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