Paclitaxel and carboplatin treatment impedes tumor growth of a genetically defined mouse ovarian cancer cell line in vivo.

  • Paclitaxel and carboplatin withdrawal results in tumor resurgence.

  • Sca-1 and CD133 expressing cells persist post-Paclitaxel and carboplatin treatment.

  • Sca-1+ and CD133+ cells form tumors rapidly, maintain tumor heterogeneity and express stem-cell related genes.

  • Sca-1+ and CD133+ expressing cells represent populations of tumor initiating cells.

Abstract

Development of recurrent platinum resistant disease following chemotherapy presents a challenge in managing ovarian cancer. Using tumors derived from genetically defined mouse ovarian cancer cells, we investigated the stem cell properties of residual cells post-chemotherapy. Utilizing CD133 and Sca-1 as markers of candidate tumor initiating cells (TIC), we determined that the relative levels of CD133+ and Sca-1+ cells were unaltered following chemotherapy. CD133+ and Sca-1+ cells exhibited increased stem cell-related gene expression, were enriched in G0/G1-early S phase and exhibited increased tumor initiating capacity, giving rise to heterogeneous tumors. Our findings suggest that residual TICs may contribute to recurrent disease.

Introduction

Ovarian cancer is the leading cause of death among women with gynecological cancers in the United States. It is estimated that in the year 2013 alone 22,240 women will be diagnosed with ovarian cancer and −14,030 women will succumb to the disease [1], [2]. Non-symptomatic onset, delayed diagnosis, and the aggressive nature of ovarian cancer make clinical management especially challenging. The primary treatment modality for women presenting with advanced stage ovarian carcinoma often includes surgical debulking immediately followed by taxane and platinum based chemotherapy [3], [4], [5], [6]. While most patients respond to first line therapy, with time a significant number will develop highly aggressive recurrent disease that is often platinum resistant [3], [7]. This resurgence is a major obstacle to successful long-term disease management in these women since the treatment options for recurrent and/or platinum resistant ovarian cancer are currently limited and less effective [8], reviewed in [9], [10]. Thus, understanding the underlying biology of the chemoresistant tumor cells that potentially lead to tumor resurgence could provide a significant advantage in therapeutic management of recurrent ovarian cancer.

Development of recurrent disease has been attributed, at least in part, to a unique population of cells often referred to as cancer stem cells (CSC), tumor initiating cells (TIC), cancer initiating cells (CIC) or tumor repopulating cells reviewed in [11], [12]. This population, in the majority of the cases, is believed to be relatively rare within the larger tumor bulk and is thought to have regained the capacity to both self-replicate and give rise to more differentiated cells resulting in a hierarchal heterogeneous tumor [13], [14], [15], [16], reviewed in [11], [17]. Unlike rapidly dividing differentiated tumor cells, TICs often evade the toxicity of standard chemotherapeutics due to their slow division rate and the ability to take advantage of efflux properties associated with multidrugresistance [18], [19], [20], [21], [22], [23], [24], [25], [26]. TICs have been identified and characterized in leukemia and several solid cancers including breast, lung, prostate, brain, liver and ovarian [15], [27], [28], [29], [30], [32], [33] reviewed in [11]. Enrichment or isolation of ovarian TICs has been successfully performed via multiple strategies including dye exclusion via efflux properties. Ovarian TICs were enriched in a side population (SP) fraction that was identified by its ability to efflux Hoechst 33342 dye [19], [22], [23], [24], [27], [28], [29], [30], [31]. Alternatively, differential expression of specific cell surface antigens or differential enzymatic activity has been utilized in the isolation andcharacterization of TICs. Expression of CD44, CD133, and CD117 and aldehyde dehydrogenase (ALDH) enzymatic activity either alone or in combination have been utilized in the identification, isolation and functional testing of TICs in human ovarian tumors, ascites and ovarian cancer cell lines [13], [21], [32], [33], [34], [35], [36], [37], [38]. Some in vitro and in vivo studies provide evidence to suggest there is an enrichment of ovarian cancer stem-like cell populations post-chemotherapy [18], [19], [23], [25], [26], [29], [38] supporting the hypothesis that these cells resist conventional cytotoxic therapies and contribute to the development of recurrent disease.

Our objective was to utilize tumors derived from a genetically defined mouse ovarian cancer cell line to assess the role of putative ovarian TICs in tumor resurgence. The T2 cell line was generated from poorly differentiated mouse ovarian tumors formed following injection of p53−/−; c-myc; myristoylated-Akt1 expressing mouse ovarian surface epithelial cells [39]. Subsequent injection of T2 cells in immunocompromised mice leads to rapid formation of poorly differentiated tumors. We used this in vivo experimental system to model tumor resurgence following treatment with paclitaxel and carboplatin. We determined whether tumor cell sub-populations identified by expression of specific stem cell markers persist post-chemotherapy, are capable of inducing tumor formation and display stem cell-like properties. Our results suggest that such TICs are present within the T2-derived tumors and possibly contribute to the resurgence of tumor growth.

Section snippets

T2 cell culture

The mouse ovarian cancer cell line T2 (p53−/−; c-myc; myristoylated-Akt1) was maintained at subconfluency in Dulbecco’s modified Eagles medium (DMEM, Mediatech Inc.) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) and 1% penicillin–streptomycin (Gibco Carlsbad, CA) and passaged 2 times a week as previously described [39].

T2 tumor generation

T2 cells (2 × 106) were resuspended in PBS:Matrigel® (BD Biosciences, Bedford, MA) in 1:1 ratio and injected subcutaneously into 6–8 week old female NOD/SCID mice

Paclitaxel and carboplatin treatment inhibits growth of T2 tumors

The effect of paclitaxel in combination with carboplatin on T2 tumor growth in vivo was assessed by administering either vehicle or paclitaxel and carboplatin to mice bearing T2-derived tumors. Although mice treated with vehicle alone exhibited a significant (p < 0.03) increase in tumor volume over the course of the treatment period, tumor volume was relatively unchanged in mice treated with paclitaxel and carboplatin (Fig. 1A, p = 0.126). The significant difference in tumor volume (p < 0.03)

Discussion

The research focus in a greater majority of cancers has expanded to include an emphasis on an often elusive and potentially rare population of tumor cells believed to possess stem cell-like properties. The specific properties of cancer stem cells are, to some degree, context specific. However, most researchers would agree that common CSC properties include the capacity to self replicate, the ability to give rise to more differentiated daughter cells and relative quiescence, and chemoresistance

Funding

This research was funded in part by grants from the Advanced Medical Research Foundation (BRR) and the Vincent Memorial Research Funds (BRR).

Conflict of interest

None declared.

Acknowledgements

The authors would like to thank Laura Prickett-Rice, Kat Folz-Donahue and Meredith Weglarz at the HSCI Flow Cytometry Core Facility for their help with cell sorting and cell cycle analysis; and colleagues at the VCRB laboratories for critical review of the manuscript.

References (63)

  • S. Yanamoto et al.

    Isolation and characterization of cancer stem-like side population cells in human oral cancer cells

    Oral Oncol.

    (2011)
  • L. Luo et al.

    Ovarian cancer cells with the CD117 phenotype are highly tumorigenic and are related to chemotherapy outcome

    Exp. Mol. Pathol.

    (2011)
  • E.J. Tokar et al.

    Stem/progenitor and intermediate cell types and the origin of human prostate cancer

    Differentiation

    (2005)
  • G.J. Spangrude et al.

    Mouse hematopoietic stem cells

    Blood

    (1991)
  • B.E. Welm et al.

    Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population

    Dev. Biol.

    (2002)
  • S.F. Altekruse et al.

    SEER Cancer Statist. Rev.

    (2010)
  • American Cancer Society

    Cancer Facts and Figures 2013

    (2013)
  • R.F. Ozols et al.

    Phase III trial of carboplatin and paclitaxel compared with cisplatin and paclitaxel in patients with optimally resected stage III ovarian cancer: a gynecologic oncology group study

    J. Clin. Oncol.

    (2003)
  • A. Berkenblit et al.

    Advances in the management of epithelial ovarian cancer

    J. Reprod. Med.

    (2005)
  • C.T. Jordan et al.

    Cancer stem cells

    N. Engl. J. Med.

    (2006)
  • M. Wintzell et al.

    Protein markers of cancer-associated fibroblasts and tumor-initiating cells reveal subpopulations in freshly isolated ovarian cancer ascites

    BMC Cancer

    (2012)
  • K.J. Hope et al.

    Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity

    Nat. Immunol.

    (2004)
  • A.T. Collins et al.

    Prospective identification of tumorigenic prostate cancer stem cells

    Cancer Res.

    (2005)
  • J. Stingl et al.

    Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis

    Nat. Rev. Cancer

    (2007)
  • A.D. Steg et al.

    Stem cell pathways contribute to clinical chemoresistance in ovarian cancer

    Clin. Cancer Res.

    (2012)
  • S. Rizzo et al.

    Ovarian cancer stem cell-like side populations are enriched following chemotherapy and overexpress EZH2

    Mol. Cancer Ther.

    (2011)
  • A.D. Steg et al.

    Stem cell pathways contribute to clinical chemoresistance in ovarian cancer

    Clin. Cancer Res.

    (2011)
  • J. Balbuena et al.

    ABCG2 is required to control the sonic hedgehog pathway in side population cells with stem-like properties

    Cytometry A

    (2011)
  • L. Hu et al.

    Ovarian cancer stem-like side-population cells are tumourigenic and chemoresistant

    Br. J. Cancer.

    (2010)
  • L. Ma et al.

    Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3

    Acta Biochim. Biophys. Sin. (Shanghai)

    (2010)
  • P.P. Szotek et al.

    Ovarian cancer side population defines cells with stem cell-like characteristics and mullerian inhibiting substance responsiveness

    Proc. Natl. Acad. Sci. USA

    (2006)
  • Cited by (24)

    • Ovarian cancer stem cell biology and chemoresistance

      2020, Overcoming Ovarian Cancer Chemoresistance
    • Ovarian cancer stem cells: What progress have we made?

      2019, International Journal of Biochemistry and Cell Biology
      Citation Excerpt :

      These CD133+ cells were highly tumorigenic which recapitulated the original tumor heterogeneity, were drug resistant and/or had vasculogenic potential (Baba et al., 2009; Ferrandina et al., 2009; Curley et al., 2009; Kusumbe et al., 2009; Stemberger-Papic et al., 2015; Cioffi et al., 2015). CD133+ cells were enriched in samples collected from platinum resistant patients and post-chemotherapy treated mouse OvCa cells, supporting the concept that CD133+ cells were chemo-resistant (Steg et al., 2012a; Kulkarni-Datar et al., 2013). However, there is discrepancy within the field as at least two studies show no difference between the tumorigenic potential of CD133+ and CD133- cells in OvCa (Stewart et al., 2011; Ishiguro et al., 2016).

    • Proteotranscriptomics Reveal Signaling Networks in the Ovarian Cancer Microenvironment

      2018, Molecular and Cellular Proteomics
      Citation Excerpt :

      Furthermore, CCL18 secreted by TAM has previously been reported to promote angiogenesis and metastasis formation in breast cancer (90, 91), consistent with our findings for HGSOC. The selective expression of KITLG by bTAM is intriguing in view of its function as a stemness-promoting factor and the previous identification of its receptor CD133 as a marker for ovarian epithelial stem cells in the mouse (92) and for cancer stem cells in ascites (93, 94). CD133 expression has also been shown to promote ovarian cancer metastasis and resistance (92, 95, 96), and is associated with a worse clinical outcome (97).

    • Ovarian cancer stem cells: Are they real and why are they important?

      2014, Gynecologic Oncology
      Citation Excerpt :

      One method to examine a subpopulation for an association with chemoresistance is to compare pre- and post-treatment specimens for changes with the exposure. Kulkarni-Datar showed that CD133/Sca-1 cells persist in tumors treated with carboplatin and paclitaxel treatment, and maintain their tumor-initiating properties [34]. Steg et al. measured expression of stem cell markers in a cohort of matched primary and recurrent tumor patient samples, on the premise that if CSCs are an important population to target, they should represent a denser population within recurrent chemoresistant tumors [35].

    View all citing articles on Scopus
    View full text