Modeling Therapy Response and Spatial Tissue Distribution of Erlotinib in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is likely the most aggressive and therapy-resistant of all cancers. The aim of this study was to investigate the emerging technology of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) as a powerful tool to study drug delivery and spatial tissue distribution in PDAC. We utilized an established genetically engineered mouse model of spontaneous PDAC to examine the distribution of the small-molecule inhibitor erlotinib in healthy pancreas and PDAC. MALDI IMS was utilized on sections of single-dose or long-term-treated mice to measure drug tissue distribution. Histologic and statistical analyses were performed to correlate morphology, drug distribution, and survival. We found that erlotinib levels were significantly lower in PDAC compared with healthy tissue (P = 0.0078). Survival of long-term-treated mice did not correlate with overall levels of erlotinib or with overall histologic tumor grade but did correlate both with the percentage of atypical glands in the cancer (P = 0.021, rs = 0.59) and the level of erlotinib in those atypical glands (P = 0.019, rs = 0.60). The results of this pilot study present MALDI IMS as a reliable technology to study drug delivery and spatial distribution of compounds in a preclinical setting and support drug imaging-based translational approaches. Mol Cancer Ther; 15(5); 1145-52. ©2016 AACR.

Early static (18)F-FET-PET scans have a higher accuracy for glioma grading than the standard 20-40 min scans.

PURPOSE: Current guidelines for glioma imaging by positron emission tomography (PET) using the amino acid analogue O-(2-[(18)F]fluoroethyl)-L-tyrosine ((18)F-FET) recommend image acquisition from 20-40 min post injection (p.i.). The maximal tumour-to-background evaluation (TBRmax) obtained in these summation images does not enable reliable differentiation between low and high grade glioma (LGG and HGG), which, however, can be achieved by dynamic (18)F-FET-PET. We investigated the accuracy of tumour grading using TBRmax values at different earlier time points after tracer injection. METHODS: Three hundred and fourteen patients with histologically proven primary diagnosis of glioma (131 LGG, 183 HGG) who had undergone 40-min dynamic (18)F-FET-PET scans were retrospectively evaluated. TBRmax was assessed in the standard 20-40 min summation images, as well as in summation images from 0-10 min, 5-15 min, 5-20 min, and 15-30 min p.i., and kinetic analysis was performed. TBRmax values and kinetic analysis were correlated with histological classification. ROC analyses were performed for each time frame and sensitivity, specificity, and accuracy were assessed. RESULTS: TBRmax values in the earlier summation images were significantly better for tumour grading (P < 0.001) when compared to standard 20-40 min scans, with best results for the early 5-15 min scan. This was due to higher TBRmax in the HGG (3.9 vs. 3.3; p < 0.001), while TBRmax remained nearly stable in the LGG (2.2 vs. 2.1). Overall, accuracy increased from 70 % in the 20-40 min analysis to 77 % in the 5-15 min images, but did not reach the accuracy of dynamic analysis (80 %). CONCLUSIONS: Early TBRmax assessment (5-15 min p.i.) is more accurate for the differentiation between LGG and HGG than the standard static scan (20-40 min p.i.) mainly caused by the characteristic high (18)F-FET uptake of HGG in the initial phase. Therefore, when dynamic (18)F-FET-PET cannot be performed, early TBRmax assessment can be considered as an alternative for tumour grading.

In vivo imaging of CT26 mouse tumours by using cmHsp70.1 monoclonal antibody.

The major stress-inducible heat shock protein 70 (Hsp70) is frequently present on the cell surface of human tumours, but not on normal cells. Herein, the binding characteristics of the cmHsp70.1 mouse monoclonal antibody (mAb) were evaluated in vitro and in a syngeneic tumour mouse model. More than 50% of the CT26 mouse colon carcinoma cells express Hsp70 on their cell surface at 4°C. After a temperature shift to 37°C, the cmHsp70.1-fluorescein isothiocyanate mAb translocates into early endosomes and lysosomes. Intraoperative and near-infrared fluorescence imaging revealed an enrichment of Cy5.5-conjugated mAb cmHsp70.1, but not an identically labelled IgG1 isotype-matched control, in i.p. and s.c. located CT26 tumours, as soon as 30 min. after i.v. injection into the tail vein. Due to the rapid turnover rate of membrane-bound Hsp70, the fluorescence-labelled cmHsp70.1 mAb became endocytosed and accumulated in the tumour, reaching a maximum after 24 hrs and remained detectable at least up to 96 hrs after a single i.v. injection. The tumour-selective internalization of mAb cmHsp70.1 at the physiological temperature of 37°C might enable a targeted uptake of toxins or radionuclides into Hsp70 membrane-positive tumours. The anti-tumoral activity of the cmHsp70.1 mAb is further supported by its capacity to mediate antibody-dependent cytotoxicity.

Flavone induces changes in intermediary metabolism that prevent microadenoma formation in colonic tissue of carcinogen-treated mice.

SCOPE: Colorectal cancer is a major cause of cancer deaths worldwide with the need for improved therapeutics and adjuvants. METHODS AND RESULTS: We here tested whether the secondary plant compound flavone affects the development of aberrant crypt foci and microadenomas triggered in C57BL/6J mice by 1,2-dimethylhydrazine. Ten weeks after the last 1,2-dimethylhydrazine injection, flavone was applied at 400 mg/kg body weight over 4 wk by gavage. Flavone was found to increase apoptosis and to reduce the rate of proliferation and aberrant crypt formation. More importantly, development of microadenomas was completely suppressed by flavone. Proteome analysis by 2-DE with mass spectrometric identification of regulated proteins suggests a downregulation of tricarboxylic acid cycle activity in colonocytes with compensation by increased FADH(2) production via a partial beta-oxidation of long-chain fatty acids to meet energy demands. Transcriptome analysis, using a Gene Chip expression array with 24,000 gene probes confirmed the proteome data and moreover revealed the increased expression of various solute transporters, suggesting increased substrate supply to be used for tricarboxylic acid cycle-independent energy production. CONCLUSION: In conclusion, changes in the levels of proteins from intermediary metabolism or their encoding mRNAs are linked to flavone-induced apoptosis and the prevention of microadenoma formation in transformed colonocytes of mice.

Proteome response in HT-29 human colorectal cancer cells to two apoptosis-inducing compounds with different mode of action.

Flavone and camptothecin were both shown to potently induce apoptosis in HT-29 human colon cancer cells. Whereas camptothecin acts on the basis of topoisomerase-I inhibition, flavone appears to burst mitochondrial production of reactive oxygen species by increasing respiratory chain activity. In our study, we searched for similarities and differences in the proteome response of HT-29 cells when treated with the two different compounds. The accessible proteome of HT-29 cells was separated subsequent to the exposure to flavone or camptothecin by 2D-polyacrylamide-gel electrophoresis using pH-gradients between 4 and 7 and 6 and 11 in the first dimension and proteins with changed expression level were identified by peptide mass fingerprints of tryptic digests of the protein spots. Whereas there was a high congruence with regard to the identities of regulated proteins and their grade of regulation, a number of spots changed specifically only in response to either flavone or camptothecin. Nuclear envelope proteins were specifically increased by camptothecin indicating the intervention of this drug with cell division processes. Increased levels of coproporphyrinogen III oxidase, involved in cytochrome synthesis, and ubiquinol-cytochrome-c reductase suggest adaptations to flavone in order to enable a higher substrate flux through the respiratory chain. In conclusion, HT-29 cells respond to camptothecin and flavone with regulations of many proteins in a similar manner suggesting those alterations to be caused by apoptosis induction. Some protein regulations, however, were specific for each compound and point to the mechanism of their action.

The suppression of aberrant crypt multiplicity in colonic tissue of 1,2-dimethylhydrazine-treated C57BL/6J mice by dietary flavone is associated with an increased expression of Krebs cycle enzymes.

Colorectal cancer is the second leading cause of cancer deaths worldwide with diet playing a prominent role in disease initiation and progression. Flavonoids are secondary plant compounds that are suggested as protective ingredients of a diet rich in fruits and vegetables. We here tested whether flavone, a flavonoid that proved to be an effective apoptosis inducer in colon cancer cells in culture, can affect the development of aberrant crypt foci (ACFs) in C57BL/6J mice in vivo when preneoplastic lesions were induced by the carcinogen 1,2-dimethylhydrazine (DMH). Flavone applied at either a low dose (15 mg/kg body wt per day) or a high dose (400 mg/kg body wt per day) reduced the numbers of ACFs significantly, independent of whether it was supplied simultaneously with the carcinogen (blocking group) or subsequent to the tumor induction phase (suppressing group). Proteome analysis performed in colonic tissue samples revealed that flavone treatment increased the expression of a number of Krebs cycle enzymes in the suppressing group and this was associated with reduced crypt multiplicity. It suggests that mitochondrial substrate oxidation is increased by flavone in colonic cells in vivo as already observed in HT-29 cells in vitro as the prime mechanism underlying tumor cell apoptosis induction by flavone. In conclusion, flavone reduces the number of ACFs in DMH-treated mice at doses that can be achieved for flavonoids by a diet rich in fruits and vegetables. Moreover, reduction in crypt multiplicity by flavone is most probably due to the preservation of a normal oxidative metabolism.

Proteomics in nutrition research: principles, technologies and applications.

The global profiling of the whole protein complement of the genome expressed in a particular cell or organ, or in plasma or serum, makes it possible to identify biomarkers that respond to alterations in diet or to treatment, and that may have predictive value for the modelling of biological processes. Proteomics has not yet been applied on a large scale in nutritional studies, yet it has advantages over transcriptome profiling techniques in that it directly assesses the entities that carry out the biological functions. The present review summarizes the different approaches in proteomics research, with special emphasis on the current technical 'workhorses': two-dimensional (2D)-PAGE with immobilized pH gradients and protein identification by MS. Using a work-flow approach, we provide information and advice on sample handling and preparation, protein solubilization and pre-fractionation, protein separation by 2D-PAGE, detection and quantification via computer-assisted analysis of gels, and protein identification and characterization techniques by means of MS. Examples from nutritional studies employing proteomics are provided to demonstrate not only the advantages but also the limitations of current proteome analysis platforms.