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Cancer stem cells are rare cells within a tumor which give rise to the diverse tumor cell population to drive tumorigenesis. To determine their malignant potential I intracranially (i.e. under the skull into underlying brain tissue) injected oligodendroglioma derived progenitor cells into the right hemisphere of FvB/N wild type mice. When injected orthotopically (i.e. grafting of tissue or cells in their natural position), as little as 1 x 104 cells reproducibly established large tumors. Transplantation experiments were done with 3 independent tumorsphere lines and 6 animals per group. Usually, mice showed severe neurological symptoms (cycling and/or partially paralyzed animals) within days after the injection and animals developed massive tumors within weeks in the forebrain close to the injection site. In contrast, even at higher cell numbers (1 x 106) normal neural stem cells isolated from the subventricular zone of wild type mice injected as control never developed tumors (n=15) (Figure 17).

Figure 17: Oligodendroglioma derived stem cells are tumorigenic. After intracranial injection of

cancer stem cells into FvB/N mice (1 x 104 cells) orthotopic tumors consistently formed within 3 to 4 weeks indicating their malignancy. Survival of animals (n=6 per group) challenged with independent CSC lines is shown. Normal stem cells from the subventricular zone of wild type mice were injected as controls and never developed tumors.

Brains of symptomatic mice were isolated, fixed and paraffin embedded followed by immunohistochemistry and hematoxylin and eosin staining (H&E, a popular staining method in histology and the most widely used stain in medical diagnosis to identify cancers). Infiltration of human glioma is a key feature that contributes to their poor prognosis and therapeutic response. Remarkably, both spontaneous (Figure 18A-C) and orthotopic murine tumors (Figure 18D-F) were infiltrative evidenced by their easily recognized nuclei invading the surrounding brain tissue. Furthermore, histopathologic analysis of orthotopic tumors demonstrated additional oligodendroglioma-like features such as (1) high cellularity, (2) high mitotic index, (3) subpial infiltration (Figure 18A) and (4) the characteristic “fried egg” appearance of cells due to a clear and swollen cytoplasm forming a perinuclear halo (Figure 18C,F). Occasionally, tumors showed rhythmic pallisading of cells (Figure 18E). Intriguingly, the orthotopic tumor (Figure 18D-F). histologically resembled the parental tumor (Figure 18A- C). H&E stainings were analyzed by Dr. Scott Vandenberg from the Neuropathology core at UCSF who confirmed the presence of high grade oligodendroglioma (WHO grade II/III).

Figure 18: Orthotopic tumors are high grade oligodendroglioma. H&E stained sections of a

representative spontaneous oligodendroglioma (A-C) and an orthotopic tumor derived from S100ß- verbB, p53-/- cancer stem cells (D-F) after formalin fixation and paraffin embedding. Histopathologic features are characteristic for high grade oligodendroglioma as indicated by a high cellularity, high mitotic index, diffuse invasion, subpial infiltration (arrow in A) and the characteristic “fried egg” appearance of cells, i.e. empty zones around the cell nuclei (black arrows in C, F). Orthotopic tumors showing rhythmic pallisading of nuclei, a pattern typical for human oligodendroglioma (E). Scale bars in (A,D) 600 µm, in (B,E) 300 µm, in (C,F) 100 µm.

Part 2 - Results To conclusively demonstrate the stemness of oligodendroglioma derived cancer stem cells, I performed sequential transplantation experiments (Figure 19). This was done in analogy to the classical repopulation experiment used to identify true hematopoietic stem cells (Bock et al, 1999). Cultured tumor derived progenitor cells (primary cancer stem cells) were transplanted into FvB/N mice to establish a tumor. At the first sign of neurological impairment animals were sacrificed and the tumor mass was dissected out taking care not to include stem cell enriched regions like the SVZ. The tumor cells were then enzymatically dissociated and cultured under conditions identical to those used to establish tumor stem cell lines from the original oligodendroglioma. This resulted in the establishment of secondary cancer stem cell lines which were intracranially injected into new recipients again developing brain tumors. As before, cancer stem cells were isolated and re-cultured (tertiary cancer stem cells) followed by transplantation into new recipients. CSCs from orthotopic tumors were sequentially transplanted for 4 passages demonstrating their malignant potential and in vivo self renewing potential (Figure20).

Figure 19: Evaluation of the tumorigenicity of oligodendroglioma derived stem cells.

Tumorigenicity was determined by injecting oligodendroglioma-derived neural stem cells in the forebrain of FvB/N mice. Secondary cancer stem cells were isolated and re-cultured from orthotopic tumors and transplanted into new recipients. CSCs were successfully transplanted for several passages demonstrating their malignancy and in vivo self-renewing potential.

It is noteworthy that tumors developed even faster (within 2 weeks; Figure 20) with CSCs isolated from tumors of later passages suggesting that these cells became more aggressive maybe due to accumulation of additional mutations during culturing and serial implantation. In addition, particularly aggressive cells could have been selected upon transplantation, tumor growth and culturing. Notably, CSCs from orthotopic tumors cultured under same conditions as their parental cell line retained their self-renewing capacity and multipotentiality after transplantation and re-culturing (data not shown). Together, the successful serial development of oligodendroglioma-like tumors provided evidence that I have indeed isolated cancer stem cells.

Figure 20: Survival upon sequential intracranial injection of tumor derived stem cells. Animals

challenged with cancer stem cells isolated from a parental tumor usually died within 3 weeks (blue line). Secondary tumorspheres were re-cultured from these orthotopic tumors and transplanted into new recipients. Note that tumor formation occurs much faster with cells from the secondary (green line) or tertiary tumor (black line). Normal stem cells from the subventricular zone of wild type mice were injected as controls and never developed tumors.