Imaging flow cytometry of tumoroids: A new method for studying GPCR expression.

Fluorescence confocal microscopy is commonly used to analyze the regulation membrane proteins expression such as G protein-coupled receptors (GPCRs). With this approach, the internal movement of GPCRs within the cell can be observed with a high degree of resolution. However, these microscopy techniques led to complex and time-consuming analysis and did not allow a large population of events to be sampled. A recent approach termed imaging flow cytometry (IFC), which combines flow cytometry and fluorescence microscopy, had two main advantages to study the regulation of GPCRs expression such as orexins receptors (OXRs): the ability (1) to analyze large numbers of cells and; (2) to visualize cell integrity and fluorescent markers localization. Here, we compare these two technologies using the orexin A (OxA) ligand coupled to rhodamine (OxA-rho) to investigate anti-tumoral OX1R expression in human digestive cancers. IFC has been adapted for cancer epithelial adherent cells and also to 3D cell culture tumoroids which partially mimic tumoral structures. In the absence of specific antibody, expression of OX1R is examined in the presence of OxA-rho. 2D-culture of colon cancer cells HT-29 exhibits a maximum level of OX1R internalization induced by OxA with 19% ± 3% colocalizing to early endosomes. In 3D-culture of HT-29 cells, internalization of OX1R/OxA-rho reached its maximum at 60 min, with 30.7% ± 6.4% of OX1R colocalizing with early endosomes. This is the first application of IFC to the analysis of the expression of a native GPCR, OX1R, in both 2D and 3D cultures of adherent cancer cells.

Ectopic expression of OX1R in ulcerative colitis mediates anti-inflammatory effect of orexin-A.

Orexins (orexin-A and orexin-B) are hypothalamic peptides that are produced by the same precursor and are involved in sleep/wake control, which is mediated by two G protein-coupled receptor subtypes, OX1R and OX2R. Ulcerative colitis (UC) is an inflammatory bowel disease, (IBD) which is characterized by long-lasting inflammation and ulcers that affect the colon and rectum mucosa and is known to be a significant risk factor for colon cancer development. Based on our recent studies showing that OX1R is aberrantly expressed in colon cancer, we wondered whether orexin-A could play a role in UC. Immunohistochemistry studies revealed that OX1R is highly expressed in the affected colonic epithelium of most UC patients, but not in the non-affected colonic mucosa. Injection of exogenous orexin-A specifically improved the inflammatory symptoms in the two colitis murine models. Conversely, injection of inactive orexin-A analog, OxB7-28 or OX1R specific antagonist SB-408124 did not have anti-inflammatory effect. Moreover, treatment with orexin-A in DSS-colitis induced OX1R-/- knockout mice did not have any protective effect. The orexin-A anti-inflammatory effect was due to the decreased expression of pro-inflammatory cytokines in immune cells and specifically in T-cells isolated from colonic mucosa. Moreover, orexin-A inhibited canonical NFκB activation in an immune cell line and in intestinal epithelial cell line. These results suggest that orexin-A might represent a promising alternative to current UC therapies.

Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2.

The protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. We investigated the expression of PAR-2 and the role of trypsin in cell proliferation in human colon cancer cell lines. A total of 10 cell lines were tested for expression of PAR-2 mRNA by Northern blot and RT-PCR. PAR-2 protein was detected by immunofluorescence. Trypsin and the peptide agonist SLIGKV (AP2) were tested for their ability to induce calcium mobilization and to promote cell proliferation on serum-deprived cells. PAR-2 mRNA was detected by Northern blot analysis in 6 out of 10 cell lines [HT-29, Cl.19A, Caco-2, SW480, HCT-8 and T84]. Other cell lines expressed low levels of transcripts, which were detected only by RT-PCR. Further results were obtained with HT-29 cells: (1) PAR-2 protein is expressed at the cell surface; (2) an increase in intracellular calcium concentration was observed upon trypsin (1-100 nM) or AP2 (10-100 microM) challenges; (3) cells grown in serum-deprived media supplemented with trypsin (0.1-1 nM) or AP2 (1-300 microM) exhibited important mitogenic responses (3-fold increase of cell number). Proliferative effects of trypsin or AP2 were also observed in other cell lines expressing PAR-2. These data show that subnanomolar concentrations of trypsin, acting at PAR-2, promoted the proliferation of human colon cancer cells. The results of this study indicate that trypsin could be considered as a growth factor and unravel a new mechanism whereby serine proteases control colon tumours.

Angiogenesis in the neoplastic sequence of Barrett's oesophagus. Correlation with VEGF expression.

The purpose of this study was to determine the angiogenic profile of human oesophageal adenocarcinoma. This study was carried out on a large series of surgically resected Barrett's adenocarcinomas and associated preneoplastic lesions. Vascularization was quantified by microvessel counting and measurement of the percentage microvessel surface area after immunohistochemistry using the CD34 antibody. The expression of vascular endothelial cell growth factor (VEGF) was also examined by immunohistochemistry. Results were correlated with clinico-pathological data and prognosis. Vascularization, assessed by both microvessel counting and measurement of the microvessel surface, was statistically higher in superficial cancers than in others. Higher vascularization was correlated with a lower rate of lymph node and distant metastasis, as well as with better survival. However, when superficial carcinomas were excluded from the study, microvessel count failed to provide any significant prognostic information. Irrespective of the inclusion or exclusion of superficial tumours, the expression of VEGF was correlated with a higher vascularisation but did not provide prognostic significance. It is concluded that high angiogenic properties are acquired in precancerous lesions and early cancers in Barrett's oesophagus. Vascularization as assessed by both microvessel counting and measurement of the microvessel surface is not informative for prognosis in infiltrative Barrett's adenocarcinomas. The expression of VEGF is correlated with vascularization, but has no independent prognostic relevance.

p53 abnormalities in adenocarcinoma of the gastric cardia and antrum.

AIM: To compare the frequency and type of p53 alterations (gene mutation and/or protein overexpression) in a consecutive series of surgically resected adenocarcinomas arising in the gastric cardia and gastric antrum, and to evaluate associations with clinicopathological findings (age, sex, and tumour histology, grade, and stage). METHODS: The series comprised 50 patients with adenocarcinoma of the cardia and 20 patients with adenocarcinoma of the antrum. p53 gene mutations (exons 5-8) were detected by denaturing gradient gel analysis and DNA sequencing. Nuclear p53 overexpression was detected by immunohistochemistry with the DO7 antibody. RESULTS: p53 gene mutations were found in 21 of 50 and five of 20 adenocarcinomas of the cardia and the antrum, respectively. Base transitions occurring at CpG dinucleotides were frequent in both types of tumour. p53 protein overexpression was seen in 32 of 50 and seven of 20 adenocarcinomas of the cardia and of the antrum, respectively. p53 gene mutation and/or protein overexpression were significantly more frequent in adenocarcinomas of the cardia (37 of 50) than in adenocarcinomas of the antrum (seven of 20). There were no differences in the clinicopathological characteristics of the tumours between p53 positive and p53 negative cases in both types of cancer. CONCLUSIONS: This study shows that p53 alterations are more frequent in adenocarcinoma of the cardia than in adenocarcinoma of the antrum. This feature is consistent with the clinical and epidemiological characteristics of these cancers, which suggest that adenocarcinoma arising in the gastric cardia might be related to oesophageal adenocarcinoma, and unrelated to adenocarcinomas of the gastric body and antrum.