In vivo PET/CT in a human glioblastoma chicken chorioallantoic membrane model: A new tool for oncology and radiotracer development.

in Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine (in press)

For many years the laboratory mouse has been used as the standard model for in vivo oncology research, particularly in the development of novel PET tracers, but the growth of tumors on chicken ... [more ▼]

For many years the laboratory mouse has been used as the standard model for in vivo oncology research, particularly in the development of novel PET tracers, but the growth of tumors on chicken chorioallantoic membrane (CAM) provides a more rapid, low cost and ethically sustainable alternative. For the first time, we demonstrate the feasibility of in vivo PET and CT imaging in a U87 glioblastoma tumor model on chicken chorioallantoic membrane (CAM), with the aim of applying this model for screening of novel PET tracers. Methods: U87 glioblastoma cells were implanted on the CAM at day 11 post-fertilization and imaged at day 18. A small animal imaging cell was used to maintain incubation and allow anesthesia using isoflurane. Radiotracers were injected directly into the exposed CAM vasculature. Sodium [18F]fluoride was used to validate the imaging protocol, demonstrating that image-degrading motion can be removed with anesthesia. Tumor glucose metabolism was imaged using [18F]fluorodeoxyglucose and tumor protein synthesis was imaged using 2-[18F]fluoro-L-tyrosine. Anatomical images were obtained by contrast enhanced CT, facilitating clear delineation of the tumor, delineation of tracer uptake in tumor versus embryo and accurate volume measurements. Results: PET imaging of tumor glucose metabolism and protein synthesis was successfully demonstrated in the CAM U87 glioblastoma model. Catheterization of CAM blood vessels facilitated dynamic imaging of glucose metabolism with [18F]fluorodeoxyglucose and demonstrated the ability to study PET tracer uptake over time in individual tumors, while CT imaging improved the accuracy of tumor volume measurements. Conclusion: In summary, we describe the novel application of PET/CT in the CAM tumor model, with optimization of typical imaging protocols. PET imaging in this valuable tumor model could prove particularly useful for rapid, high-throughput screening of novel radiotracers. [less ▲]

INTRA-TUMORAL HETEROGENEITY AND RATIONAL SELECTION OF ANTIGENS FOR TARGETED THERAPY OF LIVER METASTASES

in Acta Chirurgica Belgica (2012, May), 112(3), 8953

Objectives: Targeted therapies of liver metastases are gaining a major stake in current and future treatment options. However, the malignant lesions are heterogeneous in nature offering niches for cancer ... [more ▼]

Objectives: Targeted therapies of liver metastases are gaining a major stake in current and future treatment options. However, the malignant lesions are heterogeneous in nature offering niches for cancer cells causing treatment resistance and relapse. Therefore, a rational strategy is needed to select targetable antigens that would overcome this intra-tumoral heterogeneity. Methods: After ethical committee approval, 48 fresh liver metastases of colorectal origin were prospectively collected from patients undergoing liver resection. Here we macroscopically divided the lesion in different zones and generated a unique quantitative picture of the proteome heterogeneity in colorectal carcinoma liver metastases. Particular focus was laid on accessible proteins, a protein subclass comprising cell membrane associated and extracellular proteins. Accordingly, the tissues were ex-vivo biotinylated, affinity purified and analyzed for each zone separately using nano-UPLC-MSe proteomics technique. In total over 1500 unique proteins were statistically divided into different patterns of expression. Results: We have generated a quantitative picture of the proteome heterogeneity in colorectal carcinoma liver metastases. The study offers insight into novel targets but also antigens against which the antibodies are already involved in clinical trials or treatment of liver metastases. Extensive clustering and validation experiments highlight novel markers that offer the potential to homogeneously cover the metastatic lesion and become better targets. Conclusions: Two such antigens, LTBP2 and TGFBI were selected for functional analysis in colorectal carcinoma cells. In vitro and in vivo experiments showed that in particular TGFBI is relevant for migration and proliferation capacity of colorectal cancer cells. The suppression of this protein led to significant inhibition of tumor growth, crystalizing it as bona fide target for the development of anti-metastases therapies. [less ▲]

Novel comprehensive approach for accessible biomarker identification and absolute quantification from precious human tissues

in Journal of Proteome Research (2011), 10(7), 3160-82

The identification of specific biomarkers obtained directly from human pathological lesions remains a major challenge, because the amount of tissue available is often very limited. We have developed a ... [more ▼]

The identification of specific biomarkers obtained directly from human pathological lesions remains a major challenge, because the amount of tissue available is often very limited. We have developed a novel, comprehensive, and efficient method permitting the identification and absolute quantification of potentially accessible proteins in such precious samples. This protein subclass comprises cell membrane associated and extracellular proteins, which are reachable by systemically deliverable substances and hence especially suitable for diagnosis and targeted therapy applications. To isolate such proteins, we exploited the ability of chemically modified biotin to label ex vivo accessible proteins and the fact that most of these proteins are glycosylated. This approach consists of three successive steps involving first the linkage of potentially accessible proteins to biotin molecules followed by their purification. The remaining proteins are then subjected to glycopeptide isolation. Finally, the analysis of the nonglycosylated peptides and their involvement in an in silico method increased the confident identification of glycoproteins. The value of the technique was demonstrated on human breast cancer tissue samples originating from 5 individuals. Altogether, the method delivered quantitative data on more than 400 potentially accessible proteins (per sample and replicate). In comparison to biotinylation or glycoprotein analysis alone, the sequential method significantly increased the number (≥30% and ≥50% respectively) of potentially therapeutically and diagnostically valuable proteins. The sequential method led to the identification of 93 differentially modulated proteins, among which several were not reported to be associated with the breast cancer. One of these novel potential biomarkers was CD276, a cell membrane-associated glycoprotein. The immunohistochemistry analysis showed that CD276 is significantly differentially expressed in a series of breast cancer lesions. Due to the fact that our technology is applicable to any type of tissue biopsy, it bears the ability to accelerate the discovery of new relevant biomarkers in a broad spectrum of pathologies. [less ▲]

Proteomic evolution of s.cerevisiae during chronological aging

Poster (2010, July 19)

Opposite to the replicative aging, which refers to the exponential decline in the capacity of a single cell to divide, chronological aging of the yeast Saccharomyces cerevisiae refers to the time period a ... [more ▼]

Opposite to the replicative aging, which refers to the exponential decline in the capacity of a single cell to divide, chronological aging of the yeast Saccharomyces cerevisiae refers to the time period a yeast cell can survive in a non-dividing state. In 2006, Allen and co-workers reported that yeast cells evolve into two cell types during stationary phase: a high density population defined as quiescent cells (Q) and a low density population defined as non-quiescent (NQ) cells. These two populations mainly differ by their viability, measured as the ability to form colonies when platted on Petri dishes, and can be separated by differential centrifugation on density gradient. In this work, we used the quantitative proteomics technique of 2DDIGE (two Dimensional Differential In-Gel Electrophoresis) to compare the evolution of the yeast cellular soluble proteome during chronological aging. We also checked the impact of the carbon source on stationary-phase cell differentiation. As the ratio of Q/NQ cells is decreasing with time, we have selected three distinct periods: 0 day (32h after outset of yeast culture on glucose, 100% of Q cells), 7 days (50% of Q cells) and 14 days (100% of NQ cells) to realize 3 proteomics comparisons (fig 1). [less ▲]

Proteomic and functional characterization of a Chlamydomonas reinhardtii mutant lacking the mitochondrial alternative oxidase 1

in Journal of Proteome Research (2010)

In the present work we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase ... [more ▼]

In the present work we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase (AOX). The AOX-deficient strain displays a doubling of the cell volume and biomass without any alteration of the generation time, a significantly higher ROS production, no change in total respiration rate, and a slight decrease of the photosynthesis efficiency. In order to identify the molecular adaptation underlying these phenotypical effects, we carried out a comparative proteomic study at the level of the mitochondrial and cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways. [less ▲]