Inactivation Of Human Cancer Cells Using High-energy Protons And Heavy Ions

Part I Targeting glioblastoma-initiating cells by heavy ion irradiationPurpose:To investigate whether heavy ion irradiation represents an efficient approach to treat tumors by targeting glioblastoma-initiating cells (cancer stem cells, CSCs)Methods:The glioblastoma-initiating cells #10,#10-IR,#1095 and commercially available cells U87 were cultured in serum-free conditions (neurobasal A medium+B27+EGF+FGF) as neurospheres.#10 and #1095 were originating from different patients with glioblastoma multiforme. #10-IR is a sub-line of #10, which was radioselected by irradiation. The cells were irradiated with different heavy ion beams in GSI. X-rays were used for comparison. The applied doses ranged from OGy to 12 Gy. Neurosphere formation assay, reseeding assay, multiplex fluorescence in situ hybridization (mFISH), flow cytometry, immunofluorescence microscopy, and Human Stem Cell Signaling RT2 ProfilerTM PCR Array were performed.Results:Dose-response curves of #10,#10-IR, and #1095 showed a plateau at high doses, indicating a radioresistant subpopulation. The neurosphere formation ability was reduced in the progeny of cells irradiated with heavy ions compared to those exposed X-rays. mFISH-based chromosome analysis showed that the cells were aneuploid and carried several aberrations. The percentage of CD15-positive cells in #10-IR was 65±14 measured by flow cytometry, which was similar in immunofluorescence images. There were also CD44-positive populations in #10 and #1095 while CD133-positive cells were very less. Preliminary results on gene expression of #10-IR on RNA level showed upregulation in FGFR2 (FGF signaling pathway), LTBP4 and RGMA (TGFβ superfamily signaling pathway) and NFATC2 (Wnt signaling pathway) one month after heavy ion irradiation and downregulation in FZD6 (Wnt signaling pathway) one month after x-ray irradiation.Conclusion:Neurosphere formation assay of #10,#10-IR and #1095 after irradiation indicates the presence of a radioresistant subpopulation. The RBE a of heavy ions was elevated. Further cultivation of the irradiated cells revealed that the neurosphere formation ability is significantly reduced in the progenies of ion-irradiated cells compared to X-rays. Preliminary results on gene expression of #10-IR show that heavy ion irradiation induced more changes in human stem cell signaling pathways. Heavy ion irradiation represents a promising approach to treat tumors by targeting glioblastoma-initiating cells.Part Ⅱ Survival of chemoresistant cancer cells exposed to x-rays and heavy ionsPurpose:Human cancer cells and their chemoresistant cells were exposed to x-rays and heavy ions to compare the survival after irradiation and the karyotypes were analyzed.Materials and methods:Neuroblastoma cells(LAN-1) and glioblastoma cells (ASTRO) derived from patients and their chemoresistant cells were irradiated by x-rays and heavy ions and clonogenic survival assay were performed. Sphere formation test, giemsa-staining, multicolor fluorescence in situ hybridization technique and multicolor banding technique were performed.Results:The relative biological effectiveness (RBE) values of heavy ions beam relative to X-rays at the D10 values were from 2.3-2.6 for LAN-1 cells and 2.5-3.4 for ASTRO cells. Chemoresistant cells LAN-1 RETO are more radioresistant than untreated cells LAN-1 WT. Chemoresistant cells ASTRO RETO are more radiosensitive than untreated cells ASTRO WT. Sphere formation test showed LAN-1 RETO cells could form spheres in serum-free culture condition but other cell lines could not. Most of ASTRO WT cells had 71-90 chromosomes while ASTRO RETO had 52-83 chromosomes. Most of LAN-1 WT cells had 40-44 chromosomes and mFISH showed some stable aberrations especially on chromosome 10 with an unstained region., mBAND showed the unstained region were located on 10p.Conclusions:Heavy ion irradiation is more effective than x-ray for both untreated cancer cell lines and chemoresistant cell lines. For LAN-1 cells, chemoresistant cells (RETO) are more radioresistant than untreated cells (WT), while this effect was not found in ASTRO cells.Part Ⅲ Biophysical characterization of a relativistic proton beam for image-guided radiosurgeryPurpose:To measured the physical and radiobiological characteristics of 1 GeV protons for possible applications in stereotactic radiosurgery (image-guided plateau-proton radiosurgery).Methods:A proton beam was accelerated at 1 GeV at the Brookhaven National Laboratory (Upton, NY) and a target in polymethyl methacrylate (PMMA) was used. Clonogenic survival was measured after exposures to 1-10 Gy in three mammalian cell lines. Simulations were performed. The lateral dose profile was measured with or without the 20-cm plastic target.Results:Measurements and simulations demonstrate that the lateral scattering of the beam is very small. The lateral dose profile showed no significant differences up to 2 cm from the axis. A large number of secondary swift protons are produced in the target and this leads to an increase of approximately 40% in the measured dose on the beam axis at 20 cm depth. The relative biological effectiveness at 10% survival level ranged between 1.0 and 1.2 on the beam axis, and was slightly higher off-axis.Conclusion:The very low lateral scattering of relativistic protons and the possibility of using online proton radiography during the treatment make them attractive for image-guided plateau(non-Bragg peak) stereotactic radiosurgery.