Expression of Po66-CBP, a galectin-8, in different types of primary and secondary broncho-pulmonary tumors.

The monoclonal antibody Po66 is an IgG1 immunoglobulin isolated from a human bronchial squamous carcinoma and directed against a carbohydrate binding protein, Po66-CBP, belonging to the galectin family involved in neoplastic processes. This Po66 antibody has been shown to be useful for immunoscintigraphic detection of squamous cell carcinoma metastasis. We examined the expression of Po66-CBP in a wider range of primary or secondary malignant tumors of the lung of various histological types. We studied 52 specimens of broncho-pulmonary tumors including 41 primary squamous, glandular or neuro-endocrine tumors and 11 secondary tumors of glandular, connective tissue, melanocytic or germinal origin as well as 9 extra-pulmonary primary tumors with histological types similar to lung metastases. An immunohistochemical study was performed using an amplification system on paraffin-embedded sections. All histological types were positive for Po66 antibody, the cell origin giving no influence on the expression of Po66-CBP. There was however a relation between Po66-CBP expression and the degree of differentiation notably for squamous cell cancer and neuro-endocrine tumors. The metastatic character of the tumor tissue did not affect Po66-CBP expression.

Two messenger RNAs and five isoforms for Po66-CBP, a galectin-8 homolog in a human lung carcinoma cell line.

Galectins are animal proteins which specifically bind beta-D-galactoside residues and their specific cellular function is not yet clearly established. However, these proteins seem to play a role in neoplastic transformations. Po66 is a murine monoclonal antibody directed against a protein from human lung carcinoma, Po66 Carbohydrate-Binding-Protein (Po66-CBP), which belongs to the galectin-8 family. Our results show that the Po66-CBP gene generates five transcripts by alternative splicing, which could give rise to five proteins: two proteins belong to the tandemly repeated galectin family and three belong to the single carbohydrate recognition domain galectins. All these proteins are encoded by a unique gene located in 1q42. Experiments carried out by reverse transcriptase-polymerase chain reaction show that the levels of expression of these five galectin-8 isoforms are variable during the culture time in SK-MES-1, a human lung squamous carcinoma cell line. Cancer Genome Anatomy Project database analysis confirms the presence of Po66-CBP in lung cancer and its absence in healthy lung.

Galectin-8: a complex sub-family of galectins (Review).

Galectins are animal lectins, that can specifically bind beta-galactosides. Twelve galectins have been described in vertebrates, belonging to three different groups: prototype, tandem-repeat and chimeric. These proteins seem to be involved in cellular interactions and neoplastic transformations. We present an overview of a particular galectin member: galectin-8. This galectin, which has been intensively studied over the last six years, presents a particular type of gene regulation. It is widely expressed in tumoral tissues and seems to be involved in integrin-like cell interactions. Studies show that the LGALS8 gene encodes for almost seven mRNAs by alternative splicing pathways and various polyadenylation sites. These mRNAs could encode for six isoforms of galectin-8, of which three belong to the tandem-repeat galectin group (with two carbohydrate binding domains) and the three others to the prototype group (one carbohydrate binding domain). All these isoforms seem to be differentially expressed in various tumoral cells. This untypical galectin-8 subfamily seems to have a complex expression regulation, that could be involved in cancer phenomena.

Sodium butyrate induces growth inhibition and modulates galectin-8 expression in human lung carcinoma cells.

Galectins are animal lectins, which may play a role in neoplastic transformation. Po66-Carbohydrate Binding Protein (Po66-CBP) belongs to the galectin-8 family and is expressed in lung tumor cells but not in normal ones. Recent studies showed that galectin-8 could be used for human lung squamous cell carcinoma radioimmunotherapy. To optimize this method of treatment, we attempted to increase galectin-8 expression in human lung tumor cells. A human lung squamous (SK-MES-1) or adeno (A 549) carcinoma cell line was grown with or without sodium butyrate. Cell growth, morphology, transcriptional, expression translational expression and cellular localization of galectin-8 were studied. 3 mM of sodium butyrate inhibited the two cell lines' growth after 48 hours of treatment, but only in SK-MES-1 cells galectin-8 expression is modulated without any secretion and cellular localization modifications, apoptosis or necrosis. Sodium butyrate could be an interesting tool in optimizing the radioimmunotherapy of human lung squamous carcinoma, but not of adenocarcinoma.

Immunohistochemical expression of the intracellular component of galectin-8 in squamous cell metaplasia of the bronchial epithelium in neoplastic and benign processes.

Specified galectins are known to play a role in regulating cell proliferation, differentiation, adhesion and migration. Po66, a mouse IgG1 monoclonal antibody produced by immunization against squamous cell cancer, reacts against a carbohydrate-binding protein (Po66-CBP), recently shown to be a member of the galectin family with a strong homology with galectin-8 (PCTA-1), identified as a human tumor-associated antigen. We studied Po66 in squamous metaplasia of the bronchi in order to determine whether it could be specifically involved in neoplastic conditions and if so, if it would be helpful in distinguishing metaplasia at risk of cancer. Twenty-eight formalin-fixed, paraffin-embedded archival tissues of 17 metaplasias with SCC, 3 metaplasia with distant neoplastic disease and 8 metaplasias with an inflammatory process, were immunostained using a streptavidin biotin peroxydase method. The squamous metaplasias were positively stained in non-neoplastic disease as well as in neoplastic processes. Expression was also observed in stromal and normal cells. Po66-CBP was not associated with a pre-neoplastic character. We discussed the expression of this intra-cellular component of galectin-8 according to the functions of galectins in cellular differentiation, host reaction against tumor, and inflammation.

Is magnetic resonance imaging texture analysis a useful tool for cell therapy in vivo monitoring?

Assessment of anti-tumor treatment efficiency is usually done by measuring tumor size. Treatment may however induce changes in the tumor other than tumor size. Magnetic Resonance Imaging Texture Analysis (MRI-TA) is presently used to follow activated lymphocyte cell therapy. We used a 7T microimager to acquire high-resolution MR images of an experimental liver metastasis from colon carcinoma in rats treated (n = 4) or not (n = 3) with a cell therapy product. MRI-TA was then performed with Linear Discriminant Analysis and showed: i) a significant variation of tumor texture with tumor growth and ii) a significant modification in the texture of tumors treated with activated lymphocytes compared with untreated tumors. T2-weighted images or volume calculation did not evidence any difference. MRI-TA appears as a promising method for early detection and follow-up of response to cell therapy.

[Vertebrate galectins: structure and function, role in tumoral process]. / Les galectines animales: structure et fonctions, rôle dans les processus tumoraux.

Galectins are proteins structurally related to the lectin family. They share, with lectins, the ability to bind carbohydrate residues. Galectins are suspected to mediate several biological functions such as embryonic development growth, immune response and apoptosis. Their role is similar to that of adhesion molecules in cell to cell or to matrix interactions. Their contribution to human carcinogenesis has been suggested from experimental studies. In clinical research, they could be used as a differentiation marker, particularly in thyroid carcinomas and in certain lymphomas.

Somatostatin receptor in breast cancer and axillary nodes: study with scintigraphy, histopathology and receptor autoradiography.

UNLABELLED: We conducted a prospective analysis of somatostatin receptor scintigraphy using (111)In radiolabeled pentetreotide, a somatostatin analog, in patients with breast cancer in the aim to visualize the primary tumor and axillary or parasternal metastatic extension because some malignant breast tumors express somatostatin receptors (SS-R) in 50%, approximately. An analysis of SS-R was performed by autoradiography. PATIENTS AND METHODS: Thirteen patients with clinically suspected breast tumors (T1, T2), and at least one palpable axillary node (N1) were included. In vivo planar scintigrams were acquired 1, 4, and 24 h after subcutaneous, then after intravenous injections (24 h delay between injections). Improved (111)In-pentetreotide uptake in invaded nodes after subcutaneous injection was hypothesized. Ex vivo scintigrams of surgical specimens were also acquired immediately after tumor resection and axillary dissection. Pathological examination and receptor autoradiography were performed on all surgical specimens. RESULTS: Among 11 pathologically proven malignant tumors (9 ductal and 2 lobular carcinomas), only four were scintigraphically visible although six expressed SS-R receptors in vitro. Among six pathologically proven malignant nodes, four expressed SS-R, including two visualized scintigraphically. Scintigrams acquired after subcutaneous injections were less sensitive than after intravenous injections. There were no false positive. False negatives occurred in cases with small tumors with low-density or heterogeneously distributed SS-R. There was no significant difference by histological type or prognostic factors. CONCLUSION: Somatostatin receptor scintigraphy does not appear to be sensitive enough to evaluate axillary node extension of breast cancer or even to confirm the presence of tumoral tissue, and this whatever the administration route for (111)In-pentetreotide.

Studies of technetium-99m nitridobisdithiocarboxylate leucocyte specific radiopharmaceutical: [99mTcN(DTCX)2], DTCX = CH3(CH2)8CS2. The cellular and subcellular distribution in human blood cells, and chemical behaviour. Synthesis of the analogous rhenium-188 radiopharmaceutical.

The distribution of the radiopharmaceutical ([99mTcN(DTCX)2], DTCX = CH3(CH2)8CS2) in the leucocyte population determined by a density separation with double gradient Polymorphprep was studied. Microautoradiographic analysis showed a subcellular distribution of the radiomarker in human blood cells. This technique confirmed the observed lymphocyte selectivity (69%) and revealed that the uptake was predominantly cytoplasmic around the nucleus. A labeling mechanism by passive endocytosis could be proposed involving a required lipophilicity of the radiopharmaceutical for lymphocyte targeting. Finally, we describe the new synthesis with an efficient yield and radiochemical purity of the analogous radiopharmaceutical [188ReN(DTCX)2].

Expression of Po66-CBP, a type-8 galectin, in different healthy, tumoral and peritumoral tissues.

UNLABELLED: A monoclonal antibody, Po66, recognized an antigen named Po66 carbohydrate binding protein (PO66-CBP), which was homologous to the galectin-8 protein. Two additional isoforms previously not described were characterized. The aim of this study was to compare the expression of Po66-CBP and its isoforms in different healthy, tumoral and peritumoral tissues and at last to determine the localization of the protein in tumors and distant tissues. MATERIALS AND METHODS: Reverse transcriptase PCR of Po66-CBP was performed on total RNA extract from eleven healthy and eleven tumoral and peritumoral tissue specimens. Antibody Po66 was used to localize the protein in the tumors and distant tissues by an immunohistochemistry method. RESULTS: Po66-CBP was expressed by half of the healthy tissues. One of the isoforms, the last often present in healthy tissues, was found in all tumoral and peritumoral tissues studied. Immunohistochemistry evidenced a gradient of protein expression in normal cells depending on the vicinity of tumoral tissue. Po66-CBP expression was modified in cancerous tissue, suggesting the implication of galectins in carcinogenesis.

Rhenium-188 and technetium-99m nitridobis(N-ethoxy-N-ethyldithiocarbamate) leucocyte labelling radiopharmaceuticals: [188ReN(NOET)2] and [99mTcN(NOET)2], NOET = Et(EtO)NCS2: their in vitro localization and chemical behaviour.

In this study, we have investigated the preparation of rhenium-188 nitridobis(N-ethoxy-N-ethyldithiocarbamate) [188ReN(NOET)2] (NOET = Et(EtO)NCS2), analogous to the known technetium-99m radiopharmaceutical. The new 188Re complex was synthesized in good yield with a satisfactory radiochemical purity, using a kit method. The subcellular localization of both radiopharmaceuticals in granulocytes was observed by microautoradiography. The uptake was independent of the radionuclide and predominantly nuclear. Furthermore, HPLC was used to characterize the 99mTc complex before and after blood cell labelling and revealed that the intact radiopharmaceutical was involved.

The uptake of a monoclonal antibody (Po66) in lung tumour cell aggregates involves a phagocytose-like phenomenon.

The efficacy of radioimmunotherapy with radiolabelled monoclonal antibody (Mab) depends on the amount of antibody taken up by the tumour and on its intratumoral distribution. Multicellular spheroids (MTS) of lung cell carcinoma was investigated to study cellular and subcellular distribution of a Mab Po66 labelled with I-125. We have shown that the incorporation of Po66 in MTS regularly increases during 3 days while Py, a non specific Mab, remains low. The distribution of radiolabelled studied by light and electron microscopy autoradiography have demonstrated that Po66 first localized on extra cellular debris, is then phagocyted and observed in the cytoplasm of viable cells. This mechanism of penetration and distribution of Mab Po66 is a new and interesting phenomenon, and emphasizes contribution of transmission electron microscopy in nuclear medicine.

Treatment of human lung carcinoma xenografts with a combination of 131I-labelled monoclonal antibody Po66 and doxorubicin.

Po66, a mouse monoclonal antibody, is directed against an intracytoplasmic antigen present in human lung squamous cell carcinoma cells. In previous work it was found that the co-administration of 125I-radiolabelled Po66 and doxorubicin strongly enhanced the uptake of radioactivity by the tumour. The present-work was designed to evaluate, in a tumour-bearing mouse model of lung carcinoma, the ability of 131I-labelled Po66 to retard tumour growth when injected alone, or in combination with doxorubicin (8 mg kg-1 at 1-week intervals). A single dose of 550 microCi 131I-Po66 alone had no effect on tumour growth, whereas three fractionated doses of 250 microCi 131I-Po66 decreased it over two doubling times from 14.5 +/- 1.5 days for untreated control mice to 24.8 +/- 2.7 days. Mice treated with doxorubicin alone had a double tumour doubling time of 22.6 +/- 4.9 days, compared to 35.2 +/- 2.9 days (1.55-fold increase) in mice treated with doxorubicin and a single dose of 550 microCi 131I-Po66. Doxorubicin combined with three fractionated doses of 250 microCi 131I-Po66 provoked a twofold decrease in tumour growth compared to mice treated with doxorubicin alone. The administration of fractionated doses of 131I-Po66 simultaneously with doxorubicin resulted in a highly delayed mortality, which was not observed when 131I-Po66 was administered after doxorubicin. Thus, in a non-small-cell lung tumour model, a 131I-radiolabelled monoclonal antibody, directed against an intracellular antigen, significantly potentiated the effect of chemotherapy. Such a therapeutic approach could be used as an adjuvant therapy and improve the effect of chemotherapy on distant small metastases.

Purification of a tumoral marker recognized by monoclonal antibody Po66 and associated with human lung squamous cell carcinoma.

Monoclonal antibody (MAb) Po66, a murine IgG1, was raised by immunization against human lung squamous cell carcinoma. When injected intravenously, Po66 showed prolonged retention in the tumor. It recognized an intracellular antigen. The human lung squamous carcinoma cell line SK-MES-1 expresses the antigen recognized by MAb Po66 and was used as a source of biological material for its purification. The SK-MES-1 cell line was labeled in culture with [35S]methionine and its lysate was immunoprecipitated with Po66 immobilized on Protein G-Sepharose. The precipitate contained three proteins (47, 50 and 69 kDa) absent in the controls. The 69 kDa polypeptide was further purified by anion exchange and immunoaffinity chromatographies. To date, no other tumor marker expressed in non-small cell lung cancer with these characteristics has been described and as such this marker is interesting for future use in immunotherapy and in diagnosis.

Monoclonal antibody Po66 uptake by human lung tumours implanted in nude mice: effect of co-administration with doxorubicin.

The efficacy of radioimmunotherapy of tumours with radiolabelled monoclonal antibodies (MAbs) depends on the amount of antibody taken up by the tumour and on its intratumoral distribution. In the case of MAbs directed against intracellular antigens, increasing the permeability of the cytoplasmic membrane may augment the bioavailability of the antigen for the antibody. This raises the question whether the induction of tumour necrosis by chemotherapy can enhance the tumour uptake of radiolabelled monoclonal antibodies. In this work, the effect of doxorubicin on the biodistribution of Po66, an MAb directed against an intracellular antigen, was studied in nude mice grafted with the human non-small-cell lung carcinoma cell line SK-MES-1. After injection on day 0 of 125I-labelled Po66, tumour radioactivity increased up to days 3-5, and then remained unchanged to day 14. The combined administration of 125I-labelled Po66 with 8 mg kg-1 doxorubicin, in two doses separated by 7 days, doubled the radioactivity retained by the tumour. Histological and historadiographic analysis showed, however, that the drug induced cellular damage. In the absence of doxorubicin, the accumulation of Po66 was restricted to some necrotic areas, whereas with doxorubicin the necrosis was more extensive and the antibody more evenly distributed. These results suggest that chemotherapy and immunoradiotherapy combined would enhance tumour uptake of radioisotope and promote more homogenous distribution of the radiolabelled MAb. This would promote eradication of the remaining drug-resistant cells in tumours.

Serum and tissue distribution of a fragment of cytokeratin 19 (cyfra 21-1) in lung cancer patients.

A sensitive and relatively specific tumoral marker for lung epidermoid carcinomas could be used to identify patients likely to benefit from new therapeutic protocols. The cyfra 21-1 fragment of cytokeratin 19 has raised much hope in this regard amongst both technologists and clinicians. In a study of 195 subjects, we have shown by means of a serum assay that the usual cut-off value for this marker (3.3 ng/ml) can be lowered to 1.5 ng/ml without loss of specificity, and with an increase in sensitivity. There was a good correlation between serum marker level and tumor extension, but though cyfra 21-1 was not predictive of the suitability of a patient for surgery. A decrease of cyfra-21-1 was observed after complete resection of the tumor. There was no relation between serum assay results and immunohistochemical findings.

Biodistribution of monoclonal antibody Po66 in a human lung tumour-bearing mouse model: effect of blood exchange on tumour antibody uptake.

We report a method designed to improve the specificity of tumour uptake after intravenous injection of an anti-tumour monoclonal antibody (MAb). It consists in increasing the blood clearance of the MAb injected in order to diminish its tissue activity, without altering tumour binding. Po66, an MAb directed against lung squamous cell carcinoma, was radiolabelled with 125I and injected i.v. into tumour-bearing nude mice. Radioactivity uptake by the tumour reached a plateau on days 3-5 which persisted up to day 14 after antibody injection. The radiolabelled Po66 remaining in the circulation on day 5 after injection was removed by means of exsanguination and blood transfusion. This blood exchange technique depleted circulating radiolabelled MAb by 60%, whenever mice had been injected with Po66 or an unrelated control IgG1. The proportion of radiolabelled Po66 taken up by the tumour 5 days after blood exchange did not differ substantially from that of non-exsanguinated controls (96.1% of controls). In contrast, there was a significant decrease in blood radioactivity (46% of control values on day 5). Blood exchange provoked a 1.8 fold increase in the tumour/blood and a 1.5-1.8 fold increase of the tumour/organ radioactivity ratios. After injection of unrelated radiolabelled IgG1, blood exchange reduced by 50% both blood and tumour radioactivity, and did not increase the tumour/blood or tumour/organ ratios. Hence, removal of 60% of circulating Po66, 5 days after its injection, did not affect the binding or retention of the antibody by the tumour, but would probably constitute a marked improvement if the antibody is used for two-phase radioimmunotherapy.

Per operative localization of a carcinoid tumour of the breast using indium-111 pentetreotide and a nuclear surgical probe.

A 69-year-old woman presented with a single enlarged lymph node in the left axilla. Clinical examination and other investigations, including various imaging methods, failed to reveal the primary tumour. However, indium-111 pentetreotide scan revealed a site of uptake in the anterior region of the left thorax. Peroperative imaging with 111In-pentetreotide confirmed the tumour uptake and use of a nuclear surgical probe allowed precise localization of the tumour, which was completely resected.

Interactions between human macrophages and tumor cells in three-dimensional cultures.

Human blood mononuclear cells were cultured for 7 days in hydrophobic plastic bags. Macrophages differentiated from monocytes and purified by elutriation were then cocultured with round-shaped aggregates of epithelial cells (spheroids). Spheroids prepared from the SK-MES-1 carcinoma cell line were cultured individually, under constant stirring, in multiwell plates coated with agarose. Macrophage/spheroid interactions were investigated under various experimental conditions. Macrophages activated with interferon gamma aggregated to each other and to spheroids, in contrast to control unactivated macrophages. Histological examination, after staining with a macrophage-specific monoclonal antibody, showed that both control and interferon-gamma-activated macrophages migrated between epithelial tumor cells and infiltrated the spheroids. The addition of anti-ICAM-1 monoclonal antibody inhibited macrophage homotypic aggregation as well as aggregation to and penetration into spheroids. The macrophages did not exert cytolytic effects, as judged by a chromium-51 release assay, but provoked a diminution of tritiated thymidine incorporation by tumor cells. Cytostatic activity was observed with effector: target ratios as low as 1:16, and was maximal (99% at a 1:1 E:T ratio) with macrophages differentiated in the presence of granulocyte/macrophage-colony-stimulating factor. The cytostatic effect was not related to tumor necrosis factor alpha secretion.

No effect of a low-frequency pulsed magnetic field on the brain blood flow among mice.

For thirty minutes Swiss mice were exposed to a 60 G 12 Hz or a 460 Hz pulsed magnetic field. The blood-brain flow was then calculated thanks to an i.v. injection of 99mTc HM-PAO, 0, 3 or 24 hours after the end of the exposure. In the end, it was in fact impossible to discriminate between the exposed mice and the controls.