<i>In vivo</i> imaging of therapeutic response using contrast enhanced diffusion weighted MRI.

<p>(A) MR gadolinium (Gd) contrast-enhanced T1-weighted images and ADC color overlay maps demonstrate effects of <i>in vivo</i> effects of treatment with obtained from representative control, perifosine, CCI-779 and combination (perifosine + CCI-779) treated glioma mice at 5-7 days post-initiation of therapy. (B) Plot of mean MRI-determined tumor volumes based upon Gd-contrast enhanced regions versus time post-treatment initiation for control, perifosine, CCI-779 and combination (perifosine + CCI-779) treated mice. (Error bars ± SEM). (C) Plot of percent change of mean ADC values versus time post-treatment initiation for control, perifosine, CCI-779 and combination (perifosine + CCI-779) treated mice. (Error bars ± SEM).</p

PDGF-B-driven glimoas treated with the combination of perifosine and CCI-779 undergo cell death and have decreased proliferation.

<p>Images of H&E, IHC with anti-p-S6RP, anti-PCNA, anti-Ki67, and TUNEL of (A) PTEN +/+ and (B) PTEN -/- GBMs after glioma-bearing mice were treated <i>in vivo</i> for 5 days with either vehicle, 30 mg/Kg perifosine, 40 mg/Kg CCI-779, or a combination of 30 mg/Kg perifosine with 40 mg/Kg CCI-779. PCNA and Ki67 images are 200x with the black bar indicating 100 microns. The graph on the right shows quantification of Ki67 and TUNEL staining for 3-4 independent tumors. *,**, and *** represent significance determined by ANOVA analysis for p>0.05, p>0.01, and p>0.001.</p

Combing perifosine and CCI-779 effectively decreases the levels of pAKT and pS6RP of gliomas treated <i>in vivo</i>.

<p>Immunoblot analysis of (A) PTEN +/+ and (B) PTEN -/- tumors after mice with GBMs were treated for 5 days with either vehicle, 30 mg/kg perifosine, 40 mg/kg CCI-779, or a combination of 30 mg/Kg perifosine with 40 mg/Kg CCI-779. The right panel shows the quantification of the changes in pAkt and pS6RP based on comparing the average of each group to the vehicle treated tumors (n = 3 mice per group).</p

Combing perifosine and CCI-779 effectively inhibits both the Akt and mTOR pathways in primary glioma cultures treated <i>in vitro</i>.

<p>(A) A representative immunoblot of PTEN +/+ primary GBM cell cultures after 4 hours with no treatment (NT), vehicle (V), 30 µM perifosine (P), 1 nM CCI-779 (C) or combination of 30 µM perifosine and 1 nM CCI-779 (P+C). (B) Quantification of the changes in pAkt and pS6RP based on three independent primary PTEN +/+ GBM cell cultures, with 20 µg protein per well. (C) A representative immunblot of PTEN -/- primary GBM cell cultures after 4 hours with no treatment (NT), vehicle (V), 30 µM perifosine (P), 1 nM CCI-779 (C) or combination of 30 µM perifosine and 1 nM CCI-779 (P+C). (D) Quantification of the change in pAKT and pS6RP based on three independent primary PTEN -/- GBM cultures, with 20 µg protein per well.</p

Generation of PTEN wt and PTEN null gliomas using the RCAS/tv-a system.

<p>(A) Four-to-six week old <i>nestin-tv-a/ink4a-arf-/-/pten^fl/fl</i> mice are stereotactically injected with DF-1 cells expressing the RCAS-PDGF-B virus and from high-grade gliomas with PTEN intact. (B) Four-to-six week old <i>nestin-tv-a/ink4a-arf-/-/pten^fl/fl</i> mice are stereotactically injected with DF-1 cells expressing the RCAS-PDGF-B virus and DF-1 cells expressing RCAS-Cre virus and from high-grade tumors with PTEN deleted.</p

MR, histological images and western blots are presented from representative animals in <i>Study 1</i> treatment groups.

<p>(A) MRI data consists of anatomical contrast-enhancing T1-weighted images and ADC maps. Histological stains provide information on tumor cellularity (H&E) and apoptosis (cleaved Caspase-3). All data were acquired at day 7 post-treatment initiation. (B) Representative western blot for the detection of cleaved Caspase 3 in tumor tissue from all treatment groups. B-Actin was used as a loading control to ensure proper loading of the protein samples. The tumor tissue from all groups was acquired at day 2 post-treatment initiation.</p

Plots of the percent change in tumor volume and normalized ADC (ADC) for each of the treatment groups in <i>Study 2</i>.

<p>Presented are treatment groups (A) Control, (B) irradiation (IR), (C) gemcitabine (GEM) and (D) combination gemcitabine and irradiation (GEM+IR). Data is presented over the first week of the study as the mean ± SEM.</p

MR and histological images and western blots are presented from representative animals in <i>Study 2</i> treatment groups.

<p>(A) MRI data consists of anatomical contrast-enhancing T1-weighted images and ADC maps. Histological stains provide information on tumor cellularity (H&E) and apoptosis (caspase-3). All data were acquired at day 7 post-treatment initiation. (B) Tumor tissue from animals left untreated or treated with GEM, IR and GEM+IR at day two post-treatment initiation was assessed for cleaved Caspase 3. Western blot of representative animal tissue is shown and proper loading of protein samples was ensured by probing for Gapdh.</p

Treatment schedule and Kaplan-Meier survival plots are presented for each therapy in <i>Study 2</i>.

<p>(A) Treatment schedule schematic for <i>Study 2</i>. Animals were randomized into four groups: control, IR, GEM and GEM+IR. Animals of the control group received vehicle 2 days a week for 2 weeks. Animals in the IR group received 1 Gy for 5 days as week with a two day break between treatment blocks for 2 weeks. The GEM group received 10 mg/kg GEM in saline i.p., and GEM+IR received GEM i.p. followed by 1 Gy with a 3 hour lag time between treatments. Control vehicle and GEM administration occurred every third day for a total of four doses. Arrows indicate the day of treatment. (B) Treatment groups are Controls, irradiation (IR), gemcitabine (GEM) and combination gemcitabine and irradiation (GEM+IR).</p

Results from clinical trial.

<p>(A) Representative PRM overlays show stable disease (top) and progressive disease (bottom) for PRMHU (left) and PRMADC+ (right). Blue represents regions of decreased value, red increased value, and green statistically unchanged value. (B) The bar plot shows significant differences (marked with *) between stable disease (SD, gray, n = 8) and progressive disease (PD, black, n = 4) groups in volume fractions of bone PRM results (labeled PRM_HU-, volume fraction of decreased attenuation at about 10 weeks post-treatment) and DW-MRI (volume fraction of increased ADC at about 2 weeks post-treatment).</p