Clinically relevant doses of chemotherapy agents reversibly block formation of glioblastoma neurospheres

Abstract

Glioblastoma patients have a poor prognosis, even after surgery, radiotherapy, and chemotherapy with temozolomide or 1,3-bis(2-chloroethy)-1-nitrosourea. We developed an in vitro recovery model using neurosphere cultures to analyze the efficacy of chemotherapy treatments, and tested whether glioblastoma neurosphere-initiating cells are resistant. Concentrations of chemotherapy drugs that inhibit neurosphere formation are similar to clinically relevant doses. Some lines underwent a transient cell cycle arrest and a robust recovery of neurosphere formation. These results indicate that glioblastoma neurospheres can regrow after treatment with chemotherapy drugs. This neurosphere recovery assay will facilitate studies of chemo-resistant subpopulations and methods to enhance glioblastoma therapy.

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.