Timing effects of combined radioimmunotherapy and radiotherapy on a human solid tumor in nude mice.

Timing effects of radioimmunotherapy (RIT) combined with external-beam radiotherapy (RT) were assessed in human colon carcinoma xenografts. Initially, dose effects of fractionated RT and RIT were evaluated separately. Then, 30 Gy RT (10 fractions over 12 days) were combined with three weekly i.v. injections of 200 microCi of 131I-labeled anti-carcinoembryonic antigen monoclonal antibodies in four different treatment schedules. RIT was given either prior to, concurrently, immediately after, or 2 weeks after RT administration. The longest regrowth delay (RD) of 105 days was observed in mice treated by concurrent administration of RT and RIT, whereas the RDs of RT and RIT alone were 34 and 20 days, respectively. The three sequential combination treatments produced significantly shorter RDs ranging from 62 to 70 days. The tumor response represented by the minimal volume (MV) also showed that concurrent administration of RT and RIT gave the best result, with a mean MV of 4.5% as compared to MVs from 26 to 53% for the three sequential treatments. The results were confirmed in a second experiment, in which a RT of 40 Gy was combined with an identical RIT as above (three injections of 200 microCi of 131I-labeled monoclonal antibodies). At comparable toxicity levels, the maximum tolerated RT or RIT alone gave shorter RDs and less tumor shrinkage compared to simultaneous RT+ RIT. These results may be useful for designing clinical protocols of combined RIT and RT.

Human Prominin-1 (CD133) Is Detected in Both Neoplastic and Non-Neoplastic Salivary Gland Diseases and Released into Saliva in a Ubiquitinated Form.

Prominin-1 (CD133) is physiologically expressed at the apical membranes of secretory (serous and mucous) and duct cells of major salivary glands. We investigated its expression in various human salivary gland lesions using two distinct anti-prominin-1 monoclonal antibodies (80B258 and AC133) applied on paraffin-embedded sections and characterized its occurrence in saliva. The 80B258 epitope was extensively expressed in adenoid cystic carcinoma, in lesser extent in acinic cell carcinoma and pleomorphic adenoma, and rarely in mucoepidermoid carcinoma. The 80B258 immunoreactivity was predominately detected at the apical membrane of tumor cells showing acinar or intercalated duct cell differentiation, which lined duct- or cyst-like structures, and in luminal secretions. It was observed on the whole cell membrane in non-luminal structures present in the vicinity of thin-walled blood vessels and hemorrhagic areas in adenoid cystic carcinoma. Of note, AC133 labeled only a subset of 80B258-positive structures. In peritumoral salivary gland tissues as well as in obstructive sialadenitis, an up-regulation of prominin-1 (both 80B258 and AC133 immunoreactivities) was observed in intercalated duct cells. In most tissues, prominin-1 was partially co-expressed with two cancer markers: carcinoembryonic antigen (CEA) and mucin-1 (MUC1). Differential centrifugation of saliva followed by immunoblotting indicated that all three markers were released in association with small membrane vesicles. Immuno-isolated prominin-1-positive vesicles contained CEA and MUC1, but also exosome-related proteins CD63, flotillin-1, flotillin-2 and the adaptor protein syntenin-1. The latter protein was shown to interact with prominin-1 as demonstrated by its co-immunoisolation. A fraction of saliva-associated prominin-1 appeared to be ubiquitinated. Collectively, our findings bring new insights into the biochemistry and trafficking of prominin-1 as well as its immunohistochemical profile in certain types of salivary gland tumors and inflammatory diseases.

Different behaviour of mouse-human chimeric antibody F(ab')2 fragments of IgG1, IgG2 and IgG4 sub-class in vivo.

Mouse-human chimeric monoclonal antibodies (MAbs) of 3 different human IgG sub-classes directed against carcinoembryonic antigen (CEA) have been produced in SP-0 cells transfected with genomic chimeric DNA. F(ab')2 fragments were obtained by pepsin digestion of the purified chimeric MAbs of human IgG1, IgG2 and IgG4 sub-class and of parental mouse MAb IgG1. The 4 F(ab')2 fragments exhibit similar molecular weight by SDS-PAGE. They were labelled with 125I or 131I and high binding (80 to 87%) to purified unsolubilized CEA was observed. In vivo, double labelling experiments indicate that the longest biological half-life and the highest tumour-localization capacity is obtained with F(ab')2 from chimeric MAb of human IgG2 sub-class, whereas F(ab')2 from chimeric MAb IgG4 give very low values for these 2 parameters. F(ab')2 from chimeric MAb IgG1 and from parental mouse MAb yield intermediate results in vivo. Our findings should help to select the appropriate human IgG sub-class to produce chimeric or reshaped MAb F(ab')2 to be used for tumour detection by immunoscintigraphy and for radioimmunotherapy.

In vivo localisation of radiolabelled antibodies to carcinoembryonic antigen in human colon carcinoma grafted into nude mice.

SEVERAL attempts have been made to show the specific localisation in vivo of anti-tumour antibodies. Most of these studies, however, either in experimental animals1,2 or in humans3 were performed with antibodies obtained by adsorption and elution from poorly characterised crude tumour fractions.

Ablation of human colon carcinoma in nude mice by 131I-labeled monoclonal anti-carcinoembryonic antigen antibody F(ab')2 fragments.

Pooled F(ab')2 fragments of three MAbs against distinct epitopes of carcinoembryonic antigen (CEA) were used for radioimmunotherapy of nude mice bearing a subcutaneous human colon carcinoma xenograft. 9-10 d after transplantation when tumor nodules were in exponential growth, 36 mice were treated by intravenous injection of different amounts of 131I-labeled MAb F(ab')2. All 14 mice injected with a single dose of 2,200 (n = 10) or 2,800 microCi (n = 4) showed complete tumor remission. 8 of the 10 mice treated with 2,200 microCi survived in good health for 1 yr when they were killed and shown to be tumor free. Four of nine other mice treated with four fractionated doses of 400 microCi showed no tumor relapse for more than 9 mo. In contrast, all 15 mice injected with 1,600-3,000 microCi 131I-control IgG F(ab')2 showed tumor growth retardation of only 1-4 wk, and 15 of 16 mice injected with unlabeled anti-CEA MAb F(ab')2 showed unmodified tumor progression as compared with untreated mice. From tissue radioactivity distributions it was calculated that by an injection of 2,200 microCi 131I-MAb F(ab')2 a mean dose of 8,335 rad was selectively delivered to the tumor, while the tissue-absorbed radiation doses for the normal organs were: peripheral blood, 2,093; stomach, 1,668; kidney, 1,289; lung, 1,185; liver, 617; spleen, 501; small intestine, 427; large intestine, 367; bone, 337; and muscle, 198. These treatments were well tolerated since out of 19 mice with complete tumor remission only 4 required bone marrow transplantation and 17 were in good health for 6-12 mo of observation. The results demonstrate the selective destruction of established human colon carcinoma transplants by intravenous injection of either single or fractionated doses of 131I-MAb F(ab')2.

Antibody-fluorescein conjugates for photoimmunodiagnosis of human colon carcinoma in nude mice.

To improve the detectability of tumors by light-induced fluorescence, the use of monoclonal antibodies (MoAb) as carriers of fluorescent molecules was studied. As a model for this approach, the biodistribution of an anticarcinoembryonic antigen (CEA) MoAb coupled to fluorescein was studied in mice bearing a human colon carcinoma xenograft. In vitro, such conjugates with fluorescein-MoAb molar ratios ranging from four to 19, doubly labeled with 125I, showed more than 82% binding to immobilized CEA. In vivo, conjugates with a fluorescein-MoAb molar ratio of ten or less resulted in a tumor uptake of more than 30% of the injected dose of radioactivity per gram tumor at 24 hours. Tumor to liver, kidney, and muscle ratios of 20, 30 and 72, respectively, were obtained 48 hours after injection of the 125I-MoAb-(fluorescein)10 conjugate. The highest fluorescence intensity was always obtained for the tumor with the anti-CEA MoAb conjugate; whereas in control mice injected with fluoresceinated control immunoglobulin G1, no detectable increase in tumor fluorescence was observed. To compare these results with a classically used dye, mice bearing the same xenografts received 60 micrograms of Photofrin II. The intensity of the fluorescence signal of the tumor with this amount of Photofrin II was eight times lower than that obtained after an injection of 442 ng of fluorescein coupled with 20 micrograms of MoAb, which gave an absolute amount of fluorescein localized in the tumor of up to 125 ng/g of tumor. These results illustrate the possibility of improving the specificity of in vivo tumor localization of dyes for laser-induced fluorescence photodetection and phototherapy by coupling them to MoAb directed against tumor markers.

Combined radioimmunotherapy and chemotherapy of human colon carcinoma grafted in nude mice, advantages and limitations.

In order to determine if 5-fluorouracil (5FU) could potentiate the effect of radioimmunotherapy (RIT), nude mice bearing subcutaneous human colon carcinoma xenografts were treated by 1 or 2 intravenous injection(s) of subtherapeutic doses of 131I labeled F(ab')2 from anti-carcinoembryonic antigen monoclonal antibodies combined with 5 daily intraperitoneal injections of 5FU. Control mice received either 131I F(ab')2 alone, 5FU alone or no treatment. RIT alone induced significant tumor regression, while 5FU alone gave only minimal tumor growth inhibition. The combined treatment group also resulted in long-term tumor regression with tumors remaining significantly smaller than in the RIT alone group. There was however, no significant difference in tumor recurrence time between the groups treated with RIT alone or with RIT + 5FU. Myelotoxicity, the major side effect of RIT, detected by the decrease of peripheral white blood cells (WBC), was shown to be almost identical between the groups receiving only RIT or only 5FU. Surprisingly, there was no cumulative bone marrow toxicity in animals which received 5FU before RIT. Furthermore, in the latter group, the WBC levels after RIT were significantly higher than in the control group receiving only RIT. Taken together, the results demonstrate the higher therapeutic efficiency of RIT as compared to 5FU in this model. They do not show, however, that the combination of the two forms of treatment can induce longer tumor remission. Interestingly, the WBC results suggest that 5FU given before RIT can have a radioprotective effect on bone marrow, possibly by selecting radioresistant bone marrow stem cells.

Aberrant crypt foci in patients with neoplastic and nonneoplastic colonic disease.

Aberrant crypt foci (ACF) are putative preneoplastic lesions that might represent the earliest morphological lesion visible in colonic carcinogenesis. However, findings concerning the growth and morphological features of these lesions in human studies suggest that ACF are highly heterogeneous in nature. In this study, we evaluated the morphological features of a large number of ACF in colon mucosa of 26 patients with colorectal carcinoma (CRC), four patients with adenoma as well as seven patients with nonneoplastic colonic diseases. By dissecting microscope, 508 ACF were identified, and of these, 378 were sampled for histological examination. The median ACF density (number of ACF/cm2) was significantly higher in the left colon than in the right colon (0.047 v 0.014 ACF/cm2). Unexpectedly, in our series, the overall ACF density was higher in the nonneoplastic colonic diseases than in CRC (0.13 v 0.032 ACF/cm2, P=.0087), cases of nonneoplastic diseases, however, being limited to 7 patients. ACF were significantly larger in colons with CRC or adenoma than in colons with nonneoplastic disease (P < .03). On histological examination, we observed 133 ACF with normal epithelium, 189 ACF with hyperplasia, 27 ACF with atypical hyperplasia, and 29 ACF with dysplasia. We noted a progressive increase of median ACF size from normal mucosa to hyperplasia, atypical hyperplasia, and dysplasia. Dysplastic ACF were more frequently observed in patients with CRC or adenoma and showed predominantly elongated crypt orifices (P < .0001). We conclude that ACF are histologically heterogeneous, encompass a spectrum of lesions of which only a subset are associated with dysplasia and then represent an early step in colorectal carcinogenesis. ACF with dysplasia are characterized by larger size, elongated crypt orifices, and an association with CRC.

Carcinoembryonic antigen expression, antibody localisation and immunophotodetection of human colon cancer liver metastases in nude mice: a model for radioimmunotherapy.

Colorectal cancer frequently disseminates through the portal vein into the liver. In this study, outbred Swiss nude mice were adapted to facilitate the induction of liver metastases by a pre-grafting treatment with 6 Gy total body irradiation and i.v. injection of anti-asialo GM1 antibody. One day later, cultured LS 174T human colon cancer cells were injected into the surgically exposed spleen, which was resected 3 min later. In 48 of 65 mice, a few to several hundred liver metastases were macroscopically observed at dissection 3 to 4 weeks after transplantation. Ten of 10 mice, followed-up for survival, died with multiple large confluent liver metastases. By reducing the radiation dose to 4 or 0 Gy, or omitting the anti-asialo GM1 antibody injection, only 60%, 37% or 50% of mice, respectively, had visible metastases 3 weeks after transplantation. Carcinoembryonic antigen (CEA) measured in tumour extracts was in the mean 25.6 micrograms/g in liver metastases compared with 9.2 micrograms/g in s.c. tumours. Uptake of radiolabelled anti-CEA monoclonal antibody (MAb) in the metastases 12, 24 and 48 hr after injection gave a mean value of 39% of the injected dose per gram of tissue (ID/g). In comparison, MAb uptake in s.c. and intrasplenic tumours or lung metastases gave a mean percentage ID/g of 20, 18 and 15, respectively. Laser-induced fluorescence after injection of indocyanin-MAb conjugate allowed direct visual detection of small liver metastases, including some that were not visible under normal light. Preliminary results showed that mice, pre-treated with 4 Gy irradiation and the anti-asialo GM1 injection, were tolerant to radioimmunotherapy with a total dose of 500 muCi 131I labeled anti-CEA intact MAbs given in 3 injections.

Direct comparison of a radioiodinated intact chimeric anti-CEA MAb with its F(ab')2 fragment in nude mice bearing different human colon cancer xenografts.

Tumour localisation and tumour to normal tissue ratios of a chimeric anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb), in intact form and as an F(ab')2 fragment labelled with 125I and 131I, were compared in groups of nude mice bearing four different colon cancer xenografts, T380, Co112 or LoVo, of human origin, or a rat colon cancer transfected with human CEA cDNA, called '3G7'. For each tumour, three to four mice per time point were analysed 6, 12, 24, 48 and 96 h after MAb injection. In the different tumours, maximal localisation of intact MAb was obtained at 24 to 48 h, and of F(ab')2 fragment 12 to 24 h after injection. Among the different tumours, localisation was highest with colon cancer T380, with 64% of the injected dose per gram (% ID/g) for the intact MAb and 57% for its F(ab')2 fragment, while in the three other tumours, maximal localisation ranged from 14 to 22% ID g-1 for the intact MAb and was about 11% for the F(ab')2. Tumour to normal tissue ratios of intact MAb increased rapidly until 24 h after injection and remained stable or showed only a minor increase thereafter. In contrast, for the F(ab')2 fragment, the tumour to normal tissue ratios increased steadily up to 4 days after injection reaching markedly higher values than those obtained with intact MAb. For the four different xenografts, tumour to blood ratios of F(ab')2 were about 2, 3 and 5 to 16 times higher than those of intact antibodies at 12, 24 and 96 h after injection, respectively.

Tumor-necrosis factor can enhance radio-antibody uptake in human colon carcinoma xenografts by increasing vascular permeability.

Marked differences in the tumor uptake of a 125I-labeled monoclonal antibody (MAb) directed against carcinoembryonic antigen (CEA) were observed in 4 serially transplanted human colorectal carcinomas in nude mice. A comparative study showed that elevated values of measurable tumor vascular parameters, such as permeability, blood flow and blood volume, correlated better with high MAb tumor uptake than the concentration of target antigen in the tumor. In an attempt to modify the vascular parameters and to determine if this could increase antibody uptake by the tumor, rhTNF alpha (TNF) was injected i.t. or i.v. and antibody localization experiments were performed immediately thereafter. Results showed that the permeability of the tumor vessels increased 8 to 10 fold 1 hr after i.t. injection of TNF as compared to control tumors injected with saline. Tumor uptake of 125I-labeled anti-CEA MAb, was 3 times higher 2 hr after i.v. injection and still 27% higher 22 hr later, as compared to results from controls. Intravenous injection of TNF simultaneously with the 125I-labeled anti-CEA MAb also resulted in a 2-fold increase in tumor uptake 4 hr after injection, but the increase was no longer significant 24 hr after injection. Interestingly after i.v. injection of TNF, the MAb concentration in the blood and other normal tissues, such as liver, kidneys, lungs and heart was decreased, resulting in significantly higher ratios of tumor to normal tissue. Taken together the results demonstrate that injection of TNF can increase tumor vascular permeability and improve radio-antibody uptake. This raises the possibility of increasing the radiation dose delivered by antibody to the tumor in the course of radioimmunotherapy.

Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells.

To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.

Caveolin-1-mediated post-transcriptional regulation of inducible nitric oxide synthase in human colon carcinoma cells.

Reactive oxygen species are now widely recognized as important players contributing both to cell homeostasis and the development of disease. In this respect nitric oxide (NO) is no exception. The discussion here will center on regulation of the inducible form of nitric oxide synthase (iNOS) for two reasons. First, only iNOS produces micromolar NO concentrations, amounts that are high by comparison with the picomolar to nanomolar concentrations resulting from Ca2(+)-controlled NO production by endothelial eNOS or neuronal nNOS. Second, iNOS is not constitutively expressed in cells and regulation of this isoenzyme, in contrast to endothelial eNOS or neuronal nNOS, is widely considered to occur at the transcriptional level only. In particular, we were interested in the possibility that caveolin-1, a protein that functions as a tumor suppressor in colon carcinoma cells (Bender et al., 2002; this issue), might regulate iNOS activity. Our results provide evidence for the existence of a post-transcriptional mechanism controlling iNOS protein levels that involves caveolin-1-dependent sequestration of iNOS within a detergent-insoluble compartment. Interestingly, despite the high degree of conservation of the caveolin-1 scaffolding domain binding motif within all NOS enzymes, the interaction detected between caveolin-1 and iNOS in vitro is crucially dependent on presence of a caveolin-1 sequence element immediately adjacent to the scaffolding domain. A model is presented summarizing the salient aspects of these results. These observations are important in the context of tumor biology, since down-regulation of caveolin-1 is predicted to promote uncontrolled iNOS activity, genotoxic damage and thereby facilitate tumor development in humans.

Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression.

An increased expression of nitric oxide synthase (NOS) has been observed in human colon carcinoma cell lines as well as in human gynecological, breast, and central nervous system tumors. This observation suggests a pathobiological role of tumor-associated NO production. Hence, we investigated NOS expression in human colon cancer in respect to tumor staging, NOS-expressing cell type(s), nitrotyrosine formation, inflammation, and vascular endothelial growth factor expression. Ca2+-dependent NOS activity was found in normal colon and in tumors but was significantly decreased in adenomas (P < 0.001) and carcinomas (Dukes' stages A-D: P < 0.002). Ca2+-independent NOS activity, indicating inducible NOS (NOS2), is markedly expressed in approximately 60% of human colon adenomas (P < 0.001 versus normal tissues) and in 20-25% of colon carcinomas (P < 0.01 versus normal tissues). Only low levels were found in the surrounding normal tissue. NOS2 activity decreased with increasing tumor stage (Dukes' A-D) and was lowest in colon metastases to liver and lung. NOS2 was detected in tissue mononuclear cells (TMCs), endothelium, and tumor epithelium. There was a statistically significant correlation between NOS2 enzymatic activity and the level of NOS2 protein detected by immunohistochemistry (P < 0.01). Western blot analysis of tumor extracts with Ca2+-independent NOS activity showed up to three distinct NOS2 protein bands at Mr 125,000-Mr 138,000. The same protein bands were heavily tyrosine-phosphorylated in some tumor tissues. TMCs, but not the tumor epithelium, were immunopositive using a polyclonal anti-nitrotyrosine antibody. However, only a subset of the NOS2-expressing TMCs stained positively for 3-nitrotyrosine, which is a marker for peroxynitrite formation. Furthermore, vascular endothelial growth factor expression was detected in adenomas expressing NOS2. These data are consistent with the hypothesis that excessive NO production by NOS2 may contribute to the pathogenesis of colon cancer progression at the transition of colon adenoma to carcinoma in situ.