Radioimmunotherapy of the GW-39 human colonic tumor xenograft with 131I-labeled murine monoclonal antibody to carcinoembryonic antigen

We have investigated the therapeutic efficacy of a single injection of 131I-labeled murine mouse monoclonal antibody (NP-4) against carcinoembryonic antigen using the human colonic tumor xenograft, GW-39, grown in the cheek pouches of adult hamsters. Therapeutic efficacy was dependent on the dose of radioactivity, the specificity of the antibody for the tumor, and the size of the tumor when the radioantibody was administered. A dose of 1 mCi of 131I-labeled NP-4 given 1 day after tumor transplantation completely inhibited the growth of 6 of 11 tumors over a 12-week period, and histological evidence indicated that viable tumor was absent in the tissue remaining at the injection site. Lower doses (0.5 mCi) of 131I-labeled NP-4 inhibited tumor growth over 90% in comparison to untreated animals, but the tumors eventually resumed growth. Delaying the administration of radioantibody for 4 or 7 days after tumor transplantation significantly reduced the therapeutic efficacy. Although the same dose of 131I-labeled irrelevant immunoglobulin G also inhibited tumor growth, 131I-labeled NP-4 was generally 2-3 times more effective in reducing tumor growth than was the control IgG. There was a 13% loss in body weight within 7 days after treatment with 1 mCi, but all the animals regained their weight by day 14, indicating that the level of radioactivity was tolerated well. Dosimetric calculations predicted that over 14 days a dose of nearly 2400 rads was delivered to the tumors with 131I-labeled NP-4. These results confirm our previous studies that 131I-labeled antibody can effectively inhibit tumor growth, but suggest that radioantibody therapy is most effectively administered when there is a low tumor burden.     

Comparison of therapeutic efficacy and host toxicity of two different 131I-labelled antibodies and their fragments in the GW-39 colonic cancer xenograft model

The in vivo uptake, therapeutic potential, and host toxicity were evaluated for both the intact IgG and F(ab')2 fragment of 2 murine monoclonal antibodies (MAbs) directed against either carcino-embryonic antigen (CEA), called NP-4, or colon-specific antigen-p (CSAp), called Mu-9, in the GW-39 human colorectal carcinoma grown in the hamster cheek pouch. Mu-9 IgG and F(ab')2 were retained longer by the tumor than was the NP-4 IgG or F(ab')2, respectively. Localization of the two antibodies by micro-autoradiography revealed two distinct patterns. Mu-9 was seen densely around whole acini of tumor cells, whereas NP-4 was found around each individual cell, albeit less densely. The anti-tumor effects of 131I-labelled Mu-9 and NP-4 IgG were equal, but the therapeutic effectiveness of Mu-9 F(ab')2 was significantly higher than NP-4 F(ab')2, as measured by change in tumor size. A dose-dependent increase in host toxicity, as measured by change in body weight and change in peripheral white blood cells (p-WBCs), was observed with 0.5 to 3.0 mCi doses of 131I-IgG regardless of the MAb used. A 10-22% loss in body weight lasting 3-7 weeks and a 50-80% loss in pWBCs lasting 6-8 weeks were observed in these animals. In contrast, 3 x 2 mCi doses of 131I-NP-4 or Mu-9 F(ab')2 given at 3-day intervals, a schedule which was equally therapeutic to a single 2-mCi dose of 131I-IgG, resulted in only a 9% loss in body weight and a 50% loss in pWBCs that lasted only 1 week. This was followed by a complete recovery over the next 2-3 weeks. These data suggest that multiple doses of F(ab')2 can be as tumoricidal as a single dose of an intact MAb IgG, but significantly less toxic to the host.     

Purification and characterization of carcinoembryonic antigen from GW-39, a xenografted human colonic tumor system

Carcinoembryonic antigen (CEA) was purified from GW-39 human tumor xenografts in hamsters by immunoaffinity chromatography. Binding of the antigen to immobilized monoclonal antibody provided a high degree of purification of CEA in a single step. A recovery of 79% and a 750-fold purification were obtained. The purified CEA has a molecular size of 180 kilodaltons, an isoelectric point of 4.4, and a specific activity of 0.94. About 73% of the radiolabeled GW-39 CEA reacted with goat anti-CEA serum. The amino acid and carbohydrate compositions of the GW-39 CEA were comparable to those of CEA preparations from other sources.     

Carcinoembryonic antigen and glucose phosphate isomerase in a human colonic cancer model (GW-39).

Levels of carcinoembryonic antigen (CEA) and glucose phosphate isomerase (GPI) have been compared in the circulating blood of hamsters bearing intra-muscular grafts of GW-39 human colonic tumour. CEA in the sera of GW-39 tumour-bearing hamsters ranged from 2-6 to 8-4 ng/ml (mean = 4-5 +/- 1-7 ng/ml). GPI in the sera of normal hamsters ranged from 332 to 749 iu/1 (mean = 602 +/- 110 iu/1) while those with 14-week-old intra-muscular grafts of a hamster amelanotic melanoma, (A.Mel.3), or GW-39 human colonic carcinoma had a range of 664 to 1267 iu/1 (mean = 1024 +/- 220 iu/1) and 1430 to 4719 iu/1 (mean = 2065 +/- 601 iu/1) respectively. Thus, the ratio of enzyme activity in GW-39, A.Mel.3, and normal hamsters was 3-4:1-7:1, indicating a significant elevation (P less than 0-01) in animals bearing a human colon carcinoma or a hamster melanoma, with particularly high values obtained in hamsters with GW-39. Sequential determinations of CEA and GPI in a group of hamsters transplanted intra-muscularly with GW-39 tumours revealed that both markers increased proportionately with duration of tumour growth, suggesting that both serum CEA and GPI may be used as measures of tumour growth. The concentration of GPI in GW-39 human colonic carcinoma xenografts was also significantly higher than that measured in normal human colon, primary human colonic cancer, or normal hamster tissues. These results support the view that GPI, in addition to CEA, is a quantitatively increased marker in this tumour model, and is liberated into the circulation in proportion to the increase in tumour mass.     

Antibody protein dose and radioimmunodetection of GW-39 human colon tumor xenografts

This study investigates the influence of antibody protein dose on the radioimmunodetection of a CEA-producing, human colonic tumor xenograft (GW-39) using affinity-purified goat anti-carcinoembryonic antigen (CEA) antibody and a murine monoclonal anti-CEA antibody (NP-2). Hamsters bearing GW-39 tumors were given an equal mixture of 131I-labelled antibody and 125I-labelled irrelevant IgG at doses varying from 0.01 to 1.0 mg (0.1 to 10 mg/kg body weight) for each antibody preparation. No differences were found in the percentage of antibody in the tumor or the clearance rate from the blood as the antibody dose was increased. The concentration of antibody in the tumor increased proportionally with the antibody dose and maintained a linear relationship with increasing dose, indicating that antigenic sites in the tumor were not saturated with antibody. The concentration of irrelevant IgG in the tumor also increased proportionally as the dose was increased, but the concentration of irrelevant IgG in the tumor was less than the antibody and was removed more rapidly from the tumor than the antibody. By considering the amount of irrelevant IgG in the tumor as a measure of the amount of antibody non-specifically bound, we determined that there was no change in the amount of antibody in the tumor between days 3 and 7, and the amount of antibody increased uniformly for both the tumor and non-tumor tissues, resulting in no improvement in the tumor/non-tumor ratios with increasing antibody dose. External scintigraphy verified that the dose of the antibody did not influence tumor imaging. Thus, tumor accretion of antibody in this model is not dependent principally on the antibody protein dose, and other factors such as accessibility to antigen and antibody metabolism may play important roles in regulating the uptake of antibody in tumor.     

Comparison of tumor targeting of mouse monoclonal and goat polyclonal antibodies to carcinoembryonic antigen in the GW-39 human tumor-hamster host model

We have evaluated 4 radioiodinated mouse monoclonal anticarcinoembryonic antigen antibodies (MAbs) by using the GW-39 human colorectal tumor xenograft transplanted i.m. in immunocompetent hamsters to determine whether there were any differences in their tumor localization properties. Additional comparisons were made to affinity-purified goat anticarcinoembryonic antigen antibody. Statistically significant differences were found in the percentage/g of tumor uptake and tumor/nontumor ratios among the antibodies, so that the antibodies could be ranked according to their tumor localization properties (NP-2 greater than NP-4 = goat antibody greater than NP-1 greater than NP-3). Although statistical differences were found, tumor/nontumor values generally were not distinguished by a factor of more than 1.5, suggesting that these differences may not be biologically significant. F(ab')2 fragments of NP-2 were found to be superior to NP-4 F(ab')2 fragments, giving tumor/liver and tumor/blood ratios of 16 and 11.5, respectively, within 3 days, in comparison to 5.4 and 3.8 for NP-4 F(ab')2 fragments. Mixtures of all of the MAbs or a mixture of NP-2 and NP-4 did not improve tumor localization, in comparison to NP-2 alone. These studies suggest that mixtures of these anticarcinoembryonic antigen MAbs may not afford better tumor imaging than the use of a certain single antitumor MAb.     

Targeted therapy of athymic mice bearing GW-39 human colonic cancer micrometastases with 131I-labeled monoclonal antibodies.

The therapeutic potential of radiolabeled antibodies is usually evaluated in experimental animal models bearing s.c. xenografts. We have established a micrometastatic model of the GW-39 human colonic carcinoma in the nude mouse lung (J. Natl. Cancer Inst., 83: 627-632, 1991) and presented preliminary findings on the efficacy of a 131I-anticarcinoembryonic antigen (CEA) antibody in this model. We now extend our observations on the use of radioiodinated labeled monoclonal antibodies (MAbs) to treat multiple small tumor nodules. Biodistribution and dosimetry analysis was performed for intact and F(ab')2 of NP-4 anti-CEA IgG, Mu-9 anti-colon-specific antigen IgG, isotype-matched irrelevant anti-AFP IgG, and intact MAb 34A anti-lung endothelial IgG antibody. Comparisons were made for rad dose delivered to small s.c. tumors, normal lung, lung with tumor nodules, and isolated tumor nodules. Survival curves were generated for tumor-bearing animals treated 1, 7, or 14 days after tumor cell implantation with these antibodies using the maximal tolerated dose for intact antibodies (275 microCi) and for F(ab')2 fragments (1.2 mCi). The studies established the following observations: (a) in contrast to previous results in a bulky tumor model in hamsters, intact antibodies are more therapeutic than MAb fragments for both NP-4 and Mu-9; (b) tumor nodule size, even on the microscopic level, affects therapeutic outcome; antibodies were more effective when administered 7 days postimplantation (mean nodule diameter, 150 microns) compared with treatment 14 days postimplantation (mean nodule diameter, 750 microns); (c) administration of radioiodinated Mu-9 was exquisitely effective on single avascular tumor cells that had seeded in lung; irrelevant antibody was minimally radiotoxic; (d) as in the bulky disease model, the anti-colon-specific antigen p antibody delivers a higher rad dose than the anti-CEA antibody and is significantly more therapeutic in the micrometastasis model; (e) a higher affinity anti-CEA antibody (MN-14) recognizing the same epitope on CEA as NP-4 was equally therapeutic; (f) the use of MAb directed against the lung endothelium was not as therapeutic as a tumor-associated antibody; and (g) all tumor-associated antibodies were more efficacious than administration of the maximal tolerated dose of 5-fluorouracil and leucovorin in this human tumor-xenograft model. These results provide further support for the use of radioimmunotherapy in the handling of minimal disease, probably as part of an adjuvant treatment regimen.     

Influence of implantation site on growth, antigen expression and metastatic potential of human colonic cancer HT29 and 5583 xenografts in nude mice

In order to study the interaction between tumors and host environmental factors, we xenografted cells from the human colonic carcinoma cell lines HT29 and 5583-S in the subcutis, cecum, spleen and liver of nude mice and compared growth characteristics, metastatic potential and some phenotypic features of the xenografts in these sites. No remarkable differences were observed between the tumors at different inoculation sites in regard of their expression of carcinoembryonic antigen and secretory component or type of mucin produced. Also the proportion of DNA synthesizing cells as determined by bromodeoxyuridine incorporation appeared to be comparable in the studied implantation sites. Local invasive growth characteristics and metastatic potential, however, showed marked differences. Subcutaneous and cecal xenografts frequently showed tumor cell invasion into the surrounding tissue, vasoinvasive growth and discontinuous basement membrane deposition, whereas splenic and hepatic implants demonstrated more encapsulation, no invasion of blood vessels and more continuous basement membrane deposition. Subcutaneous xenografts produced no metastasis. With HT29 cells liver and lymph node metastases occurred frequently from the splenic as well as the cecal xenografts. 5583 cells regularly produced liver metastases from the splenic xenografts, whereas no metastasis from cecal xenografts were observed. We conclude that although the patterns of invasive growth and the metastatic potential differ for various implantation sites, antigen expression and cell kinetic features of tumor implants are hardly influenced by the site of inoculation.     

Karyology of a human colonic carcinoma (GW-39) serially xenografted in hamsters and in nude mice.

A karyological analysis of the G-banded chromosomes of the serially transplantable GW-39 human colonic carcinoma was undertaken. GW-39 cells harvested from hamsters, nude (athymic) mice and cell cultures from 1972 to 1976 revealed a hypodiploid stemline of 45 human chromosomes, with a consistent loss of a 21-chromosome. No apparent alterations of the near-diploid stemline resulted during long-term xenogeneic propagation of this human cancer line.     

Influence of the site of human gallbladder xenograft (Mz-ChA-1) on angiogenesis at the distant site

We reported that orthotopic xenograft of human gallbladder cancer (Mz-ChA-2) produced a greater amount of endogenous angiogenic inhibitory factors, however, only TGFbeta1 suppressed angiogenesis and tumor growth at the distant site (intracranium). The aim of this study was to confirm the validity of our previous findings that the site of the primary tumor would influence the angiogenesis in the distant site in a different xenograft of human gallbladder cancer (Mz-ChA-1). The growth rates, histology of the ectopic (flank) and orthotopic (gallbladder) xenografts, the plasma level of TGFbeta1, micro-circulation and angiogenesis in the distant site (intracranium) were estimated by size-measurement, hematoxylin and eosin staining, ELISA, intravital fluorescence microscopic observation and cranial window gel assay for angiogenesis. All experiments were performed in severe combined immunodeficient (SCID) mice. Orthotopic tumors grew faster and were less necrotic than ectopic tumors. Angiogenesis, vessel diameters, vessel density and leukocyte-rolling count in the distant site were significantly decreased in orthotopic tumor-bearing mice compared to those in either ectopic or no tumor-bearing mice. The plasma level of TGFbeta1 was significantly elevated in mice bearing orthotopic tumor as compared with ectopic and no tumor-bearing mice. Angiogenesis at the distant site was inhibited by the orthotopic xenograft of Mz-ChA-1 by the greatest amount of TGFbeta1 production. The results of the present study together with our previous study imply that the primary tumor microenvironment is conducive to the angiogenesis at a distant site by the production of the endogenous angiogenesis inhibitor TGFbeta1.     

The effect of glucose administration on the uptake of photofrin II in a human tumor xenograft.

Athymic BALB/c nude mice, bearing a human melanoma LOX, were given the photosensitizing drug Photofrin II (10 mg/kg body wt.) intraperitoneally. The mice were also given one of the following chemicals intraperitoneally: glucose, galactose and glucose plus nordihydroguaiaretic acid (NDGA) which is an inhibitor of glycolysis. Multiple injections of glucose (3 g/kg body wt. given at -1, 0, +1 and +3 h relative to the injection of 10 mg/kg of Photofrin II at time 0) resulted in a significant increase in the uptake of Photofrin II in the tumor 4 h after a Photofrin II injection, while the uptake of Photofrin II in the other tissues remained unchanged. Administration of galactose had no significant effect on the uptake of Photofrin II in the tissues studied (tumor, muscle, skin and liver). NDGA seemed to abolish the effect of glucose injection.     

A fluorouridine-anti-CEA immunoconjugate is therapeutically effective in a human colonic cancer xenograft model.

5-fluorouridine (FUR), an antineoplastic agent, was site-specifically conjugated to the carbohydrate moiety of a anticarcinoembryonic antigen (CEA) monoclonal antibody (MAb) by using amino-dextran as the intermediate carrier. The final immunoconjugate contains approximately 30-35 molecules of FUR per molecule of immunoglobulin, has immunoreactivity retained as examined by flow cytometry, and is cytotoxic to the target cells as examined by 75selenomethionine incorporation studies. In the GW-39/nude mouse model, the conjugate remained efficient in targeting the human colonic tumor and possessed greater inhibitory growth effects on the subcutaneous tumor than free FUR or an irrelevant antibody conjugate. In addition, the reduced host toxicity of the conjugate may permit the use of this agent in a high-dose therapy of this tumor system.     

ProstaCaid inhibits tumor growth in a xenograft model of human prostate cancer

We have recently demonstrated that the dietary supplement ProstaCaid (PC) inhibits growth and invasive behavior of PC-3 human prostate cancer cells in vitro. In the present study, we evaluated toxicity and whether PC suppresses growth of prostate cancer in a xenograft model of human prostate cancer cells implanted in mice. Here, we show that an oral administration of PC (100, 200 and 400 mg/kg) did not affect body weight or activity of liver enzymes (ALT, AST) and did not show any sign of toxicity in liver, spleen, kidney, lung and heart tissues in mice. In addition, PC treatment resulted in the inhibition of tumor volumes (1024.6 ± 378.6 vs. 749.3 ± 234.3, P<0.001) in a xenograft model of prostate cancer with human hormone refractory (independent) PC-3 prostate cancer cells. Moreover, qRT-PCR analysis demonstrated significant upregulation of expression of CDKN1A (p21) and inhibition of expression of IGF2, NR2F2 and PLAU (uPA) genes by an oral administration of PC in prostate cancer xenografts. Our study demonstrates that the concentrations of the dietary supplement ProstaCaid tested did not show signs of toxicity, and its oral application has significant anticancer activity in vivo and can be considered as an alternative treatment for prostate cancer patients.     

The effect of second antibody clearance on the distribution and dosimetry of radiolabelled anti-CEA antibody in a human colonic tumor xenograft model

Radioimmunotherapy in humans is limited by toxicity to normal tissues, caused by circulating radio-antibody. Second antibody directed against the first (anti-tumor) antibody accelerates clearance of first antibody from normal tissues, and may thus improve the therapeutic ratio. The effect of second antibody both on anti-tumor antibody distribution and on tumor and normal tissue radiation doses, has been investigated in nude mice bearing colonic tumor xenografts. Second antibody, given either 6 or 24 hr after the first, rapidly cleared circulating activity and reduced the calculated radiation dose to all tissues except the spleen, where it rose by 11 and 43% respectively. The dose received by the blood fell by 87% and 71%, while that to the tumor was reduced by 81% and 58%, after 6 or 24 hr second antibody. Administration of second antibody therefore improved the tumor to blood ratios. Tumor identification by gamma camera was greatly facilitated by the use of second antibody, and required no background subtraction. Results obtained from this system demonstrate the utility of second antibody in protecting normal tissues from prolonged circulating radioactivity during radioimmunotherapy.     

Influence of implantation site on formation of metastases

It has been suggested that local factors at the site of growth of a primary tumor might influence the outcome of the metastatic process. Compilation of the data from the literature revealed that growth of tumor cells in the selective medium of the intraperitoneal cavity, of the lymph node and/or of the spleen leads to progression towards a population of cells with a higher metastatic capacity. In search for an experimental model with transplantable rodent tumors that could be used to study the influence of the anatomic site of an implant on the formation of spontaneous metastases, we have considered heterogeneity of microenvironmental conditions in the subcutaneous milieu. For the MO4 mouse fibrosarcoma, a primary tumor growing subcutaneously in the tail was highly metastatic to lymph nodes and lungs while it failed to produce metastases when growing in the pinna. Implantation of a spheroidal aggregate of MO4 tumor cells, alternatively in the tail and in the pinna of syngeneic C3H/He mice, might be an appropriate model, which is discussed in this review.     

Astragalus saponins induce growth inhibition and apoptosis in human colon cancer cells and tumor xenograft

Astragalus memebranaceus is used as immunomodulating agent in treating immunodeficiency diseases and to alleviate the adverse effects of chemotherapeutic drugs. In recent years, it has been proposed that Astragalus may possess anti-tumorigenic potential in certain cancer cell types. In this study, the anti-carcinogenic effects of Astragalus saponin extract were investigated in HT-29 human colon cancer cells and tumor xenograft. Our findings have shown that Astragalus saponins (AST) inhibit cell proliferation through accumulation in S phase and G2/M arrest, with concomitant suppression of p21 expression and inhibition of cyclin-dependent kinase activity. Besides, AST promotes apoptosis in HT-29 cells through caspase 3 activation and poly(ADP-ribose) polymerase cleavage, which is indicated by DNA fragmentation and nuclear chromatin condensation. Nevertheless, we also demonstrate the anti-tumorigenic effects of AST in vivo, of which the reduction of tumor volume as well as pro-apoptotic and anti-proliferative effects in HT-29 nude mice xenograft are comparable with that produced by the conventional chemotherapeutic drug 5-fluorouracil (5-FU). In addition, the side effects (body weight drop and mortality) associated with the drug combo 5-FU and oxaliplatin are not induced by AST. These results indicate that AST could be an effective chemotherapeutic agent in colon cancer treatment, which might also be used as an adjuvant in combination with other orthodox chemotherapeutic drugs to reduce the side effects of the latter compounds.     

Dietary protein restriction inhibits tumor growth in human xenograft models of prostate and breast cancer

Purpose: Data from epidemiological and experimental studies suggest that dietary protein intake may play a role in inhibiting prostate and breast cancer by modulating the IGF/AKT/mTOR pathway. In this study we investigated the effects of diets with different protein content or quality on prostate and breast cancer.Experimental Design: To test our hypothesis we assessed the inhibitory effect of protein diet restriction on prostate and breast cancer growth, serum PSA and IGF-1 concentrations, mTOR activity and epigenetic markers, by using human xenograft cancer models.Results: Our results showed a 70% inhibition of tumor growth in the castrate-resistant LuCaP23.1 prostate cancer model and a 56% inhibition in the WHIM16 breast cancer model fed with a 7% protein diet when compared to an isocaloric 21% protein diet. Inhibition of tumor growth correlated, in the LuCaP23.1 model, with decreased serum PSA and IGF-1 levels, down-regulation of mTORC1 activity, decreased cell proliferation as indicated by Ki67 staining, and reduction in epigenetic markers of prostate cancer progression, including the histone methyltransferase EZH2 and the associated histone mark H3K27me3. In addition, we observed that modifications of dietary protein quality, independently of protein quantity, decreased tumor growth. A diet containing 20% plant protein inhibited tumor weight by 37% as compared to a 20% animal dairy protein diet.Conclusions: Our findings suggest that a reduction in dietary protein intake is highly effective in inhibiting tumor growth in human xenograft prostate and breast cancer models, possibly through the inhibition of the IGF/AKT/mTOR pathway and epigenetic modifications.