Histolopathology.

<p>Histologic features of liver PHEO lesions stained with H&E 24 hours after <i>i.p</i>. vehicle alone (left) or with <i>i.p.</i> LB1 at 1.5 mg/kg plus TMZ by gavage at 80 mg/kg (right). Exposure to a single <i>i.p.</i> injection of vehicle showed a homogeneous field of healthy appearing tumor cells, whereas combination treatment resulted in extensive necrosis of tumor cells.</p

Western blot on MPC cells.

<p>Changes in pAKT, p53, pMDM2, and pPlk-1 after 24 hours treatment on MPC cells with 5 µM of LB1, 50 µM of TMZ and combination of both drugs. (A) Western blots show that LB1 exposure increases pAKT compared to control MPC cells (untreated, only vehicle). TMZ does not noticeably change the expression of pAKT and combination of LB1 and TMZ highly increases pAKT expression. (B) A demonstration of markedly increased expression of p53 after TMZ treatment but inhibition of expression by exposure to LB1. Noticeable increase in the expression of pMDM2 in MPC cells treated with LB1 alone or in combination with TMZ. (C) Noticeable increases in expression of pPlk1 in MPC cells after exposure to the combination of drugs.</p

<i>In vivo</i> anti-tumor activity of LB1 and TMZ and histological examination.

<p>Effects of treatment upon growth and molecular changes in hepatic tumors: (A) MRI images of untreated mice at weeks 5, 6, and 7 following intravenous injection of MPC cells and a photomicrograph showing the growth hepatic lesions and Alzet minipump. Barbed arrow indicates the gall bladder. Plain arrows indicate the same tumor nodules over time. (B) Inhibition of total hepatic tumor volume by LB1 alone at 1.5 mg/kg daily for 14 days by <i>c.i.</i> administered at 5^th day after MPC cells injection; TMZ alone at 80 mg/kg for 3 doses administered at 15^th day after MPC cells injection; and, combination of both drugs. (C) Survival curve combining the data from the study depicted in (B) a total of 10 control animals, 10 animals with combination treatment LB1 and TMZ, 5 animals with TMZ alone, and 5 animals with LB1 alone. Kaplan-Meier analysis revealed that survival following LB1 plus TMZ was significantly greater than with LB1 alone and TMZ alone (log rank, <i>P</i><0.0001). (D) MRI images of mice, treated with the combination of LB1 by <i>c.i</i>. for 14 days and 3 doses of TMZ, at week 7, 9, and 12. Partial response of treatment is presented with delayed appearance of hepatic tumors compared to untreated group. Complete response presents absence of hepatic tumors after treatment. A photomicrograph of the liver of one treated animal at week 12, showing the absence of gross tumor, and the presence of fibrous scar tissue (arrows). (E) Inhibition of estimated total hepatic tumor volume by combination treatment of LB1 and TMZ. LB1 at 1.5 mg/kg daily for 14 days by <i>c.i</i>. administered at 5^th day after MPC cells injection and TMZ at 80 mg/kg every 3 days for 14 doses beginning on 15^th day after MPC cells injection (with combination or alone). (F) Survival curve combining the data described in E. Total of 12 control animals, 5 animals for TMZ and 7 animals for combination treatment LB1 and TMZ. Survival of animals with combined treatment were significantly greater compared to controls (log rank, <i>P</i><0.0001). (G) Serial MRI images of mice, treated with the combination of LB1 and 14 doses TMZ with partial and complete responses. (H) Histologic features of liver PHEO at week 12 stained with H&E receiving no treatment or LB1 by <i>c.i.</i> and three doses of TMZ as described in D. Untreated animals showed intrahepatic deposits of cancer cells whereas the liver of an animal receiving both drugs that had no gross evidence of tumor revealed normal parenchyma and fibrous tissue, believed to be scarring at former sites of tumor masses. (I) Survival curves of animals treated with LB1 and 14 doses of TMZ when administration started at the same time, on day 5 after MPC cells injection. (n = 5 treated animals with LB1 plus TMZ, n = 5 for controls; log rank, <i>P</i> = 0.0035).</p

<i>In vitro</i> anti- proliferative activity of LB1 and TMZ and their combination.

<p>Inhibition of growth of MPC cells in culture: (A and B) Exposure for 3 days to increasing concentrations of LB1 or TMZ. (C–E) Exposure to increasing concentrations of LB1 plus TMZ. (F) Synergy analysis was done based on data from (A–E) using CalsuSyn software. CI values: C = 1 as additivity; C<1 as synergy; C>1 as antagonism. Combo 1 presents combination of 5 µM of LB1 and 100, 200, 300 µM of TMZ; Combo 2 presents combination of 7.5 µM of LB1 and 100, 200, 300 µM of TMZ; and Combo 3 10 µM of LB1 and 100, 200, 300 µM of TMZ.</p

Effect of LB1 and TMZ on tumor cell cycle and apoptosis.

<p>(A) Cell cycle analysis of MPC cells in exponential growth exposed for 48 hours to vehicle alone; LB1 alone at 5 µM; TMZ at 50 µM and; LB1 at 5 µM and TMZ at 50 µM. (B) PARP expression changes in 24 hours after treatment of mice bearing hepatic tumors with vehicle, LB1 alone at 1.5 mg/kg by gavage, TMZ alone by gavage at 80 mg/kg, of both drugs at the same doses.</p

Western blot on PHEO tumors.

<p>Changes in the state of phosphorylation and abundance of small pAKT, p53, pMDM2, and pPlk-1 24 hours after treatment of mice bearing hepatic tumors with vehicle, LB1 alone at 1.5 mg/kg by gavage, TMZ alone by gavage at 80 mg/kg, of both drugs at the same doses. (A) Western blots show that LB1 exposure increases pAKT in control-untreated tumors and treated tumors. TMZ does not change the expression of pAKT and combination of LB1 plus TMZ highly increases pAKT expression. (B) Western blots demonstrate marked increased expression of p53 after TMZ but complete inhibition of this induction by exposure to LB1 accompanied by an increase in the expression of pMDM2 in tumor cells exposed to LB1 alone or in combination with TMZ (C) Expression of pPlk1 shows a marked increase in tumors after exposure to the combination of drugs.</p

Optimized Brain Extraction for Pathological Brains (optiBET)

<div><p>The study of structural and functional magnetic resonance imaging data has greatly benefitted from the development of sophisticated and efficient algorithms aimed at automating and optimizing the analysis of brain data. We address, in the context of the segmentation of brain from non-brain tissue (i.e., brain extraction, also known as skull-stripping), the tension between the increased theoretical and clinical interest in patient data, and the difficulty of conventional algorithms to function optimally in the presence of gross brain pathology. Indeed, because of the reliance of many algorithms on priors derived from healthy volunteers, images with gross pathology can severely affect their ability to correctly trace the boundaries between brain and non-brain tissue, potentially biasing subsequent analysis. We describe and make available an optimized brain extraction script for the pathological brain (optiBET) robust to the presence of pathology. Rather than attempting to trace the boundary between tissues, optiBET performs brain extraction by (i) calculating an initial approximate brain extraction; (ii) employing linear and non-linear registration to project the approximate extraction into the MNI template space; (iii) back-projecting a standard brain-only mask from template space to the subject’s original space; and (iv) employing the back-projected brain-only mask to mask-out non-brain tissue. The script results in up to 94% improvement of the quality of extractions over those obtained with conventional software across a large set of severely pathological brains. Since optiBET makes use of freely available algorithms included in FSL, it should be readily employable by anyone having access to such tools.</p></div

Optimization results.

<p>Box plot depiction of the mismatch between each tested pipeline and the manually brain-extracted “benchmark” images. Increased sum of squares implies lower extraction quality – as compared to the benchmark extractions. (RBX: Robex; 3dSS: 3dSkullStrip; us: useskull; L: linear transformation; NL: non-linear transformation; M152: MNI152; A152: Average152. The boxplot depicts the 25^th, 50^th (i.e., median) and 75^th percentiles, and top and bottom whiskers depict the maximum and minimum values, respectively).</p

Sample extraction results.

<p>Rendering of brain extractions obtained from different algorithms/options (shown in grey) compared to the manually-traced benchmark (shown in green wireframe) for three sample patients with different degrees of brain pathology (little, medium, and high, for the left, middle and right columns respectively; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115551#pone-0115551-g003" target="_blank">Fig. 3</a>). The first row depicts the rendering of the benchmark volume (in grey) and the benchmark wireframe to illustrate a case of “perfect fit”. (Renderings obtained with ParaView <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115551#pone.0115551-Hendrson1" target="_blank">[19]</a>).</p

OptiBET workflow.

<p>Visual representation of the optiBET script workflow: (1) initial approximate brain extraction is performed using BET (and options ‘B’ and ‘f’, as suggested previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115551#pone.0115551-Popescu1" target="_blank">[5]</a>); (2) sequential application of a linear and non-linear transformation from native space to MNI template space; (3) back-projection of a standard brain-only mask from MNI to native space; and (4) mask-out brain extraction.</p