Demonstration of iNOS-mRNA and iNOS in human monocytes stimulated with cancer cells in vitro.

Synthesis and localization of inducible nitric oxide synthase mRNA (iNOS-mRNA) and iNOS protein in the cultures of human monocytes (Mphi) and colon carcinoma cell line (DeTa) that resulted in nitric oxide (NO) synthesis has been studied. The iNOS-mRNA was observed around the sixth hour of culture and peaked at the twelfth hour. The iNOS-mRNA, as determined by the in situ hybridization and iNOS protein, as detected by staining with specific anti-iNOS monoclonal antibodies, were observed preferentially in the cytoplasm of some Mphi, but not in cancer cells. Mphi cultured alone did not show detectable iNOS-mRNA expression and iNOS protein. Mphi sorted out from tumor cells after 8 h of co-culture expressed iNOS protein and iNOS-mRNA, which were not detected in Mphi without previous contact with cancer cells. Prevention of NO synthesis by (L-N5-1-iminoethyl)-ornithine (L-NIO) partly inhibited Mphi cytotoxic activity against DeTa (NO-inducing cancer cell line) but not against the human pancreatic cancer (HPC-4) cell line that does not induce NO production in Mphi. This suggests that Mphi cytotoxic activity, at least in some cases, may be NO dependent. These observations provide further evidence that Mphi can be directly stimulated by cancer cells for de novo production of NO and suggest that iNOS occurring in the tumor-infiltrating macrophages may arise as a result of their interactions with tumor cells. However, because only some tumor cells are able to induce NO production in a small proportion of Mphi, its role in the anti-tumor response of the host is probably limited.

Preferential overexpression of CD44v5 in advanced gastric carcinoma Goseki grades I and III.

Tumor progression is associated with the clonal expansion of surviving cell variants. These results in cancer cell heterogeneity and selection of cells with a high malignant potential reflected also by the ability to metastasize. In seeding and implantation of cancer cells at the distant site cell adhesion molecules play a crucial role. Of particular interest is CD44 adhesion molecule, which possibly is involved in tumor metastasis development. Forty cases of an advanced gastric cancer were studied. Paraffin block were collected from the files. In addition to routine tumor typing, grading (Lauren and Goseki classifications) and staging, CD44 (standard, v5 and v6) was studied by immunohistochemistry and RT-PCR. CD44 immunoreactivity was found in 36 of the 40 studied cases. A significant overexpression of CD44v5 was noticed in gastric cancer. This was especially seen in Goseki's grades I and III (72.7% of cases) and was less common in Goseki's grades II and IV (44.4% of cases). CD44v6 was less commonly expressed. In some cases CD44 heterogeneity of neoplastic intravascular emboli was noticed and in some other cases stronger expression of CD44 was present in deeper parts of cancer infiltrate. Immunohistochemical expression was mostly in concert with CD44 gene expression as shown by RT-PCR results. Some discrepancies are discussed. These findings are interesting in view of better prognosis and different route of dissemination of Goseki's grades I + III compared with Goseki's grades II + IV of the gastric cancer. We have shown an overexpression of CD44v5 in an advanced gastric cancer, especially in Goseki's grades I and III. This could reflect a different malignant potential and a different route of dissemination of gastric well differentiated adenocarcinomas.

Evaluation of circulating tumour cells expressing CD44 variants in the blood of gastric cancer patients by flow cytometry.

The occurrence of circulating tumour cells in the blood of 51 patients with gastric cancer (stages I-IV) was studied using flow cytometry, cell sorting, immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The lysed whole blood samples were stained with monoclonal antibodies against common leukocyte antigen (CD45), epithelial membrane antigen (EMA), tumour associated glycoprotein 72kD (TAG72), CD44 variants (v5 and v6) and analysed by flow cytometry within ungated or CD45-gated populations. The frequency of detection of TAG72+, CD44v5+ and v6+ cells within CD45- gate was considerably increased in comparison to the ungated population. Furthermore, the presence of tumour cells was directly demonstrated by immunostaining for cytokeratin 18 of sorted CD45- population. The presence of CD44v5+, v6- cells and CD44v-mRNA in the blood was compared to their expression in the primary tumour. The occurrence of circulating CD44v+ cells was associated with their presence in the primary tumour and CD44v-mRNA in the blood. The method described may provide a sensitive tumour marker-independent tool for detection of circulating tumour cells in cancer patients.

Detection of cytokine gene expression in human monocytes and lymphocytes by fluorescent in situ hybridization in cell suspension and flow cytometry.

The use of digoxigenin (DIG)- and biotin-labelled dsDNA probes to detect TNFalpha-mRNA accumulation in human peripheral blood mononuclear cells (PBMC) and isolated monocytes is described. The fragment of the glyceraldehyde-3-phosphate dehydrogenase GAPDH-cDNA was used as a control probe. The hybridization signals were detected by staining with fluorescein isothiocyanate (FITC)-labelled anti-DIG antibody and avidin-FITC, respectively. The cells were stimulated in vitro with lipopolysaccharide (LPS) for 0.5-6 h. The TNFalpha-mRNA was detected in monocytes 1 h after stimulation with LPS, and the highest accumulation was seen around 2 h. The TNFalpha-mRNA in stimulated PBMC was detected at the lower level peaking around 4 h. The TNFalpha-mRNA accumulation was lower in lymphocytes than in monocytes when PBMC were studied. There was no difference in the level of GAPDH-mRNA between unstimulated and stimulated cells. Finally, an enhanced accumulation of TNFalpha-mRNA was observed in PBMC from some patients with sepsis or cancer. Thus, this study shows that cytokine gene expression may be detected in cells ex vivo. This opens the possibility of studying the level of cytokine gene activation in PBMC of patients with diseases where the role of cytokines in their pathophysiology is implicated.

Detection of cancer cells in the blood by FACS sorting of CD45- cells.

We describe a simple and sensitive method for detection of low number of cancer cells in the blood. The method is based on FACS sorting of leukocytes labelled with anti-CD45 monoclonal antibody and examining CD45- cells by conventional cytology and immunostaining for cytokeratin 18. In a model study, cancer cells seeded at the frequency of 1 per 106 and 1 per 107 leukocytes were detected in CD45- population. Sensitivity of this method was comparable to reverse transcription polymerase chain reaction (RT-PCR) used for detection of cancer cells expressing CD44 variants-mRNA. In a pilot study, cancer cells were also isolated from the blood of some patients with locally advanced gastric cancer. This method may be useful for detection of circulating tumour cells in cancer patients.