Clinically relevant doses of chemotherapy agents reversibly block formation of glioblastoma neurospheres
Introduction
Temozolomide (TMZ) is the most common chemotherapy drug used to treat glioblastomas [1]. 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, carmustine) is an older drug that surgeons now deposit in the tumor bed as dissolvable wafers [2]. Both of these drugs alkylate DNA at multiple sites, including the O6 position of guanine, which can result in futile cycles of DNA repair and, ultimately, cell death [3].
Even after aggressive treatment that eliminates most of the tumor load, these tumors recur in an average of 6.9 months after initial treatment [4]. Tumor regrowth implies that glioblastomas include a population of cells that are resistant to therapy and maintain the ability to proliferate. Recently, a population of cells with stem cell-like properties was identified in brain tumors [5], and several laboratories have reported that these cells are relatively resistant to chemotherapy and radiation [6], [7].
Neurosphere cultures are heterogeneous, and only a fraction of cells are capable of sphere formation. These cells are classified as neurosphere-initiating cells (NICs) [8], [9], [10]. To quantify the NICs, normal or transformed cells are plated at low density in defined medium and after 1–2 weeks, the numbers of neurospheres are scored [11], [12]. For normal neural cells, this assay was developed to assess stem cells [8], but is now thought to also detect early progenitor cells [13], [14], [15]. It is not known whether there is a similar ambiguity for tumor NICs. Spheroid-based drug screens for other tumor types are under development [16].
The glioblastoma cells used for this assay are grown as neurospheres in defined medium to prevent differentiation. Unlike serum-supplemented cultures, glioblastoma neurosphere cells form invasive brain tumors in immunodeficient mice [17]. In addition, based on expression profiling, neurosphere cultures resemble glioblastoma tumors from patients more closely than serum-supplemented cultures [17].
Using the neurosphere-formation assay, we found that clinically relevant doses of BCNU or TMZ inhibit neurosphere formation. For four of five cell cultures, neurosphere formation resumes following a recovery period; dissociation of these initial spheres allows robust formation of secondary spheres. BCNU and TMZ induce S and/or G2/M cell cycle arrest, which partially reverses by 7 days post-treatment. Collectively, these results indicate that neurosphere formation is highly sensitive to chemotherapy drugs, and in some cases, the NICs may enter a reversible cell cycle arrest. This reversible cell cycle arrest may protect the NICs from chemotoxicity [18], [19], [20], [21], allowing regrowth of some cultures after chemotherapy. In addition, an ex vivo TMZ-treated culture that resumes sphere formation is also capable of tumor initiation as subcutaneous xenografts. This model for the survival and recovery of cultured glioblastoma neurosphere cells may provide insights for tumor recurrence in vivo.
Section snippets
Cell culture
In a previous study, PTEN −/− neural precursor cells [22] were infected with a retrovirus bearing the human mutant receptor EGFRvIII, and we refer to these as EGFRvIII PTEN −/− cells [23]. These transformed cells formed glioblastoma-like tumors in immunodeficient mice, and we established the aggressive PET2 line from one of these tumors. As a control, PTEN +/+ neural precursor cells were infected with an empty GFP MSCV-XZ066 virus [24]. We refer to these as GFP PTEN +/+ cells. A major advantage
Neurosphere formation is inhibited at lower concentrations of chemotherapy drugs than those required to inhibit bulk cell proliferation
To determine how chemotherapy drugs affect neurosphere formation, we measured neurosphere formation in non-transformed and glioblastoma neurosphere cultures without or with treatment with BCNU or TMZ. Because of their rapid decay in aqueous solution [1], [30], these drugs likely persist only a few hours, but their effects on the tumor cells last longer [31]. After 7 or 10 days, we counted the number of neurospheres that formed without or with drug treatment. For all of these cultures, BCNU and
Discussion
To more effectively treat glioblastomas, we must target the tumor-initiating cells that are resistant to current therapies. To determine the effects of chemotherapy drugs on normal and glioblastoma neurosphere cells, we measured neurosphere formation and bulk cell proliferation after treatment with BCNU or TMZ. The MTT/MTS IC50s were 2.5–240-fold greater than the neurosphere IC50s, and the MTT/MTS IC90 values for which there was substantial cell death were 16–400-fold greater than the
Conflict of interest
None declared.
Acknowledgments
We thank Dr. Michael Glantz for the gift of the temozolomide and Drs. Stephen Lyle and Lyndon Kim for their comments on this manuscript. The University of Massachusetts Medical School Tumor Bank and Dr. Julian Wu of Tufts University School of Medicine supplied freshly excised glioblastomas. The University of Massachusetts Flow Cytometry Core analyzed marker expression and cell cycle profiles. We thank the National Institutes of Health for support (Grant NS021716).
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