Sebastian Foersch

In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor.

BACKGROUND & AIMS Epidermal growth factor receptor (EGFR) is a therapeutic target for colorectal cancer (CRC). However, technical challenges have limited in vivo imaging of EGFR in CRCs. Confocal laser endomicroscopy (CLE) enables accurate microscopic visualization of CRC in patients during endoscopy. We evaluated the ability to use CLE in vivo for instantaneous molecular imaging of EGFR in CRC models. METHODS Tumors were grown in mice (n = 68) from human CRC cell lines that expressed high (SW480 cells) or low (SW620 cells) levels of EGFR. Tumors were visualized in vivo with a handheld CLE probe after injection of fluorescently labeled EGFR antibodies. EGFR-specific fluorescence was graded from 0 to 3+. Neoplastic and non-neoplastic specimens from human colorectal mucosa were examined. In vivo findings were correlated with histopathology, immunohistochemistry, and fluorescence microscopy analyses. RESULTS CLE analysis of cell cultures confirmed the different expression levels of EGFR between cell lines. In living animals, CLE differentiated EGFR expression levels between tumor cell limes (mean fluorescence, 1.92 +/- 0.22 [SW480] and 0.59 +/- 0.21 [SW620], P = .0004). CLE analysis of EGFR expression in human specimens allowed distinction of neoplastic from non-neoplastic tissues (mean fluorescence, 2.0 +/- 0.37 vs 0.25 +/- 0.16, respectively, P = .0035). CONCLUSIONS CLE can be used for in vivo, molecular analysis of CRC and to differentiate EGFR expression patterns in xenograft tumors and human tissue samples. Because CLE can be performed during endoscopy, in vivo molecular imaging might be used in diagnosis of CRC and to predict response to targeted therapies.

Interleukin-6 - A Key Regulator of Colorectal Cancer Development

Growing evidence proposes an important role for pro-inflammatory cytokines during tumor development. Several experimental and clinical studies have linked the pleiotropic cytokine interleukin-6 (IL-6) to the pathogenesis of sporadic and inflammation-associated colorectal cancer (CRC). Increased IL-6 expression has been related to advanced stage of disease and decreased survival in CRC patients. According to experimental studies, these effects are mediated through IL-6 trans-signaling promoting tumor cell proliferation and inhibiting apoptosis through gp130 activation on tumor cells with subsequent signaling through Janus kinases (JAKs) and signal transducer and activator of transcription 3 (STAT3). During recent years, several therapeutics targeting the IL-6/STAT3 pathway have been developed and pose a promising strategy for the treatment of CRC. This review discusses the molecular mechanisms and possible therapeutic targets involved in IL-6 signaling in CRC.

Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy

Background Vascular endothelial growth factor (VEGF) is a therapeutic target in gastrointestinal cancer (GiC). However, its in vivo visualisation could not be achieved to date with endoscopic techniques. Confocal laser endomicroscopy (CLE) is a novel imaging technique for gastrointestinal endoscopy providing in vivo microscopy at subcellular resolution. The aim of the study was to evaluate CLE for in vivo molecular imaging of VEGF in GiC. Methods Molecular imaging of tumours in APCmin mice, in xenograft models and in surgical specimens of patients with colorectal cancer (CRC) was achieved after application of labelled antibodies. The tumour sites were scanned with the probe for the strongest specific fluorescent signal. From all tumour sites examined with CLE in vivo, targeted specimens were obtained for histology, immunohistochemistry (IHC) and fluorescence microscopy. Results A VEGF-specific signal was visualised in vivo in 13/15 APCmin mice and in 9/10 xenograft tumours. CLE enabled the cytoplasmatic distribution of VEGF to be displayed due to its subcellular resolution. In human tissue, a VEGF-specific signal was observed in 12/13 malignant specimens and in 10/11 samples from healthy mucosa from the patients (p<0.03). CLE findings correlated well with ex vivo microscopy. Conclusion In vivo molecular imaging with specific targeting of VEGF is possible in murine tumours, human xenografts and tissue specimens using CLE. CLE with similar probes can be performed in human colonoscopy. Therefore—from a technical point of view—in vivo molecular imaging is transferable to stratification of patients with CRC during endoscopy even today. CLE could contribute to the identification of lesions at risk and potentially predict response to targeted treatment.

Confocal Laser Endomicroscopy for Diagnosis and Histomorphologic Imaging of Brain Tumors In Vivo

Early detection and evaluation of brain tumors during surgery is crucial for accurate resection. Currently cryosections during surgery are regularly performed. Confocal laser endomicroscopy (CLE) is a novel technique permitting in vivo histologic imaging with miniaturized endoscopic probes at excellent resolution. Aim of the current study was to evaluate CLE for in vivo diagnosis in different types and models of intracranial neoplasia. In vivo histomorphology of healthy brains and two different C6 glioma cell line allografts was evaluated in rats. One cell line expressed EYFP, the other cell line was used for staining with fluorescent dyes (fluorescein, acriflavine, FITC-dextran and Indocyanine green). To evaluate future application in patients, fresh surgical resection specimen of human intracranial tumors (n = 15) were examined (glioblastoma multiforme, meningioma, craniopharyngioma, acoustic neurinoma, brain metastasis, medulloblastoma, epidermoid tumor). Healthy brain tissue adjacent to the samples served as control. CLE yielded high-quality histomorphology of normal brain tissue and tumors. Different fluorescent agents revealed distinct aspects of tissue and cell structure (nuclear pattern, axonal pathways, hemorrhages). CLE discrimination of neoplastic from healthy brain tissue was easy to perform based on tissue and cellular architecture and resemblance with histopathology was excellent. Confocal laser endomicroscopy allows immediate in vivo imaging of normal and neoplastic brain tissue at high resolution. The technology might be transferred to scientific and clinical application in neurosurgery and neuropathology. It may become helpful to screen for tumor free margins and to improve the surgical resection of malignant brain tumors, and opens the door to in vivo molecular imaging of tumors and other neurologic disorders.

Colitis and Colorectal Cancer

Inflammatory bowel diseases (IBD) are accompanied by an increased risk of developing colitis-associated carcinoma (CAC). These tumors are one of the most important causes of morbidity and mortality in patients with IBD and distinctly differ from sporadic colorectal cancer in their biology and underlying mechanisms. First, this review discusses risk factors for the development of CAC and summarizes some of the most important genetic alterations and molecular pathways involved in inflammatory carcinogenesis. Then, new endoscopic techniques, such as chromoendoscopy and confocal laser endomicroscopy, and their contribution to surveillance and early detection of CAC are presented. Last, we briefly address different types of concepts for prevention (i.e. anti-inflammatory agents) and treatment (i.e. surgical resection) of CAC and give an outlook on this important aspect of IBD.

Innate and Adaptive Immunity in Inflammatory Bowel Diseases

While barrier function and the effects of the intestinal microbiome have only recently moved into the focus of inflammatory bowel disease research, the role of the innate and the adaptive immune system in these gastrointestinal disorders has extensively been studied. Although still not completely understood, the increasing knowledge about the immune systems contribution to the pathophysiology of inflammatory bowel diseases has led to new diagnostic and therapeutic approaches. This review gives a compact overview on this important topic.

Endomicroscopic Imaging of COX-2 Activity in Murine Sporadic and Colitis-Associated Colorectal Cancer.

Although several studies propose a chemopreventive effect of aspirin for colorectal cancer (CRC) development, the general use of aspirin cannot be recommended due to its adverse side effects. As the protective effect of aspirin has been associated with an increased expression of COX-2, molecular imaging of COX-2, for instance, during confocal endomicroscopy could enable the identification of patients who would possibly benefit from aspirin treatment. In this pilot trial, we used a COX-2-specific fluorescent probe for detection of colitis-associated and sporadic CRC in mice using confocal microscopy. Following the injection of the COX-2 probe into tumor-bearing APCmin mice or mice exposed to the AOM + DSS model of colitis-associated cancer, the tumor-specific upregulation of COX-2 could be validated with in vivo fluorescence imaging. Subsequent confocal imaging of tumor tissue showed an increased number of COX-2 expressing cells when compared to the normal mucosa of healthy controls. COX-2-expression was detectable with subcellular resolution in tumor cells and infiltrating stroma cells. These findings pose a proof of concept and suggest the use of CLE for the detection of COX-2 expression during colorectal cancer surveillance endoscopy. This could improve early detection and stratification of chemoprevention in patients with CRC.

High Accumulation of Metformin in Colonic Tissue of Subjects With Diabetes or the Metabolic Syndrome

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 Dear Editors: The recognition that the hyperinsulinemic state is associated with the metabolic syndrome or type II diabetes mellitus is linked with increased colorectal cancer risk has led to studies of antidiabetes drugs to prevent cancer. Metformin has multiple actions that contribute to its potential anticancer effects, and has been associated with reduced cancer incidence and mortality in diabetic patients and patients with colorectal cancer specifically. In a recent double-blind, placebo-controlled, randomized phase III clinical trial in 151 patients, a low dose of 250 mg/d of metformin was associated with a decrease of approximately 40% in colorectal adenoma recurrence 1 year after initial polypectomy. Although the liver is recognized as the major site of metformin pharmacodynamics, the gut as an important site of action. To date, confirmation that metformin can reach the colonic tissue and attain adequate levels to exert direct potential antitumor effects in humans is lacking. We performed a pilot study in 12 subjects with diabetes or the metabolic syndrome to compare metformin concentrations in plasma with colonic tissue according to colon site. The median metformin plasma concentration was

Prognostic relevance of androgen receptor expression in renal cell carcinomas

Background Despite rapid discoveries in molecular biology of renal cell carcinoma (RCC) and advances in systemic targeted therapies, development of new diagnostic and therapeutic strategies is urgently needed. The androgen receptor (AR) has been shown to hold prognostic and predicitve value in several malignancies. Here, we studied a possible association between AR expression and prognosis in patients with RCCs. Results Low AR expression levels were associated with occurrence of distant metastasis and higher tumor stage in papillary and clear-cell RCCs. Importantly, multivariate Cox regression analyses revealed that AR is an independent prognostic factor for cancer-specific survival. Materials and Methods The expression of AR was measured by immunohistochemistry and assessed by digital image analysis using a tissue microarray containing tumor tissue of a large and well-documented series of RCC patients with long-term follow-up information. Chi-squared tests, Kaplan-Meier curves and Cox regression models were used to investigate the possible relationship between AR expression and clinico-pathological characteristics and patient survival. Conclusions Patients affected by AR-positive tumors exhibit a favorable prognosis by multiple Cox regression, while loss of AR expression is related to aggressive disease. Therefore, assessing AR expression offers valuable prognostic information that could improve treatment selection for metastatic disease. Moreover, our findings highlight a potential therapeutic use of AR pharmaceuticals in patients with RCCs.

In Vivo Immunohistochemistry With Labeled Bevacizumab Using Confocal Laser Endomicroscopy in Models of Human Colorectal Cancer

Helicobacter pylori CagA is delivered into H. pylori-adherent gastric epithelial cells through a bacterial type IV secretion system. The delivered CagA is tyrosine-phosphorylated at the EPIYA motifs by the host cell kinases, and then binds and aberrantly-activate Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2) and partitioning-defective 1 (PAR1), thereby acting as an epigenetic oncoprotein that promotes gastric cancer development in its early phase. Recently, Ishikawa et al. reported that the biological half-life of CagA is about 200 min in the gastric epithelial cells (FEBS Lett.,583:2414,2009). This report showed that the action of CagA as an epigenetic oncoprotein was not sustained for a prolonged period of time in the normal condition. In fact, the stability of CagA in the gastric epithelial cells is important for its further oncogenesis. The present study was designed to examine the degradation mechanisms of CagA in the gastric epithelial cells. Methods: MKN28 cells were transfected with CagA expression vectors (the pTet-Off and pTRE2hyg-Wild-typecagA-HA) (WT-A10 cells), and then CagA in the WT-A10 cells was induced for 24 h. H. pylori ATCC700392 was infected with AGS cells for 5 h. At 5 h after the infection, the AGS cells were incubated with 400 μg/mL kanamycin and harvested at 4, 12 and 24 h. 50 μM Z-Leu-Leu-Leu-al (MG132) and 20 μM lactacystin were used as the 26S-proteasome inhibitor, 5 mM3-methyladenine and 50 nMwortmannin as the inhibitors of autophagy, and rapamycin as the inducer of autophagy. Intracellular CagA was evaluated by westernblotting and immunocytochemistry. Results: After the induction of CagA in the WT-A10 cells for 24 h, significantly increase by 2-fold of the intracellular level of CagA was observed following treatment with the proteasome inhibitors, but not following that with the inhibitors of autophagy. On the other hand, degradation of CagA in the WT-A10 cells was promoted in a dose-dependent manner by induction of autophagy with rapamycin. In addition, ubiquitinated-CagA was detected in the WT-A10 cells by immunoprecipitation and the TUBE reaction method. Although the levels of intracellular CagA in the AGS cells decreased in a timedependent manner after H. pylori infection, the rate of CagA degradation in the AGS cells was attenuated to 60% under treatment with proteasome inhibitors at 4 h, and further was attenuated to 70% under treatment with the autophagy inhibitors at 24 h. In addition, conversion of LC3-I to LC3-II, which shows autophagy, was detected in the AGS cells at 24 h. Conclusion: Intracellular CagA was initially targeted for proteasomal degradation, and then it was extensively degraded by the induction of autophagy.