At three and eight weeks post-intracranial injection, pcGBM39 and pcGBM2-bearing mice were randomized into treatment groups: TMZ (200 mg/kg; cumulative dose) was orally administered either alone or in combination with ICT/CLIO-ICT (80mg/kg; cumulative dose), twice per week for three consecutive weeks
At three and eight weeks post-intracranial injection, pcGBM39 and pcGBM2-bearing mice were randomized into treatment groups: TMZ (200 mg/kg; cumulative dose) was orally administered either alone or in combination with ICT/CLIO-ICT (80mg/kg; cumulative dose), twice per week for three consecutive weeks. potential for clinical translation. MRI studies were conducted on a 7T MR scanner (Bruker Biospin, Billerica, MA). The transverse relaxation times (T2) of ferumoxytol and CLIO-ICT in water with various of Fe concentrations (0, 2.5, 5, 10, 20, 40 g/mL) were measured individually using a fast spin echo sequence with a repetition time (TR) of 3000 ms, multiple echo times (TE) of 6.8, 13.6, 20.4, 27.3, 34 and 40.9 ms. The T2 relaxivity values (r2) was obtained from linear least-squares determination of the slope of 1/T2 relaxation rate (s?1) versus the Fe concentration plot. The concentration of iron (Fe) was determined using inductively coupled plasma-mass spectrometry (ICP-MS) and the iron (Fe) atoms in each iron oxide core was Rabbit Polyclonal to Caspase 7 (Cleaved-Asp198) estimated to be 6200 using Diamond? crystal structure analysis software. The molar concentration of iron (Fe) in CLIO-ICT stock solution was then calculated. Since ICT is linked GW 9662 with FITC, the average amount of ICT covalently immobilized to a single CLIO-ICT nanoparticle was GW 9662 determined by the emission intensity of FITC. In brief, the emission profile of FITC of a diluted CLIO-ICT solution was recorded upon excitation at 495 nm. The concentration of FITC was then estimated using a calibration plot obtained from a set of standard FITC solutions. The average number of ICT on each CLIO-ICT was then obtained. To determine the sizes of ferumoxytol and CLIO-ICT, the samples in DI water at a Fe concentration of 100 g/mL were analyzed using a Zetasizer Nano ZS equipment. Cell Culture Two patient-derived GBM cell lines (pcGBM2, pcGBM39) and three commercially available GBM cell lines (U87-MG, U138 and A172 from ATCC, Manassas, VA, USA) were used for studies. Both pcGBM2 and pcGBM39 were kindly provided by Dr. Sanjiv Sam Gambhir, Stanford University. HCN2, normal cortical neuron cells were purchased from ATCC. U87-MG, U138, A172 cells were grown in DMEM (Life Technologies) containing 10% FBS and 1% GW 9662 Penicillin/Streptomycin (Life Technologies). HCN2 cells were grown in DMEM with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 90%; fetal bovine serum, 10%. pcGBM2 and pcGBM39 were cultured as floating cellular spheres as previously described (30). All cell lines used were authentic and confirmed to be mycoplasma negative using the MycoAlert Mycoplasma Activity Kit GW 9662 (Lonza). To test for mycoplasma activity, MycoAlert substrate was added to the test media (cell supernatant) which catalyzes the conversion of ADP to ATP by mycoplasma enzymes in the cell supernatant. This was followed by addition of a luciferase reaction mix that cleaved ATP to produce a bioluminescent signal as an indication of mycoplasma activity. All cell lines used were from early passages. Assessment of MMP-14 gene expression of different patient-derived GBM neurospheres (pcGBM2 and pcGBM39) and GBM cell lines (U87-MG, U138 and A172) as well as human neuronal cortical cells (HCN2) as controls was determined by qPCR as previously described (29). qPCR expression analysis for MMP-14 and the control marker GAPDH was done and the total cellular RNA was extracted from each sample with the QIAGEN RNeasy? mini kit. cDNA was prepared from total RNA and quantitative real-time PCRs (qPCRs) were carried out and analyzed on an Applied Biosystems StepOne? Real-Time PCR System. The formation of double-stranded DNA product was monitored by TaqMan? gene expression primers. evaluation of the therapeutic efficacy of CLIO ICT against GBM cancer cells and GIC Patient-derived GBM neurospheres (pcGBM2 and pcGBM39) and GBM cell lines (U87-MG, U138 and A172) as well as human neuronal cortical cells (HCN2) cells were plated in 96 well plates at 2 104 cells/well and treated with CLIO-ICT (10 nM), ICT (10 nM), CLIO (0.01 mM) and DMSO GW 9662 at 37C, 5% CO2. Cell viability after 96 hours was assessed using MTS assay kit (Promega) as per the manufacturer’s instruction. At the end of the incubation period, the absorbance was measured at 490 nm in a microplate reader (Expert Plus V1.4 ASYS). Cell viability was also verified with cell counting following addition of trypan blue (Sigma) at a final.