Abstract
The tumor-initiating cell (TIC) frequency of bulk tumor cell populations is one of the criteria used to distinguish malignancies that follow the cancer stem cell model from those that do not. However, tumor-initiating cell frequencies may be influenced by experimental conditions and the extent to which tumors have progressed, parameters that are not always addressed in studies of these cells. We employed limiting dilution cell transplantation of minimally manipulated tumor cells from mammary tumors of several transgenic mouse models to determine their tumor-initiating cell frequency. We determined whether the tumors that formed following tumor cell transplantation phenocopied the primary tumors from which they were isolated and whether they could be serially transplanted. Finally we investigated whether propagating primary tumor cells in different tissue culture conditions affected their resident tumor-initiating cell frequency. We found that tumor-initiating cells comprised between 15% and 50% of the bulk tumor cell population in multiple independent mammary tumors from three different transgenic mouse models of breast cancer. Culture of primary mammary tumor cells in chemically-defined, serum-free medium as non-adherent tumorspheres preserved TIC frequency to levels similar to that of the primary tumors from which they were established. By contrast, propagating the primary tumor cells in serum-containing medium as adherent populations resulted in a several thousand-fold reduction in their tumor-initiating cell fraction. Our findings suggest that experimental conditions, including the sensitivity of the transplantation assay, can dramatically affect estimates of tumor initiating cell frequency. Moreover, conditional on cell culture conditions, the tumor-initiating cell fraction of bulk mouse mammary tumor cell preparations can either be maintained at high or low frequency in vitro thus permitting comparative studies of tumorigenic and non-tumorigenic cancer cells.
Highlights
Tumor-initiating cells (TICs), often termed cancer stem cells, are functionally defined by their capacity to re-grow a new tumor after transplant into experimental animals that recapitulates the phenotype of the primary tumor from which the cells were isolated, and which can be serially transplanted demonstrating their capacity to differentiate and self-renew [1]
The mammary tumors we investigated may have progressed to a greater extent than those studied by others and this parameter may account for the higher TIC fraction that we observed
Vaillant et al reported that whereas the tumors from the mammary tumor virus (MMTV)-Neu model, identical to that which we investigated, comprised a relatively minor TIC fraction (,1%), the TIC could not be separated from the bulk tumor cells using antibodies to CD61, CD29 and CD24, leading these investigators to suggest that these tumors do not follow the cancer stem cell model [19]
Summary
Tumor-initiating cells (TICs), often termed cancer stem cells, are functionally defined by their capacity to re-grow a new tumor after transplant into experimental animals that recapitulates the phenotype of the primary tumor from which the cells were isolated, and which can be serially transplanted demonstrating their capacity to differentiate and self-renew [1]. TICs are infrequent in most human tumors, rarely exceeding 0.01% of the bulk tumor cell population [3,4,5,6,8,12,13]. Various parameters influence TIC frequency in bulk tumor cell preparations including the methods used to isolate and process tumor samples, the site of tumor cell injection, the extent of the immunedeficiency of the recipient host, the duration of the observational period following tumor cell transplant, and whether agents that facilitate tumor cell engraftment such as Matrigel or stromal cells are co-injected with the tumor cells [21]. The frequency of TICs in tumors is insufficient to distinguish malignancies that follow the cancer stem cell model from those that do not
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