Brain Tumor Stem Cells

The Neurosphere Assay and the Discovery of Adult Neurogenesis

The advent of the neurosphere assay, originally developed for the isolation of adult NSCs, was critical to the identification of cancer-initiating stem cells from human brain tumors. It was originally believed that the adult brain did not retain the ability to produce new neurons in the postnatal period, but rather was a static organ. ^, Altman was first to challenge this view in 1962 when he used tritiated thymidine to follow dividing cells in restricted areas of the neonatal rat brain. Altman was able to show that these dividing cells eventually differentiated into cells that morphologically resembled neurons. Kaplan and Hinds confirmed these observations in the 1970s by using electron microscopy to show that the postnatally dividing cells in the brain contained dendrites and axons. The introduction of the neurosphere assay developed by Reynolds and Weiss allowed for the in vitro isolation of normal stem cells from adult and fetal brain tissue ( Fig. 135.1 ). In this assay, brain tissue is dissociated into single cells and cultured in special media containing various mitogens (e.g., basic fibroblast growth factor, epidermal growth factor). Under these conditions, NSCs form a ball of cells called neurospheres that contain the parent NSC and its differentiated progeny: neurons, oligodendrocytes, and astrocytes. The self-renewal capacity of these NSCs is confirmed by dissociating the neurospheres and plating individual cells per well with neurosphere media. The neurosphere-forming ability of each cell from the original neurosphere is then assessed to determine “stemness.” This assay allowed for the identification and isolation of adult NSCs in the subventricular zone (SVZ) of the lateral ventricles and the subgranular layer of the hippocampal dentate gyrus. ^,

It is now recognized that there is a strict hierarchy found in the largest of these germinal centers, the SVZ, where adult stem cells systematically differentiate into more dedicated progenitor cells. NSCs are found as quiescent type B cells that give rise to the surrounding and more rapidly dividing type C cells. These cells subsequently differentiate into type A cells (neuroblasts) that surround the ventricles, separated only by a single layer of ependymal cells, and directly divide to form new neurons in the adult brain. Adult neurogenesis is implicated to be involved in the formation of new neuronal connections and to contribute to memory formation in the normal adult brain. More important for our discussion, because adult NSCs and physiologic somatic stem cells must maintain a balance between self-renewal and differentiation, it has been hypothesized that cancer and, in the specific case of gliomas, the existence of BTSCs may be the result of unregulated self-renewal.

Development of the Brain Tumor Stem Cell Hypothesis

The traditional belief in oncology for decades maintained that human tumors represented a collection of cells of which most (or all) had the intrinsic ability to initiate tumorigenesis. The concept that only a small fraction of cells are ultimately responsible for tumorigenesis was originally proposed in 1963, when Bruce and Van Der Gaag recognized the ability of a small number of lymphoma cells to rapidly proliferate and differentiate in vivo. The theory was not supported until the advent of more sophisticated research tools that allowed scientists to recognize not only that leukemic cells were clonal in origin but also that cancer cells maintained a hierarchical organization similar to embryonic tissue; only a small subset of proliferating progenitors replenished the nonregenerating leukemia cells. ^, This concept quickly spread to other types of malignancies, and it was noted that only a subset of cells from human breast, pancreas, prostate, head and neck, and colon cancer tissue had the ability to initiate tumorigenesis on transplantation.

Rosenblum and colleagues were first to intuit the potential of the clonogenic assay applied to some peculiar cells isolated from malignant gliomas ; their results were later confirmed by Ignatova and associates, who later described how to isolate neurosphere-forming, bipotent (neuronal and astroglial) precursors from glioblastoma multiforme (GBM). This was subsequently validated and extended by Singh and colleagues, who also demonstrated that bipotent lineage-restricted progenitors from CNS tumors displayed short-term self-renewal (three passages in culture). Similar findings were later reported by Hemmati and associates, who described the absence of oligodendrocytes in these cells obtained from pediatric brain tumors and also addressed the issue of tumorigenicity, demonstrating tumor formation following intracerebral transplantation of tumor-derived cells in an animal model.

Collectively these studies demonstrated that cells endowed with some of the characteristics expected from stem cells can be found within brain tumors and opened novel scenarios of investigation in this area. In this view, these studies also raised some intriguing issues. First and foremost, can aberrant neural precursors be involved in the establishment, expansion, and recurrence of CNS cancers (i.e., are tumorigenic), or are they a by-product of uncontrolled proliferation of developmentally deranged actual tumorigenic cells? Second, if these precursors are tumorigenic, what are their actual identity and properties? Do they possess all of the features to qualify as BTSCs?

Galli and colleagues took these simple observations further by showing that neurosphere-forming cells can also act as cancer-initiating cells and establish tumors that closely resemble the main histologic, cytologic, and architectural features of the human disease, even with multiple serial transplantations. This was later confirmed by Singh and colleagues using a different method of isolating BTSCs (using the NSC marker, CD133, to be discussed later). Wakimoto and associates further demonstrated in a mouse model that BTSC xenografts are able to preserve the distinctive genotype, cytologic hallmarks, and diverse histologic variants displayed by the patient GBM from which the cells were isolated.

Unlike NSC neurospheres, however, BTSC neurospheres consisted of abnormally differentiated cells with a genetic profile similar to the original tumor and a significantly increased proliferation rate. ^{, , ,} The number of in vitro neurospheres formed in a particular sample of human brain cancer was shown to be correlated with a more rapidly growing, aggressive tumor in vivo, suggesting that poor clinical outcome was associated with the number of brain tumor–perpetuating stem cells. These experiments suggested that not all cells within brain tumors had the ability to initiate tumorigenesis. ^{, ,} Rather, brain tumor cells are hierarchically organized, with stem cells at the top of the hierarchy and “differentiating” into the aberrant tumor stromal cells at the bottom. This basic organization was similar to normal adult neurogenesis, with a small colony of NSCs replenishing the cells of the SVZ and hippocampus. ^{, ,}

Despite the neurosphere representing a functional assay to test the self-renewal capacity and therefore isolate BTSCs, some limitations must be underlined: this method is not able to detect quiescent BTSC populations; cultures (performed in nonadherent medium condition) contain populations with stem cell properties as well as more differentiated cells; and the amount of BTSCs may vary depending on passage, sphere diameter, and oxygen concentration.