Investigations on the usefulness of CEACAMs as potential imaging targets for molecular imaging purposes

Members of the carcinoembryonic antigen cell adhesion molecules (CEACAMs) family are the prototype of tumour markers. Classically they are used as serum markers, however, CEACAMs could serve as targets for molecular imaging as well.In order to test the anti CEACAM monoclonal antibody T84.1 for imaging purposes, CEACAM expression was analysed using this antibody. Twelve human cancer cell lines from different entities were screened for their CEACAM expression using qPCR, Western Blot and FACS analysis. In addition, CEACAM expression was analyzed in primary tumour xenografts of these cells. Nine of 12 tumour cell lines expressed CEACAM mRNA and protein when grown in vitro. Pancreatic and colon cancer cell lines showed the highest expression levels with good correlation of mRNA and protein level. However, when grown in vivo, the CEACAM expression was generally downregulated except for the melanoma cell lines. As the CEACAM expression showed pronounced expression in FemX-1 primary tumours, this model system was used for further experiments. As the accessibility of the antibody after i.v. application is critical for its use in molecular imaging, the binding of the T84.1 monoclonal antibody was assessed after i.v. injection into SCID mice harbouring a FemX-1 primary tumour. When applied i.v., the CEACAM specific T84.1 antibody bound to tumour cells in the vicinity of blood vessels. This binding pattern was particularly pronounced in the periphery of the tumour xenograft, however, some antibody binding was also observed in the central areas of the tumour around blood vessels. Still, a general penetration of the tumour by i.v. application of the anti CEACAM antibody could not be achieved despite homogenous CEACAM expression of all melanoma cells when analysed in tissue sections. This lack of penetration is probably due to the increased interstitial fluid pressure in tumours caused by the absence of functional lymphatic vessels.     

Epithelial Glycoprotein-2 Expression is Subject to Regulatory Processes in Epithelial–mesenchymal Transitions During Metastases: an Investigation of Human Cancers Transplanted into Severe Combined Immunodeficient Mice

The human cell-surface antigen epithelial glycoprotein-2 recognized by the monoclonal antibody MOC-31 is an epithelial tumour-associated glycoprotein expressed in non-squamous carcinomas. MOC-31 immunoreactivity was investigated in human breast, colon, ovarian and lung cancer cell lines, grown either in vitro or in severe combined immunodeficient (SCID) mice as solid tumours and/or metastases. Three of four small-cell lung cancer cell lines (NCI-H69, OH3 and SW2) and three of four ovarian cancer cell lines (SoTü 1, 3 and 4) expressed epithelial glycoprotein-2. In contrast, all three breast (MCF-7, BT20, T47D) and all three colon (HT29, CACO2, SW480) cancer cell lines strongly reacted with monoclonal antibody MOC-31. A notable difference in MOC-31 immunoreactivity was observed in spontaneously formed lung metastases of HT29 colon cancer cells. Whereas larger metastases (> 30 cells) re acted with a similar staining pattern to the primary tumour, smaller metastases did not. These findings indicate that differentiation processes during the epithelial–mesenchymal transition occur in metastases, which lead to a transient loss of epithelial glycoprotein-2 expression during the migratory and early post- migratory period. This loss of antigen expression indicates that the process of metastases formation is a regulatory event, and this transient loss of antigen expression might represent a potential obstacle to antibody-based therapy in the setting of minimal residual disease.     

The interaction between CD44 on tumour cells and hyaluronan under physiologic flow conditions: implications for metastasis formation

The adhesion of tumour cells to the endothelial cells of blood vessels of the microcirculation represents a crucial step in haematogenous metastasis formation. Similar to leukocyte extravasation, selectins mediate initial tumour cell rolling on endothelium. An additional mechanism of leukocyte adhesion to endothelial cells is mediated by hyaluronan (HA). However, data on the interaction of tumour cells with hyaluronan under shear stress are lacking. The expression of the hyaluronan binding protein CD44 on tumour cell surfaces was evaluated using flow cytometry. The adhesion of tumour cells to HA with regard to adhesive events and rolling velocity was determined in flow assays in the human small cell lung cancer (SCLC) cell lines SW2, H69, H82, OH1 and OH3, the colon carcinoma cell line HT29 and the melanoma cell line MeWo. Hyaluronan deposition in human and mouse lung blood vessels was histochemically determined. MeWo adhered best to HA followed by HT29. SCLC cell lines showed the lowest CD44 expression on the cell surface and lowest number of adhesive events. While hyaluronan was deposited in patches in the microvasculature of the alveolar septum in the human lung, it was only present in the periarterial space in the mouse lung. Certain tumour entities bind to HA under physiological shear stresses so that HA can be considered a further ligand for cell extravasation in haematogenous metastasis. As hyaluronan is deposited within the pulmonary microvasculature, it may well serve as a ligand for its binding partner CD44, which is expressed by many tumour cells.     

Interleukin-8 as an autocrine growth factor for human colon carcinoma cells in vitro

Cell lines derived from human colon carcinomas secrete interleukin 8 (IL-8) in vitro and this chemokine has also been detected immunohistochemically in human colon carcinoma specimens, in which it is tumour cell associated. In these experiments, IL-8 was shown to comprise an important component of the angiogenic activity of colon carcinoma cell line supernatants. The effect of modulating IL-8 activity upon the growth of the colon carcinoma cell lines HCT116A, HT29 and CaCo2 was investigated. Supplementing endogenously produced IL-8 by recombinant chemokine led to stimulation of cell growth. Neutralization of the effect of endogenously produced IL-8, either with the specific antagonist peptide AcRRWWCR or with blocking anti-IL-8 antibody, resulted in around 50% inhibition of cell growth (P<0.05). All of the colon carcinoma cell lines tested expressed mRNA for both IL-8RA and RB when grown at confluence. At the protein level, all cell lines expressed IL-8RA. Expression of IL-8RB was weak, although increased expression was seen in HCT116A cells as they approached confluence. Antibodies to IL-8RA and RB did not affect proliferation at low cell density but were strongly inhibitory when cells were cultured at a higher density. These data suggest that IL-8 acts as an autocrine growth factor for colon carcinoma cell lines and would support the concept that a similar autocrine loop operates in vivo.     

Differential regulation of CDX1 and CDX2 gene expression by deficiency in methyl group donors

The CDX2 and CDX1 homeobox genes have respectively a tumour suppressor and proliferative role in the intestinal epithelium. We analyzed DNA methylation and histones modifications associated with CDX2 and CDX1 promoters in two human colon cancer cell lines expressing differentially these genes, Caco2/TC7 [CDX2 positive-CDX1 negative] and HT29 [CDX2 negative-CDX1 negative] cells. Chromatin immunoprecipitation experiments indicated that CDX2 and CDX1 gene expression correlated with a histone modifications pattern characterizing active chromatin (H3K4 trimethylated and H3 acetylated). Bisulfite DNA sequencing and methylation-specific PCR showed that CDX2 and CDX1 promoters display no methylation in HT29 cells even though both genes are not expressed. In contrast, the CDX1 promoter is methylated in Caco2/TC7. DNA demethylation by 5aza-dC or the combination of 5aza-dC plus SAHA, an inhibitor of histone deacetylases, restored CDX1 expression in Caco2/TC7 cells but these treatments were inefficient on both CDX2 and CDX1 in HT29 cells. Thus, in colon cancer cells the changes in chromatin conformation are heterogeneous and repression of CDX2 and CDX1 in HT29 cells is not due to epigenetic mechanisms. In vivo, dietary deprivation of methyl groups in rats upregulated CDX1 mRNA and downregulated to a lesser extent CDX2 mRNA expression. Moreover, methyl group deprivation downregulated CDX2 protein by changing its phosphorylation pattern. The changes in CDX2 and CDX1 expression determined by methyl group deprivation may constitute one of the mechanisms sustaining the protective role attributed to folate in colon cancer.     

Tumour cell‐intrinsic CTLA4 regulates PD‐L1 expression in non‐small cell lung cancer

Cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD‐1) are immune checkpoint proteins expressed in T cells. Although CTLA4 expression was found in multiple tumours including non‐small cell lung cancer (NSCLC) tissues and cells, its function in tumour cells is unknown. Recently, PD‐1 was found to be expressed in melanoma cells and to promote tumorigenesis. We found that CTLA4 was expressed in a subset of NSCLC cell lines and in a subgroup of cancer cells within the lung cancer tissues. We further found that in NSCLC cells, anti‐CTLA4 antibody can induce PD‐L1 expression, which is mediated by CTLA4 and the EGFR pathway involving phosphorylation of MEK and ERK. In CTLA4 knockout cells, EGFR knockout cells or in the presence of an EGFR tyrosine kinase inhibitor, anti‐CTLA4 antibody was not able to induce PD‐L1 expression in NSCLC cells. Moreover, anti‐CTLA4 antibody promoted NSCLC cell proliferation in vitro and tumour growth in vivo in the absence of adaptive immunity. These results suggest that tumour cell‐intrinsic CTLA4 can regulate PD‐L1 expression and cell proliferation, and that anti‐CTLA4 antibody, by binding to the tumour cell‐intrinsic CTLA4, may result in the activation of the EGFR pathway in cancer cells.     

UBE2Q1 expression in human colorectal tumors and cell lines

Colorectal cancer is the third most common cancer in the world. Ubiquitin–proteasome system has shown to be activated in colorectal and other malignancies. UBE2Q1 is a novel human gene that encodes a putative E2 ubiquitin conjugating enzyme. Here, we investigated the expression pattern of UBE2Q1 gene in cell lines and tissues from human colorectal tumors. Quantitative (q) RT-PCR were employed to evaluate the expression levels of the mRNA for UBE2Q1 in colorectal cancer cell lines (HT29/219, LS180, SW742, Caco2, HTC116, SW48, SW480 and SW1116). Expression of UBE2Q1 at the protein levels were assessed by Western blotting in cell lines as well as in 43 human colorectal tumor tissues. All cell lines tested expressed UBE2Q1 gene at the level of both mRNA and protein, with the SW1116 line representing the lowest level of expression. The cell lines HT29/219 and SW742 showed the highest levels of UBE2Q1 protein and mRNA respectively. When compared to corresponding normal tissues, malignant parts of colorectal tumors showed increased levels of UBE2Q1 immunoreactivity in 32 (74.42 %) of cases. These data suggest that UBE2Q1 is differentially expressed in colorectal cell lines and shows overexpression in colorectal tumors.     

Human carcinoma cell lines xenografted in athymic mice: biological and antigenic characteristics of an intraabdominal model

Summary In order to investigate in vivo clinical applications of murine monoclonal antibodies directed against human ovarian carcinoma a preclinical in vivo model was developed using BALB/c athymic mice. Three human carcinoma cell lines (MCF7, HT29, and SW626) were injected into the peritoneal cavity of pristane-primed animals and the biological and antigenic characteristics of the i.p. grown tumors were studied. The animals were killed when moribund or 6–8 weeks after tumor injection. At autopsy tumor take was observed in 85% of the injected animals, whereas palpable nodules were evident in only 83%. Examination of the peritoneal cavity revealed intraabdominal carcinomatosis with tumor masses varying in size between 0.2 and 0.5 cm in diameter and tumor sheets. The most frequently affected organs were the diaphragm, the liver, and the reproductive system. Ascitic fluid formation was rare and no animal developed tumors outside the peritoneal cavity. To determine whether the in vivo tumors retained the same antigenic characteristics as the in vitro cell lines, four monoclonal antibodies (MBrl, MOv2, MOv8, and MOv15) directed against ovarian carcinoma-associated antigens and two different experimental approaches (immunofluorescence and immunoblotting) were used. Variations at either a quantitative or a qualitative level were observed for some antigens, whereas no evident changes were apparent for others. In particular, the antigens detected by MBr1 and MOv15 on the MCF7 line both maintained high levels of expression and immunoblotting staining pattern, whereas the antigens detected by MOv2 on the HT29 and SW626 lines, although present at a high level, clearly changed their staining pattern. As regards the antigens recognized by MOv8 and MOv15 on the HT29 and SW626 lines, we observed a drastic decrease in the level of their expression and in many cases a drop below the threshold of detectability of the test. The intraabdominal carcinomatosis described partially mimics the growth characteristics of human ovarian cancer and maintains the expression of some antigenic markers associated with epithelial tumors of the ovary and may therefore be useful in devising immunodiagnostic and/or immunotherapeutic strategies for ovarian carcinoma.     

Tumour necrosis factor receptor I (p55) is upregulated on endothelial cells by exposure to the tumour-derived cytokine endothelial monocyte- activating polypeptide II (EMAP-II)

Endothelial monocyte activating polypeptide-II (EMAP-II) is an inflammatory cytokine known to have a role in neutrophil and macrophage chemotaxis and in apoptosis. It is a tumour-derived cytokine that sensitizes tumour vasculature to the effects of systemic TNF. In order to gain insight into the mechanism by which EMAP-II sensitizes vessels to TNF, we focused on its effects on TNF receptor expression. In human umbilical vein endothelial cells (HUVEC), TNF-R1 mRNA is increased four-fold following incubation with recombinant EMAP-II. Conditioned media from cell lines known to produce high levels of EMAP-II upregulated TNF-R1 but not TNF-R2 by up to twenty-fold compared to media controls and low expressing cell lines; this effect was blocked by anti-EMAP-II antibody. Recombinant EMAP-II upregulated TNF-R1 expression by approximately six-fold. Analysis of HUVEC lysates by ELISA showed increased expression of TNF-R1 within 2 h; TNF-R2 expression was unaffected by recombinant EMAP-II. Finally, immunohistochemistry of human melanomas in vivo showed that TNF-R1 staining is increased on the vessels of tumours known to express high levels of EMAP-II compared to low EMAP-II expressing tumours. These results suggest that EMAP-II upregulates TNF-R1 expression by endothelial cells both in vitro and in vivo. This induction of TNF-R1 expression may be the mechanism by which EMAP-II sensitizes tumour endothelium to the effects of TNF leading to haemorrhagic necrosis.     

Estrogen receptor beta mRNA in colon cancer cells: growth effects of estrogen and genistein

Knowledge regarding the expression of the recently cloned estrogen receptor beta (ERbeta) in colonic mucosa is limited. In this study, we demonstrated that five human colon cancer cell lines, HT29, Colo320, Lovo, SW480, and HCT116, expressed ERbeta mRNA, but lacked ERalpha mRNA. Results from a cell growth assay demonstrated that these colon cancer cells were not influenced by estrogen, while genistein possessed slight growth inhibitory effects on HT29, Colo320 and Lovo cells at 10 microM, at which concentration is stimulated the growth of ERalpha-positive human breast cancer MCF-7 cells. Tamoxifen inhibited the growth of HT29 and Colo320 cells, dose-dependently, as well as MCF-7 cells. A transfected reporter plasmid containing a vitellogenin estrogen response element could be activated by estradiol in Colo320 cells. Taken together with previous reports, these data suggest that ERalpha and ERbeta may have different biological functions in colon cells.     

Gene expression profiles in human peripheral blood mononuclear cells as biomarkers for nutritional in vitro and in vivo investigations

Identification of chemopreventive substances may be achieved by measuring biological endpoints in human cells in vitro. Since generally only tumour cells are available for such investigations, our aim was to test the applicability of peripheral blood mononuclear cells (PBMC) as an in vitro primary cell model since they mimic the human in vivo situation and are relatively easily available. Cell culture conditions were refined, and the basal variation of gene expression related to drug metabolism and stress response was determined. Results were compared with profiles of an established human colon cell line (HT29) as standard. For biomarker development of nutritional effects, PBMC and HT29 cells were treated with potentially chemopreventive substances (chrysin and butyrate), and gene expression was determined. Key results were that relevant stress response genes, such as glutathione S-transferase T2 (GSTT2) and GSTM2, were modulated by butyrate in PBMC as in HT29 cells, but the blood cells were less sensitive and responded with high individual differences. We conclude that these cells may serve as a surrogate tissue in dietary investigations and the identified differentially expressed genes have the potential to become marker genes for population studies on biological effects.     

Interleukin-7 receptor expression and activation in nonhaematopoietic neoplastic cell lines

The high-affinity human interleukin-7 (IL-7)R is a heterodimeric complex consisting of the IL-7Ralpha and common interleukin-2 receptor gamma (IL-2Rgamma(c)) chains. Activation of the IL-7R complex is associated with tyrosine and serine residue phosphorylation of a number of intracellular substrates leading to proliferation and induction of various cellular differentiation processes. In this study, we demonstrate, by S1 nuclease protection assay, immunoprecipitation and in vitro kinase assay that functional human (h) IL-7R is expressed in haematopoietic and nonhaematopoietic cell lines. The National Cancer Institute (NCI) tumour panel of 60 cell lines (NCI60) was screened for the expression of IL-7R mRNA by S1 nuclease protection assay, and IL-7R mRNA was detected in 9 of 12 leukemia, 3 of 7 lung, 4 of 6 CNS, 2 of 7 melanoma, 2 of 7 renal, 1 of 6 colon and 1 of 6 breast cancer cell lines. Immunoblot analysis of haematopoietic, lung cancer and brain tumour cell lines demonstrated expression of IL-7R, IL-2Rgamma(c) and p59 fyn, suggesting that the components of an IL-7R signalling network are present in nonhaematopoietic neoplastic cells. Immunoprecipitation of IL-7Ralpha followed by an in vitro kinase assay demonstrated functional receptor phosphorylation events in the lung cancer cells but not in the brain tumour cell lines. The expression of functional IL-7R on epithelial tumour cells may represent a potential target for receptor-directed therapy.     

Targeted Methylation of the Epithelial Cell Adhesion Molecule (EpCAM) Promoter to Silence Its Expression in Ovarian Cancer Cells

The Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4–8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60–80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers.     

Altered expression of sialylated carbohydrate antigens in HT29 colonic carcinoma cells

To determine whether cell growth conditions impacted carbohydrate expression, HT29 cells were gradually transferred from a conventional glucose-containing media to a glucose-free galactose containing media. Indirect immunofluorescence on acetone fixed cells showed increased expression of sialyl Lewis A antigen (CA19-9), sialyl Lewis C (DUPAN2) and Tn/sialyl-Tn on the surface of HT29 cells grown in the glucose-free galactose containing media compared to those grown in the glucose containing media. Sialyltransferases responsible for the synthesis for these sialylated epitopes were increased in the galactose-fed HT29 cells. Media overlying the cells was subjected to isopycnic ultracentrifugation in cesium chloride and the fractions derived from both glucose and galactose media with equivalent buoyant densities of 1.56 g/L, which are predicted to contain mucin glycoforms, were further separated by HPLC using a Mono-Q anion exchange column. The chromatograph of eluent from the sample derived from the cells growing in the galactose containing media showed an increased peak that reacted with the anti-sialyl Lewis A antibody, CA19-9. These results show that alteration of in vitro culture conditions may cause HT29 colonic carcinoma cells to alter the expression of sialylated carbohydrates.     

Junctional adhesion molecule-C promotes metastatic potential of HT1080 human fibrosarcoma

The junctional adhesion molecule (JAM) family is a key molecule in a process called transendothelial migration or diapedesis. Here, we report implications of JAM-C in cancer metastasis. We first determined the mRNA expression of JAMs in 19 kinds of cancer cell lines. JAM-C was expressed in most of tumors having potent metastatic properties. Especially in murine K-1735 melanoma cell lines, the highly metastatic sublines (M2 and X21) strongly expressed JAM-C when compared with the poorly metastatic ones (C-10 and C23). Next, we investigated the role of JAM-C in cancer metastasis by using human JAM-C (hJAM-C) gene-transfected HT1080 fibrosarcoma cells. In comparison with mock-transfected HT1080 cells, these cells showed a significant increase in the adhesion to various extracellular substrates and the invasion across a Matrigel-coated membrane. The knockdown of hJAM-C using small interfering RNA resulted in the suppression of both the adhesion and the invasion of HT1080 cells, suggesting that endogenous hJAM-C might be involved in tumor metastasis. Finally, we studied the role of hJAM-C in an in vivo experimental metastatic model. The results showed that the overexpression of hJAM-C in HT1080 cells significantly decreased the life spans of the tumorbearing mice. In contrast, the knockdown of hJAM-C in HT1080 cells suppressed the weight gain of the lungs with metastatic colonies. We conclude that the expression of JAM-C promotes metastasis by enhancing both the adhesion of cancer cells to extracellular matrices and the subsequent invasion.     

Activation of c-Met and Upregulation of CD44 Expression Are Associated with the Metastatic Phenotype in the Colorectal Cancer Liver Metastasis Model

Background

Liver metastasis is the most common cause of death in patients with colorectal cancer. Despite extensive research into the biology of cancer progression, the molecular mechanisms that drive colorectal cancer metastasis are not well characterized.

Methods

HT29 LM1, HT29 LM2, HT29 LM3 cell lines were derived from the human colorectal cancer cell line HT29 following multiple rounds of in vivo selection in immunodeficient mice.

Results

CD44 expression, a transmembrane glycoprotein involved in cell-cell and cell-matrix adhesions, and cancer cells adhesion to endothelial cells was increased in all in vivo selected cell lines, with maximum CD44 expression and cancer cells adhesion to endothelial cells in the highly metastatic HT29 LM3 cell line. Activation of c-Met upon hepatocyte growth factor (HGF) stimulation in the in vivo selected cell lines is CD44 independent. In vitro separation of CD44 high and low expression cells from HT29 LM3 cell line with FACS sorting confirmed that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation. Furthermore, in vivo evaluation of CD44 low and high expressing HT29 LM3 cells demonstrated no difference in liver metastasis penetrance.

Conclusions

Taken together, our findings indicate that the aggressive metastatic phenotype of in vivo selected cell lines is associated with overexpression of CD44 and activation of c-MET. We demonstrate that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation and confirm that CD44 expression in HT29 LM3 cell line is not responsible for the increase in metastatic penetrance in HT29 LM3 cell line.