Murine Colon Adenocarcinoma Cells Research Articles

Intratumoral delivery of IL-18 naked DNA induces T-cell activation and Th1 response in a mouse hepatic cancer model

BackgroundThe novel cytokine, interleukin (IL)-18, is a strong interferon-γ inducer and costimulatory factor in Th1 cell activation. IL-18 triggers IFN-γ production and enhances cytolytic activity in both T and NK cells. However, the exact mechanism of antitumor action of IL-18 remains to be clarified. To determine the effects of IL-18 plasmid DNA on hepatic cancer in mice, CT26 murine colon adenocarcinoma cells were established in mouse liver.MethodsPlasmid vectors encoding IL-18 were transferred directly into the liver 7 days after tumor injection to restrict IL-18 expression within the tumor site. The IL-18 protein level was increased in the liver 4 days after plasmid injection, and a marked antitumoral effect was observed at day 7. Antitumor effects were evaluated by measuring tumor regression, immune cell population, and IFN-γ production.ResultsThe IL-18 plasmid controlled the growth of hepatic tumors and proliferation of splenic immune cells. Moreover, treatment of CT26 tumors with the IL-18 plasmid significantly enhanced the population of the effector T and NK cells in the spleen and peripheral blood. In spleen, the population of CD4+CD62Low cells was augmented in response to IL-18 on day 7. These results are consistent with the increase in CD4+ T cells secreting IFN-γ, but not CD8+ T cells. The marked reduction of tumor growth in tumor-bearing mice was associated with the maintenance of IFN-γ production in spleen in response to IL-18. These antitumoral effects were maintained until 14 days after plasmid injection.ConclusionOur results suggest that direct plasmid DNA transfer of IL-18 with no accompanying reagents to augment transfection efficiency may be useful in tumor immunotherapy.

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase

Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse beta-glucuronidase (mbetaG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-beta-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional beta-glucuronidase (betaG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (betaG) on CT26 tumors. The fluorescent intensity in betaG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral betaG vector into carcinoma xenografts. Importantly, mbetaG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human betaG also allowed in vivo imaging, demonstrating that human betaG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.

An orthotopic colon cancer model for studying the B7-H3 antitumor effect in vivo

We established an orthotopic animal model of colon cancer in mice and applied this model to study the antitumor effects of B7-H3, the newest member of the B7 family of costimulatory molecules. Colon-26 murine colon adenocarcinoma cells were inoculated into the cecal subserosum of mice to induce colon tumor growth. The tumor growth rate and the survival time of the mice were observed. A stable B7-H3 transfected Colon-26 cell line was established and the immunogenic effect was investigated. All mice implanted with wild-type tumor cells had tumor growth in the colon and died. The mean survival rate was 23 days. Mice implanted with C26-B7-H3 had a significantly prolonged survival time of 38 days. Our data suggest that B7-H3 exerts an antitumor effect on adenocarcinoma of the colon and may be considered as an adjuvant immunotherapy in the treatment of colon cancers. Our orthotopic animal model of colon cancer in mice could be applied to in vivo experimental studies of colon cancer.

Attenuated liver tumor formation in the absence of CCR2 with a concomitant reduction in the accumulation of hepatic stellate cells, macrophages and neovascularization

The liver parenchyma is populated by hepatocytes and several nonparenchymal cell types, including Kupffer cells and hepatic stellate cells. Both Kupffer cells and hepatic stellate cells are responsive to the chemokine CCL2, but the precise roles of CCL2 and these cells in liver tumor formation remain undefined. Hence, we investigated the effects of the lack of the major CCL2 receptor, CCR2, on liver tumor formation induced by intraportal injection of the murine colon adenocarcinoma cell line, colon 26. Wild-type mice showed macroscopic tumor foci in the liver 10 days after injection of colon 26 cells. After 10 days, CCL2 proteins were detected predominantly in tumor cells, coincident with increased intratumoral macrophage and hepatic stellate cell numbers. Although tumor formation occurred at similar rates in wild-type and CCR2-deficient mice up to 10 days after tumor cell injection, the number and size of tumor foci were significantly attenuated in CCR2-deficient mice relative to wild-type mice thereafter. Moreover, neovascularization and matrix metalloproteinase 2 expression were diminished in CCR2-deficient mice with a concomitant reduction in the accumulation of macrophages and hepatic stellate cells. Furthermore, matrix metalloproteinase 2 was detected predominantly in hepatic stellate cells but not in macrophages. We provided the first definitive evidence that the absence of CCR2-mediated signals can reduce the trafficking of hepatic stellate cells, a main source of matrix metalloproteinase 2, and consequently can diminish neovascularization during liver tumor formation.

Osteopontin silencing by small interfering RNA suppresses in vitro and in vivo CT26 murine colon adenocarcinoma metastasis

Hepatic metastasis is a primary cause for failure of locoregional therapy in colorectal cancer. Increased expression of osteopontin (OPN), a ligand for alpha(v)beta3 integrin and CD44 receptors, is associated with metastasis in several types of cancer. However, the mechanism by which OPN mediates metastasis in colorectal cancer remains unknown. We hypothesized that OPN mediates invasion of colon cancer cells through basement membrane and migration through extracellular matrix (ECM). In this study, we used CT26 murine colon adenocarcinoma cells syngeneic to BALB/c mice to generate cell lines (pS-OPN) in which OPN expression was suppressed through small interfering RNA (siRNA) plasmids. CT26 wild-type cells (WT) and CT26 cells stably expressing murine-mismatch siRNA (pS-MM) served as controls. Western blotting quantified OPN protein levels and our most downregulated clone, pS-OPN-A4, demonstrated a mean 3.0-fold decrease in OPN protein expression versus WT. In vitro cell motility and invasiveness were decreased in pS-OPN-A4 by 3.6-fold (P = 0.004 versus WT) and 4.1-fold (P = 0.01 versus WT), but proliferation was similar amongst cell lines. We demonstrated that OPN suppression significantly correlates with MMP-2 downregulation. In vivo hepatic metastasis was assessed by quantifying liver weights and surface tumor nodules in 33 BALB/c mice (11/group) subjected to intrasplenic injection of tumor cells. pS-OPN-A4 resulted in a 50.4% decrease in mean liver weight compared with WT (3.79 +/- 1.49 g versus 1.88 +/- 1.34 g, P = 0.009). Only 18% of pS-OPN-A4 livers had >20 metastatic surface nodules compared with 89% for WT and 75% for pS-MM-V6. This study demonstrates that RNA interference stably reduces CT26 tumor expression of OPN and significantly attenuates CT26 colon cancer metastasis by diminishing tumor cell motility and invasiveness.

Interleukin-10 plasmid DNA inhibits subcutaneous tumor growth of Colon26 adenocarcinoma in mice

The transcription factor NF-κB is constitutively activated in many human cancers, and induces the expression of multiple proteins including antiapoptotic proteins. Recent papers indicate that NF-κB activation is inhibited by interleukin (IL)-10. In this study, we investigated the effect of IL-10 plasmid DNA on colon cancer in mice. In vitro study: Colon26 murine colon adenocarcinoma cells were either treated or untreated with IL-10 for 60 min. The cells were subsequently stimulated with TNF-α. In vivo study: to induce a high level of IL-10 in plasma, we transferred the naked plasmid vectors encoding the mouse IL-10 gene into the liver via the intravenous route. To establish tumors, we injected Colon26 cells into BALB/c mice subcutaneously. In vitro study: a 24-h incubation with TNF-α did not affect cell viabilities; however, pretreatment with IL-10 significantly enhanced the level of apoptosis induced by TNF-α. Pretreating Colon26 cells with IL-10 significantly attenuated the TNF-α-induced NF-κB activation. In vivo study: IL-10 plasmid controlled the growth of subcutaneous tumors. In subcutaneous tumor, NF-κB was activated in response to tumor growth. IL-10 plasmid markedly inhibited this activation of NF-κB in subcutaneous tumor. IL-10 plasmid induced cancer cell apoptosis linked to the down-regulation of antiapoptotic proteins, and the activation of caspase-3. These results demonstrate that IL-10 plasmid may constitute a new strategy for treating cancer growth.

Farnesyl- O-acetylhydroquinone and geranyl- O-acetylhydroquinone suppress the proliferation of murine B16 melanoma cells, human prostate and colon adenocarcinoma cells, human lung carcinoma cells, and human leukemia cells

Farnesyl- O-acetylhydroquinone (IC 50=2.5 μmol/l) suppressed the proliferation of murine B16F10 melanoma cells with a potency much greater than those of farnesol (IC 50=45 μmol/l) and farnesyl anthranilate (IC 50=46 μmol/l), its alcohol, and ester counterparts with proven anti-tumor activities in vivo. Geranyl- O-acetylhydroquinone (IC 50=5.1 μmol/l) also had a much-improved activity compared to geraniol (IC 50=160 μmol/l) and geranyl anthranilate (IC 50=30 μmol/l). The suppression by farnesyl- O-acetylhydroquinone was concentration- and time-dependent and was accompanied by arrest of cell cycle at G1 and G2/M phases as shown by flow cytometry. Farnesyl- O-acetylhydroquinone and lovastatin had additive impact on B16 cell proliferation. Farnesyl- O-acetylhydroquinone also suppressed the proliferations of human cancer cells HL-60, DU145, PC-3, LNCaP, Caco-2, and A549. Our results suggested that farnesyl derivatives, suppressors of tumor 3-hydroxy-3-methylglutaryl coenzyme A reductase activities, have potential as chemopreventive or chemotherapeutic agents.

Antitumor and antimetastatic activity of IL-23.

The structure and T cell stimulatory effects of the recently discovered cytokine IL-23 are similar to, but distinct from, those of IL-12. Although the antitumor activities of IL-12 are well characterized, the effect of IL-23 on tumor growth is not known. In this study, murine CT26 colon adenocarcinoma and B16F1 melanoma cells were engineered using retroviral vectors to release single-chain IL-23 (scIL-23) to evaluate its antitumor activity. In BALB/c mice, scIL-23-transduced CT26 cells grew progressively until day 26 to an average size of 521 +/- 333 mm(3), then the tumors started to regress in most animals, resulting in a final 70% rate of complete tumor rejection. scIL-23 transduction also significantly suppressed lung metastases of CT26 and B16F1 tumor cells. In addition, mice that rejected scIL-23-transduced tumors developed a memory response against subsequent wild-type tumor challenge. Compared with scIL-12-expressing CT26 cells, scIL-23-transduced tumors lacked the early response, but achieved comparable antitumor and antimetastatic activity. These results demonstrated that IL-23, like IL-12, provided effective protection against malignant diseases, but it probably acted by different antitumor mechanisms. As a first step in identifying these antitumor mechanisms, tumor challenge studies were performed in immunocompromised hosts and in animals selectively depleted of various lymphocyte populations. The results showed that CD8(+) T cells, but not CD4(+) T cells or NK cells, were crucial for the antitumor activity of IL-23.

GFP-transfected tumor cells are useful in examining early metastasis in vivo, but immune reaction precludes long-term tumor development studies in immunocompetent mice.

To develop effective therapeutic strategies aimed at treating tumor metastasis, critical steps in this process must be better understood. For this purpose we have established a new model to visualize and quantify early metastasis. Murine CT-26 colon adenocarcinoma cells were stably transfected with green fluorescent protein (GFP). Tumor cells were intraportally delivered to the liver of Balb/c mice and subsequently tracked by intravital fluorescence microscopy. Coinjection of fluorescent beads and in vivo propidium iodide staining allowed examination of initial tumor cell arrest, extravasation, viability and proliferation. Results showed that GFP-transfection compared to conventional labeling procedures (Calcein, cytoplasmic microspheres) did not alter early metastatic properties. However, the long-term development of liver metastases expressing GFP was markedly reduced compared to wild type CT-26 tumor cells. An increase in the size and the number of liver metastases in T- and B-cell-deficient SCID mice suggested an immune response to the GFP transfected cells responsible for the reduced metastatic growth in wild-type mice. Based on our findings, this model can be used to examine the early steps of metastasis in vivo. However, in immunocompetent mice, the use of GFP-labeled tumor cells should be limited to tracking cell arrest and extravasation, whereas evaluations of long-term metastatic growth should be performed in immunodeficient mice.

Recognition of the importance of imidazolidinone motif for cytotoxicity of 4-phenyl-1-arylsulfonylimidazolidinones using thiadiazolidine-1,1-dioxide analogs.

For probing the importance of planarity of imidazolidinone motif of 4-phenyl-1-(N-acylindoline-5-sulfonyl)imidazolidinones for their cytotoxicity, 4-phenyl-1-(N-acylindoline-5-sulfonyl)[1,2,5]thiadiazolidine-1,1-dioxides 2 were prepared and their cytotoxicity were measured against human lung carcinoma (A549), human colon carcinoma (COL0205), human ovarian cancer (SK-OV-3), human leukemic cancer (K562), and murine colon adenocarcinoma (Colon26) cell lines in vitro. Although only carbonyl moiety of imidazolidinone ring was replaced with sulfonyl group, compounds 2 do not show any activity against all five cancer cell lines unlike 1. Therefore the planarity of imidazolidinone ring of 1 should be an important factor for their cytotoxic activity.

Establishment and characterization of a rectal cancer model in mice: application to cytokine gene therapy.

We established an orthotopic animal model of rectal cancer in mice and applied this model to the study of the antitumor effects of cytokine-assisted tumor vaccine. The CT-26 murine colon adenocarcinoma cells were inoculated into the submucosa of the rectum of the mice to induce the rectal tumor. The tumor growth rate and the survival time of the mice were observed. The cDNA of granulocyte-macrophage colony-stimulating factor (GM-CSF) was transduced to the CT-26 cell line via a retroviral vector, and the therapeutic effects of irradiated GM-CSF secreting tumor vaccine on the rectal tumor were investigated. All the mice implanted with the wild-type tumor cells had tumor growth in the rectum and died. The mean survival time of the mice was 28.9 days. Two doses of irradiated GM-CSF secreting tumor vaccine administered on days 0 and 3 after tumor cell implantation significantly prolonged the survival of the mice with rectal tumor compared with that of the control groups ( P<0.0001). In contrast, no antitumor effect was observed when the treatment with GM-CSF secreting tumor vaccine was delayed to 3 days after tumor cell implantation ( P>0.17). The results suggest that cytokine gene therapy exerts an antitumor effect on small tumors and may be considered as an adjuvant immunotherapy of rectal cancers and prevention of reimplantation of tumor cells disseminated during or following surgery. The orthotopic animal model of the rectal cancer in mice could be applied to the in vivo experimental studies of rectal cancer.

Novel water insoluble lipoparticulates for gene delivery.

The objective was to design and prepare water insoluble lipoparticulates (ISLPs) for efficient gene delivery to lung tissue. Nona[(ethylenimine)-co-[(2-aminoethyl)-N-choleseteryl-oxycarbonyl-ethylenimine]] (NEACE-T) was synthesized in both its free-base and chloride salt-forms using linear polyethylenimine (PEI, Mw 423) as a headgroup and cholesteryl chloroformate as a hydrophobic lipid anchor resulting in a T-shaped lipononamer. Semitelechelic N(alpha)-cholesteryloxycarbonyl nona(ethylenimine) (st-NCNEI-L) was synthesized similarly resulting in a linear lipononamer. As confirmed by 1H-NMR, the site of conjugation was either a primary amine resulting in a linear configuration (st-NCNEI-L) or a secondary amine resulting in a T-shaped configuration (NEACE-T). ISLPs were prepared by combining NEACE-T or st-NCNEI-L with a co-lipid, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 1/1, 1/2, and 2/1 molar ratios and the lipoparticulates were hydrated and filtered. ISLP/p2CMVmIL-12 complexes were characterized for particle size, zeta potential, surface morphology, cytotoxicity, and in vitro transfection efficiency. Transgene expression was dependent on the site of cholesterol conjugation, lipononamer:colipid molar ratio, and ISLP/ p2CMVmIL-12 charge ratios. ISLP/p2CMVmIL-12 complexes were nontoxic to murine colon adenocarcinoma (CT-26) cells at 9/1 (+/-) or lower, had a mean particle size of 330-400 nm while the zeta potential varied from 36-39 mV. Atomic force microscopy (AFM) showed the surface morphology to be that of an oblate spheroid with a size comparable to that determined by dynamic light scattering. ISLP/ p2CMVmIL-12 complexes prepared using free-base NEACE-T:DOPE (1/2) at charge ratios of 3/1 and 5/1 (+/-) provided the highest levels of transgene expression, 18 times more than the levels provided by the salt-form. Secreted levels of mIL-12 p70 were 75 times higher for ISLP/p2CMVmIL-12 complexes than naked p2CMVmIL-12 and nearly 4 times higher than PEI 25 kDa/p2CMVmIL-12 complexes. The transfection efficiency of the ISLPs was dependent on the site of cholesterol conjugation, amount of colipid, and charge ratio. The highest levels of transgene expression were provided by NEACE-T:DOPE (1/2)/p2CMVmIL-12 at a 3/1 (+/-) charge ratio.

Synergistic cytotoxic effect between serine-threonine phosphatase inhibitors and 5-fluorouracil: a novel concept for modulation of cytotoxic effect.

The present study was undertaken to look for an agent or agents able to modulate the cytotoxic effect of 5-fluorouracil (FUra) and to investigate the role of serine-threonine phosphatase inhibitors on the cytotoxic effect of FUra. The cytotoxicities of FUra and protein phosphatase inhibitors (PPIs) were evaluated by two different methods: a clonogenic assay and a proliferation assay. In the clonogenic assay, cancer cells were treated with various concentration of FUra with or without PPIs for 72 h. The drug-containing medium was replaced by fresh medium, the cultures incubated for an additional 10 days, and the colonies enumerated. In the proliferation assay the cells were treated with FUra alone or in combination with PPIs for 96 h and cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay based on the uptake of the tetrazolium dye. Thymidine kinase (TK) activity was determined based on the catalytic phosphorylation of [(3)H]d-thymidine to [(3)H]dTMP. Incorporation of FUra into DNA and RNA was determined by treating the cells with [2-(14)C]fluorouracil for 72 h and measuring the radioactivity in the isolated DNA and RNA fractions. The serine-threonine phosphatase inhibitors caliculin A (CAL), okadaic acid (OA) and microcystin-LR (MCLR) dose-dependently inhibited the growth of Clone 20 and Clone 5 cells of Colon 26 murine colon adenocarcinoma cells, human cervical cancer HeLa cells, human gastric cancer MKN 7 cells, and murine sarcoma S-180 cells in vitro. Among the compounds tested, MCLR at non-toxic concentrations was found to increase FUra incorporation into RNA and DNA in Clone 20 cells by 60% and 127%, respectively, to increase TK activity alone (twofold) as well as in combination with FUra (threefold), and to potentiate the cytotoxicity of FUra synergistically and cytospecifically in vitro. The cytotoxicity of FUra alone or in combination with MCLR, but not that of PPIs alone, was abrogated almost completely by exogenous thymidine (dThd), suggesting that inhibition of thymidylate synthetase (TS) is the growth-limiting event in the cytotoxic action of FUra even in combination with MCLR. The findings presented here suggest that MCLR synergistically and cytospecifically potentiates the antitumor activity of FUra with substantial improvement in the therapeutic index of FUra via enhancement of both DNA- and RNA-directed cytotoxicity.

Complex interactions between the replicating oncolytic effect and the enzyme/prodrug effect of vaccinia-mediated tumor regression.

Replicating viruses for cancer gene therapy have beneficial antitumor effects, however, in the setting of an enzyme/prodrug system, the interactions between these viruses and the activated agents are complex. A replicating vaccinia virus expressing the cytosine deaminase gene (VVCD), which converts the prodrug 5-FC into 5-FU, was characterized in vitro and in vivo for its antitumor effects and pathogenicity. Replicating VVCD (+/-5-FC) at various MOIs was used to infect MC38 murine colon adenocarcinoma cells. At high MOIs (>0.1) virus alone was able to kill the majority (65-90%) of cells by day 5 with no additional benefit from prodrug. At low MOIs only the effect of prodrug is seen. Cell lysates demonstrated 300-fold reduced viral recovery from cells treated with both VVCD and 5-FC compared with controls treated with virus alone. Nude mice bearing subcutaneous MC38 tumors were injected with VVCD (or control) and treated with 5FC or control. Mice injected with VVCD (with or without 5FC treatment) had smaller tumors than the controls, suggesting that replicating vaccinia alone is cytotoxic to tumors in vivo. The addition of 5-FC improved the antitumor response when a low dose of virus was injected into tumors. Also, compared with mice that received virus alone, those that received VVCD and 5FC had significantly prolonged survival from virus-mediated death. In conclusion, the addition of an enzyme/prodrug system to a replicating virus can improve the antitumor response and decrease viral pathogenicity. Gene Therapy (2000) 7, 1217-1223.

AN ANTIBODY FOR MACROPHAGE MIGRATION INHIBITORY FACTOR SUPPRESSES TUMOUR GROWTH AND INHIBITS TUMOUR-ASSOCIATED ANGIOGENESIS

To verify the role of macrophage migration inhibitory factor (MIF) in tumourigenesis, we examined the effect of an anti-MIF antibody on tumour growth and angiogenesis. We inoculated murine colon adenocarcinoma cell line colon 26 cells subcutaneously into the flank in BALB/c mice. After nine days, we treated tumour-bearing mice with an anti-rat MIF antibody by intraperitoneal injection on days 9, 11, 13, 15, 17, 19 and 21. We found significant inhibition of tumour growth by this treatment from day 15 to day 22. Next, we implanted a chamber filled with colon 26 cells, which only passes soluble factors, in the subcutaneous fascia of the flank, and treated mice with the anti-rat MIF antibody at days 1, 3 and 5. By histological examination at day 6, angiogenesis within the subcutaneous fascia in contact with the chamber was markedly suppressed. In vitro, we added an anti-human MIF antibody to human umbilical vein endothelial cells (HUVEC) to evaluate its effect on cell growth by measurement of [3H]thymidine incorporation. We observed that the anti-MIF antibody significantly suppressed [3H]thymidine uptake by HUVEC. These results suggest the possibility that MIF is involved in tumourigenesis via promotion of angiogenesis.

SEREX analysis for tumor antigen identification in a mouse model of adenocarcinoma.

Evaluation of immunotherapy strategies in mouse models of carcinoma is hampered by the limited number of known murine tumor antigens (Ags). Although tumor Ags can be identified based on cytotoxic T-cell activation, this approach is not readily accomplished for many tumor types. We applied an alternative strategy based on a humoral immune response, SEREX, to the identification of tumor Ags in the murine colon adenocarcinoma cell line MC38. Immunization of syngeneic C57BL/6 mice with MC38 cells by three different methods induced a protective immune response with concomitant production of anti-MC38 antibodies. Immunoscreening of an MC38-derived expression library resulted in the identification of the endogenous ecotropic leukemia virus envelope (env) protein and the murine ATRX protein as candidate tumor Ags. Northern blot analysis demonstrated high levels of expression of the env transcript in MC38 cells and in several other murine tumor cell lines, whereas expression in normal colonic epithelium was absent. ATRX was found to be variably expressed in tumor cell lines and in normal tissue. Further analysis of the expressed env sequence indicated that it represents a nonmutated tumor Ag. Polynucleotide immunization with DNA encoding the env polypeptide resulted in strong and specific antibody responses to this self Ag in all immunized mice. Thus, SEREX offers a rapid means of identifying tumor Ags in murine cancer models.

Intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas.

Stimulation of the antitumor immune response by dendritic cells (DC) is critically dependent on their tightly regulated ability to produce interleukin-12 (IL-12). To enhance this effect artificially, bone marrow (BM)-derived DC were genetically engineered to produce high levels of functional IL-12 by ex vivo infection with a recombinant defective adenovirus (AdCMVIL-12). DC-expressing IL-12 injected into the malignant tissue eradicated 50-100% well established malignant nodules derived from the injection of two murine colon adenocarcinoma cell lines. Successful therapy was dependent on IL-12 transfection and was mediated only by syngeneic, but not allogeneic BM-derived DC, indicating that compatible antigen-presenting molecules were required. The antitumor effect was inhibited by in vivo depletion of CD8+ T cells and completely abrogated by simultaneous depletion with anti-CD4 and anti-CD8 mAbs. Mice which had undergone tumor regression remained immune to a rechallenge with tumor cells, showing the achievement of long-lasting systemic immunity that also was able to reject simultaneously induced concomitant untreated tumors. Tumor regression was associated with a detectable CTL response directed against tumor-specific antigens probably captured by DC artificially released inside tumor nodules. Our results open the possibility of similarly treating the corresponding human malignancies.

AsGM1+ NK Cells Prevent Metastasis of Invading LD-MCA-38 Tumor Cells in the Nude Mouse

Background. Although the liver is a potent tumor cell killing organ it is frequently the site of lethal metastases often signifying the endstage for patients with colorectal cancers. Enhancing hepatic-associated immunity remains elusive until the interactions among hepatic nonparenchymal cells (NPC) are deciphered. We sought to modulate the cellular components of the hepatic immune system of mice with anti-NK and anti-T-cell-neutralizing antibodies in order to determine the cell type most efficacious in preventing liver metastasis.Materials and methods. Liver-derived murine colon adenocarcinoma (LD-MCA-38) cells were injected into the ileocolic vein (ICV) of immunocompetent and immunodeficient C57BL/6 mice. Mice were pretreated 1 day prior to tumor cell injection with one of three antibodies: anti-AsGM1, Anti-NK1.1, or Anti-Thy1.2. On Day 21 laparotomy was performed to determine the extent of hepatic tumor foci. The number of hepatic tumor foci was recorded and compared by the Wilcoxon rank sum test.Results. Mice pretreated with anti-AsGM1 or Anti-NK1.1 developed a massive increase in the number of hepatic tumor foci and decreased survival compared to the control treated mice. Pretreatment with anti-Thy1.2 antibody resulted in a significant decrease in the number of hepatic tumor foci. LD-MCA-38 tumor cells were unable to colonize the liver of C57BL/6 athymic nude mice; however, anti-AsGM1 antibody abolished this antimetastatic effect. There was no difference in the extent of hepatic metastasis and survival between immunodeficient C57BL/6 bg/bg and their conventional littermates bg/+.Conclusion. AsGM1+ NK cells exhibit a significant antitumor response in the absence of T-cells. The concept of stimulating NK cell activity and suppressing T-cell function may enhance liver-associated immunity and serve as a deterrent for blood-borne tumor cells metastasizing to the liver.

Isolation, Purification, and Characterization of a Collagen-associated Serpin, Caspin, Produced by Murine Colon Adenocarcinoma Cells

A 45-kDa serpin secreted by a murine colon adenocarcinoma cell line, colon26, was isolated, purified, and characterized. It was found to bind specifically to type I collagen with high affinity and to type III collagen with lower affinity. Immunohistochemical studies of murine embryonic tissues showed a specific distribution of this collagen-associated serpin, named caspin, in relation to the formation of bone, cartilage, teeth, and basement membrane. The expression of caspin in high and low lung metastatic subclones of colon26 cell lines was inversely correlated with their metastatic capacity: low lung metastatic cells secreted higher amounts of caspin than their high lung metastatic counterparts. Caspin also demonstrated high homology with human pigment epithelium-derived factor/early population doubling level cDNA-1, which reportedly induces neuronal differentiation of human retinoblastoma cells and is expressed in association with G0 growth arrest. These findings suggest that caspin/pigment epithelium-derived factor/early population doubling level cDNA-1 is a novel factor that might play a crucial role in embryogenesis and tumor metastasis through binding to the extracellular matrix.

Immunization with a syngeneic tumor infected with recombinant vaccinia virus expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) induces tumor regression and long-lasting systemic immunity.

A recombinant vaccinia virus encoding the gene for granulocyte-macrophage colony-stimulating factor (rV-GM-CSF) was used to infect the poorly immunogenic murine colon adenocarcinoma cell line, MC-38. Infection of MC-38 tumor cells with rV-GM-CSF completely suppressed the growth of the MC-38 primary tumors, whereas progressively growing tumors were formed in mice injected with MC-38 cells infected with wild type V-Wyeth. Irradiation of the recipient B6 mice before implantation of rV-GM-CSF-infected tumor cells resulted in the development of progressively growing tumors. Moreover, in vivo T-cell depletion studies revealed that growth suppression of the rV-GM-CSF-infected tumor cells was dependent on the presence of both CD4+ and CD8+ T-cell subsets. Subsequent studies established that this immunity was long-lasting and antigen specific, as demonstrated by the protection of rV-GM-CSF-immunized mice from MC-38 tumor challenge but not from challenge with another syngeneic tumor cell type. No such effects were observed when MC-38 tumor cells were infected with recombinant vaccinia viruses expressing interleukin (IL)-2 or IL-6. The results demonstrate that paracrine release of biologically active murine GM-CSF by tumor cells infected with rV-GM-CSF enhances the intrinsic immunogenicity of a poorly immunogenic murine tumor. Presumably the augmentation of tumor immunogenicity induces an antigen-specific T-cell-dependent antitumor response that prevents the formation of primary tumors and protects mice from tumor challenge. Thus in this experimental model, GM-CSF functions as a highly effective vaccine adjuvant.