Abstract

Malignant gliomas and medulloblastomas contain a subset of self-renewing tumor progenitor cells designated brain tumor stem cells (BTSCs) that are an attractive target for T cell mediated immunotherapy. BTSCs are able to resist current therapy because they

  1. can reside in the G0 state then reenter the cell cycle,

  2. can migrate away from the tumor and infiltrate surrounding normal brain parenchyma, and

  3. express transporter molecules, which efflux chemotherapeutic agents.

To develop mouse models of BTSC immunotherapy, we have generated five murine medulloblastoma tumor lines (MM) derived from cerebellar tumors removed from Ptc+/−p53−/− transgenic mice. Mouse DCs were generated by injecting Flt3L (5 mcg) for 8 days, followed by MACS positive selection of CD11c+ splenocytes and overnight culture in medium supplemented with GM-CSF and IL-4. Electrofusion of DC and irradiated mouse medulloblastoma cells was performed by mixing DC and tumor cells at a 1:1 ratio (2 × 107 cells/ml) in electrofusion buffer [5% dextrose, 0.5 mM Mg (CH3COO)2, 0.1 mM Ca(CH3COO)2 pH 7.2]. Cells were subjected to dielectrophoresis using an ac current of 220V/cm × 9s to align the cells in a chain-like configuration between the electrodes, then pulsed with a 1200 V/cm × 25 μs to induce transient membrane breakdown. The following day, non-adherent DC were decanted and adherent cells, consisting of multinucleated heterokaryons and unfused tumor cells were harvested with a yield of 8% to 22% of cells expressing both DC and tumor markers. In previous therapy experiments DC/GL261 or DC/fibrosarcoma electrofusion vaccines were injected intrasplenically into mice bearing 7-day established intracranial tumors in combination with 5Gy local brain irradiation, and adjuvant anti-CD134 mAb. Therapy induced complete regression of established tumors, which was immunologically specific for the tumor cell component of the vaccine. Cured survivors resisted subsequent intracranial challenge. To investigate whether human glioma vaccines could be prepared, we established BTSC lines. The BTSCs were derived from tumor specimens using serum-free medium supplemented with EGF and FGF2 and they grew as non-attached neurospheres. BTSC lines were confirmed to contain genetic changes concordant with the original tumor and several lines expressed CD133 and nestin. Human DCs were prepared from normal donor PBMC cultured in GM-CSF, IL-4 and matured with PGE2 and TNF-α. Single cell suspensions of DCs and irradiated, fluorescently labeled BTSCs were mixed at a 2:1 ratio in electrofusion buffer and were subjected to ac current of 220 V/cm × 9s then dc pulse of 1200 V/cm × 99 μs. Multinucleated heterokaryons were readily apparent on cytospin preparations and FACS analysis demonstrated double positive cells containing fluorescently labeled tumor proteins and DC markers, such as HLA-DR, CD11c, CD80, and CD86. Four independent BTSC lines yielded heterokaryons incorporating between 25% and 33% of the total tumor cells. These experiments demonstrate that BTSC lines can be derived from patient samples and undergo efficient heterokaryon formation with DCs. Heterokaryons contain the entire complement of tumor antigens as well as the antigen-processing and co-stimulatory molecules found on antigen-presenting cells. Future experiments will explore whether a common set of tumor antigens are shared between BTSCs derived from GBM samples to establish whether it is feasible to prepare brain tumor vaccines.

Disclosure: No relevant conflicts of interest to declare.

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