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A striking feature of Ewing sarcoma is the presence of blood lakes lined by tumor cells. The significance of these structures, if any, is unknown. Here, we report that the extent of blood lakes correlates with poor clinical outcomes, whereas variables of angiogenesis do not. We also show that Ewing sarcoma cells form vessel-like tubes in vitro and express genes associated with vasculogenic mimicry. In tumor models, we show that there is blood flow through the blood lakes, suggesting that these structures in Ewing sarcoma contribute to the circulation. Furthermore, we present evidence that reduced oxygen tension may be instrumental in tube formation by plastic tumor cells. The abundant presence of these vasculogenic structures, in contrast to other tumor types, makes Ewing sarcoma the ideal model system to study these phenomena. The results suggest that optimal tumor treatment may require targeting of these structures in combination with prevention of angiogenesis.

A major agenda for tumor immunology is the generation of specific immune responses leading to the destruction of incipient and frank neoplasia. In this report, we show that a novel HPV16 E7 fusion protein can produce objective therapeutic responses against incipient cervical cancer in genetically engineered mice that express in the cervix the HPV16 early region genes implicated as causative agents in human cervical cancer. Although nonresponsive toward the HPV16 E7 oncoprotein in the CD8+ T-cell compartment by virtue of MHC haplotype, the mice were capable of mounting an induced CD4+ T-cell response against E7, and in addition developed spontaneous anti-E7 antibodies. HPV16/CD4-/- mice showed increased tumor burden indicative of CD4-mediated immune surveillance. Seeking to enhance the CD4 response, we immunized mice bearing incipient cervical cancer with a recombinant protein fusing E7 with a mycobacterial heat shock protein. The incidences of cervical carcinoma and of high-grade dysplasia (CIN 3) were consequently reduced by comparison to control mice. Thus, an HPV16 E7 immunogen holds promise for noninvasive treatment and prevention of human cervical cancer.

Accumulating evidence suggests that glutamate plays a key role in the proliferation and invasion of glioblastoma tumors. Astrocytic tumors have been shown to release glutamate at high levels, which may stimulate tumor cell proliferation and motility via activation of glutamate receptors. Excess glutamate has also been found to facilitate tumor invasion by causing excitotoxic damage to normal brain thereby paving a pathway for tumor migration. Results from tissue microarray analyses showed decreased excitatory amino acid transporter-2 (EAAT-2) expression in high-grade glial tumors compared with low-grade astrocytomas and normal brain. EAAT-2 expression was inversely correlated with tumor grade, implicating its potential role in glial tumor progression, which was reflected by an undetectable level of EAAT-2 protein in glioma cell lines. In this study, we sought to investigate the effect of reconstituted EAAT-2 on glioma cell growth in vitro and in vivo by adenoviral-mediated gene transfer. Infection of glioma cells with Ad-EAAT-2 resulted in a physiologic level of functional EAAT-2, and a subsequent dose-dependent reduction in cell proliferation in all glioma cell lines tested compared with controls. Interestingly, results from analyses of Annexin V staining, detection of poly(ADP-ribose)polymerase cleavage and caspase-3 activation all indicated that Ad-EAAT-2 infection elicited apoptosis in glioma cells. Ex vivo experiments in nude mice showed a total suppression of tumor growth at sites that received Ad-EAAT-2-infected cells. Collectively, our results uncovered a new function of EAAT-2 in controlling glioma proliferation. Further studies will improve our knowledge of the role of glutamate in glioma growth and may provide useful prognostic information and alternative therapeutic targets for the treatment of glioma.

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Glucocorticoids (GCs) are frequently used as cotreatment because they may have potent proapoptotic properties and reduce nausea, hyperemesis, and acute toxicity on normal tissue. In contrast to the proapoptotic effect of GCs in lymphoid cells, resistance toward cancer therapy-mediated apoptosis was induced in solid tumors of human cervix and lung carcinomas. Filter hybridization, expression data, as well as functional assays identified multiple core apoptosis molecules, which are regulated by GCs in a pro- or antiapoptotic manner. Both antiapoptotic genes such as FLIP and members of the Bcl-2 and IAP family as well as proapoptotic elements of the death receptor and mitochondrial apoptosis pathways were down-regulated in carcinomas resulting in a decreased activity of caspase-8, caspase-9, and caspase-3. In contrast, death receptor and mitochondrial apoptosis signaling as well as caspase activity was enhanced by dexamethasone in lymphoid cells. To restore apoptosis sensitivity in dexamethasone-treated carcinomas, caspase-8 and caspase-9 were transfected. This resensitized tumor cells in vitro and xenografts in vivo to cisplatin induced cell death. These data therefore raise concern about the widespread combined use of GCs with antineoplastic drugs or agents in the clinical management of cancer patients.

Angiopoietin (Ang)-1 and -2 are critical regulators of embryonic and postnatal neovascularization. Ang-1 activates the endothelial cell-specific tyrosine kinase receptor Tie-2, which in turn leads to enhanced endothelial cell survival and stabilization. The effects of Ang-1 on tumor angiogenesis remain controversial; although we have previously demonstrated that Ang-1 overexpression in colon cancer cells leads to a decrease in s.c. tumor growth, others have shown that Ang-1 may be proangiogenic. Few studies have addressed the role of the Angs in tumors growing in the organ of metastatic growth. We hypothesized that overexpression of Ang-1 may inhibit the growth of colon cancers growing in the liver by inhibition of angiogenesis. We also wanted to investigate the mechanisms by which Ang-1 affects angiogenesis in vivo. Human colon cancer cells (HT29) were stably transfected with an Ang-1 construct or an empty vector (pcDNA) and injected directly into the livers of nude mice. After 37 days, livers were harvested and weighed, and tumor sizes were measured. In an additional experiment, to validate the paracrine effect of Ang-1, various mixtures of control cells and Ang-1-transfected cells were injected into livers, and tumor growth was assessed. Direct effects of recombinant Ang-1 on angiogenesis were studied with an in vivo Gelfoam angiogenesis assay. The impact of Ang-1 on vascular permeability was investigated using an intradermal Miles assay with conditioned media from transfected cells. Liver weights (P < 0.05), tumor volumes (P < 0.05), vessel counts (P < 0.01), and tumor cell proliferation (P < 0.01) in the Ang-1 group were significantly lower than those in the control (pcDNA) group. Tumor vessels in the Ang-1 group developed a significantly higher degree of pericyte coverage (P < 0.02) than vessels in pcDNA tumors. In the cell mixture experiment, even as few as a 1:10 mixture of Ang-1-transfected cells/control cells resulted in a significant reduction of hepatic tumor volumes (P < 0.04). In the angiogenesis assay, vessel counts in Gelfoam implants were significantly decreased by the addition of Ang-1 (P < 0.01). Finally, conditioned medium from Ang-1-transfected cells decreased vascular permeability more than that from control cells (P < 0.05). Our results suggest that Ang-1 is an important regulator of angiogenesis and vascular permeability and that this effect may be secondary to increasing periendothelial support and vessel stabilization. Thus, Ang-1 could potentially serve as an antineoplastic or anti-permeability agent for patients with metastatic colorectal cancer.

Neuroblastoma is the second most common solid tumor in childhood and frequently metastasizes to the bone marrow and the bone matrix. The mechanism involved in bone metastasis and destruction in neuroblastoma is poorly understood. Using a model of bone invasion in immunodeficient mice, we demonstrated that neuroblastoma cells recruited osteoclasts to generate osteolytic lesions and invade the bone matrix. In further support of a contributory role for osteoclasts in neuroblastoma bone invasion, we demonstrated that treatment with the bisphosphonate compound, ibandronate, significantly delayed the progression of osteolytic lesions. The data suggest that bisphosphonates may be clinically effective in the treatment of bone metastases in neuroblastoma.

Resistance of breast carcinomas to hormonal therapy is a clinical obstacle for the treatment of breast cancer. The molecular mechanisms and the factors involved in the progression of tumors from an estrogen (E2)-dependent to an E2-independent phenotype are not entirely understood. Heregulin (HRG) is a pleiotropic growth factor that binds to the erbB family of receptors, which are correlated with breast cancer progression and an aggressive phenotype in the breast carcinomas overexpressing the receptors. Previous studies in transgenic mice have shown that HRG is sufficient to induce mammary gland transformation and proliferation in the presence of hormonal stimulation. However, these studies did not address the important issue of the E2 independence that is part of the progression of breast cancer. In this study, we investigated the role of HRG in E2 independence. We were able to determine that HRG up-regulation was sufficient for the development of mammary tumors in the absence of E2 stimulation, a situation that mimics the progression of the human disease. We demonstrated that in ovariectomized nude mice, HRG induced E2 independence and antiestrogen resistance and promoted metastasis and preneoplastic transformation of the adjacent mouse mammary tissue. We show that one of the mechanisms by which HRG achieves the aggressive phenotype may be mediated via an increase in activated mitogen-activated protein kinase, an increase in a matrix-degrading enzyme, MMP-9, and the overexpression of vascular endothelial growth factors. The up-regulation of these genes occurred in the absence of any additional stimulation, in an autocrine manner. Our data provide new insights into the mechanisms of breast cancer progression in vivo, and reinforce the important role that HRG plays in this process.

Interleukin (IL) 17 is a proinflammatory cytokine secreted mainly by activated human memory CD4 T cells that induces IL-6, IL-8, and nitric oxide. Because IL-6 and IL-8 have been implicated in the pathogenesis of cervical cancer, we investigated the action of IL-17 on human cervical tumor cell lines in vitro and in vivo. We showed that in vitro, IL-17 increases IL-6 and IL-8 secretion by cervical carcinoma cell lines at both protein and mRNA levels. No direct effect of IL-17 on in vitro proliferation of cervical tumor cell lines could be demonstrated. However, two cervical cell lines transfected with a cDNA encoding IL-17 exhibited a significant increase in tumor size as compared to the parent tumor when transplanted in nude mice. This enhanced tumor growth elicited by IL-17 was associated with increased expression of IL-6 and macrophage recruitment at the tumor site. A potential role of IL-17 in modulation of the human cervical tumor phenotype was also supported by its expression on the cervical tumor in patients with CD4 infiltration. IL-17 therefore behaves like a T-cell-specific cytokine with paradoxical tumor-promoting activity. This may partially explain previous reports concerning the deleterious effect of CD4 T cells in cancer.

Salmosin is a snake venom-derived novel disintegrin that antagonizes platelet aggregation. In this study, we investigated its functional specificity in tumor angiogenesis. Salmosin significantly inhibited bovine capillary endothelial cell proliferation induced by basic fibroblast growth factor but had no effect on normal growth of the cell. The basic fibroblast growth factor-induced in vivo angiogenesis in the chorioallantoic membrane was disrupted by salmosin treatment without affecting normal embryonic angiogenesis. Adhesion of the bovine capillary endothelial cells to vitronectin was also inhibited by the binding of salmosin to the alpha(v)beta3 integrin. Both the metastatic-tumor growth and the solid-tumor growth that developed in mice were effectively suppressed by salmosin treatment. Several lines of experimental evidence strongly suggest that the tumor-specific antiangiogenic activity of salmosin disrupts tumor growth by blocking the alpha(v)beta3 integrin that is expressed on the vascular endothelial cell surface.

Gene therapy transfer of angiostatin and endostatin represents an alternative method of delivering angiogenic polypeptide inhibitors. We examined whether liposomes complexed to plasmids encoding angiostatin or endostatin inhibited angiogenesis and the growth of MDA-MB-435 tumors implanted in the mammary fat pads of nude mice. We determined that plasmids expressing angiostatin (PCI-Angio) or endostatin (PCI-Endo) effectively reduced angiogenesis using an in vivo Matrigel assay. We then investigated the efficacy of these plasmids in reducing the size of tumors implanted in the mammary fat pad of nude mice. Both PCI-Angio and PCI-Endo significantly reduced tumor size when injected intratumorally (P < 0.05). Compared to the untreated control group, the mice treated with PCI-Angio and PCI-Endo exhibited a reduction in tumor size of 36% and 49%, respectively. In addition, we found that i.v. injections of liposomes complexed to PCI-Endo reduced tumor growth in the nude mice by nearly 40% when compared to either empty vector (PCI) or untreated controls (P < 0.05). These findings provide a basis for the further development of nonviral delivery of antiangiogenic genes.

We reported previously that tumor cells isolated from metastases of the in vitro transformed squamous cell carcinoma line Pam 212 exhibit an elevation in constitutive production of proinflmmatory cytokines interleukin (IL)-1alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and KC (the murine homologue of chemokine Gro-alpha). The basis for constitutive expression of these cytokines after tumor progression in vivo is unknown. Regulation of the expression of these proinflammatory cytokines involves transcription factor nuclear factor kappaB (NF-kappaB), which can be activated by cytokines such as tumor necrosis factor (TNF)-alpha. In this study, we compared the constitutive and TNF-alpha-induced expression of proinflammatory cytokines in parental Pam 212 and metastatic LY-2 and LY-8 cell lines and determined the relationship of cytokine expression to activation of NF-kappaB. We found that the metastatic cell lines exhibited an increase in constitutive and TNF-alpha-inducible expression of proinflammatory cytokines when compared with parental Pam 212 cells. The increased cytokine expression was associated with an increase in constitutive and TNF-alpha-inducible activation of NF-kappaB as demonstrated by electrophoretic mobility shift assay and luciferase-reporter gene assay. Constitutive nuclear localization of NF-kappaB p65 was observed in LY-2 and LY-8 cells in culture and in tumor specimens but rarely in Pam 212 cells, consistent with the constitutive activation of NF-kappaB in tumor cels after selection in vivo. Induction of NF-kappaB by TNF-alpha was inhibited by the addition of protease inhibitors calpain inhibitor I and N-tosyl-phechloromethyl ketone and antioxidant 1-pyrrolidinecarbodithioic acid, whereas constitutive activation of NF-kappaB and cytokine KC mRNA expression was inhibited by N-tosyl-phechloromethyl ketone alone. Overexpression of a human Ikappa(B)alpha dominant suppresser in Pam 212 cells inhibited TNF-alpha-induced NF-kappaB binding activity and KC expression. These data indicate that activation of NF-kappaB contributes to increased expression of proinflammatory cytokines during metastatic tumor progression of squamous cell carcinoma, and that distinct mechanisms may be involved in the regulation of constitutive and TNF-alpha-induced activation of NF-kappaB in squamous cell carcinoma.

We studied the effects of fragile histidine triad (FHIT) gene overexpression mediated by an adenoviral vector, Ad-FHIT, on cell proliferation, apoptosis, and cell cycle kinetics in human cancer cells and on tumorigenicity and tumor growth in nude mice. Overexpression of the FHIT gene significantly inhibited cell growth in various Ad-FHIT-transduced human lung cancer cells and head and neck carcinoma cells with FHIT gene abnormalities, but not in normal human bronchial epithelial cells. Fewer than 20% of cells in all Ad-FHIT-transduced cells survived at 7 days after transduction. Overexpression of the FHIT gene induced cell apoptosis and altered cell cycle processes. The apoptotic cell population markedly increased, and cells accumulated in S phase after Ad-FHIT transduction. The tumorigenicity of human H1299 lung cancer cells transduced by Ad-FHIT, in comparison with that of the control transductants and untreated cells, was eliminated in vivo. Subcutaneous tumor growth in nude mice who received intratumoral injections of Ad-FHIT, at a total dose of 3 x 10(10) plaque-forming units/tumor for H1299 tumors and 4 x 10(10)/tumor for A549 tumors, were suppressed by more than 85% and 90%, respectively, compared with that in nude mice who received injections of empty vector at the same dose or with PBS alone. Together, our results suggest that the FHIT gene, when delivered at high efficiency by a recombinant adenoviral vector, functions as a tumor suppressor gene both in vitro and in vivo.

Human chromosome 3p cytogenetic abnormalities and loss of heterozygosity have been observed at high frequency in the nonpapillary form of sporadic renal cell carcinoma (RCC). The von Hippel-Lindau (VHL) gene has been identified as a tumor suppressor gene for RCC at 3p25, and functional studies as well as molecular genetic and cytogenetic analyses have suggested as many as two or three additional regions of 3p that could harbor tumor suppressor genes for sporadic RCC. We have previously functionally defined a novel genetic locus nonpapillary renal carcinoma-1 (NRC-1) within chromosome 3p12, distinct from the VHL gene, that mediates tumor suppression and rapid cell death of RCC cells in vivo. We now report the suppression of tumorigenicity of RCC cells in vivo after the transfer of a defined centric 3p fragment into different histological types of RCC. Results document the functional involvement of NRC-1 in not only different cell types of RCC (i.e., clear cell, mixed granular cell/clear cell, and sarcomatoid types) but also in papillary RCC, a less frequent histological type of RCC for which chromosome 3p LOH and genetic aberrations have only rarely been observed. We also report that the tumor suppression observed in functional genetic screens was independent of the microenvironment of the tumor, further supporting a role for NRC-1 as a more general mediator of in vivo growth control. Furthermore, this report demonstrates the first functional evidence for a VHL-independent pathway to tumorigenesis in the kidney via the genetic locus NRC-1.

Potent immune responses against malignant brain tumors can be elicited by paracrine intracranial (i.c.) immunotherapy with interleukin (IL)-2. Additionally, i.c. delivery of carmustine via biodegradable polymers has been shown to significantly prolong survival in both animal models and clinical trials. In this study, we show that the combination of paracrine immunotherapy, with nonreplicating genetically engineered tumor cells that produce IL-2, and local delivery of chemotherapy by biodegradable polymers prolongs survival in a synergistic manner in mice challenged intracranially with a lethal murine brain tumor. Animals receiving IL-2-transduced cells and polymers containing 10% 1,3-bis(2-chloroethyl)-1-nitrosourea had significantly improved survival compared with animals receiving IL-2-transduced cells or 10% 1,3-bis(2-chloroethyl)-1-nitrosourea alone. Median survival for the control group was 19 days. Survival in animals receiving IL-2-transduced cells and 1% carboplatin-containing polymers was also significantly improved compared with either therapy alone. Histopathological examination on day 14 of animals receiving combination treatment showed rare degenerating tumor cells. In addition to tissue necrosis surrounding the polymer, a marked inflammatory reaction was observed. In long-term survivors (all animals receiving combination treatment), no tumor was observed and the inflammatory reaction was completely resolved. The brains of animals receiving combination therapy showed both tissue necrosis due to local chemotherapy and strong inflammation due to paracrine immunotherapy. The demonstration of synergy between paracrine IL-2 and local i.c. delivery of antineoplastic drugs is novel and may provide a combined treatment strategy for use against both primary and metastatic i.c. tumors.

We have examined the effect of synthetic low molecular weight glycoamine analogues on the metastasis of MDA-MB-435 human breast carcinoma xenografts growing in the mammary fat pads of nude mice. Initial in vitro screening of a panel of synthetic glycoamines was performed using a clonogenic growth assay in 0.9% agarose. Eight of nine compounds manifested a significant dose-dependent inhibition of colony formation by MDA-MB-435 cells in 0.9% agarose. The relative activity ranks of the compounds, based on ID50S independently determined for each synthetic glycoamine analogue, identified N-(1-deoxy-D-lactulos-1-yl)-L-leucine (Lac-L-Leu), N-(1-deoxy-D-fructos-1-yl)-D-leucine (Fru-D-Leu), N-(1-deoxy-D-fructos-1-yl)-L-phenylalanine, and N-(1-deoxy-D-fructos-1-yl)-L-leucine as the most effective inhibitors of colony formation. Two separate experimental treatment protocols were used to examine the effect of selected synthetic glycoamines on human breast cancer growth and metastasis in athymic nude mice. Group A mice were treated intraperitoneally daily from day 2 after injection of the breast cancer cells until the end of the experiment (17 weeks). In group B, the mice were untreated until the mean tumor diameter was 10 mm, at which time daily i.p. treatment began. After 7 days, the primary tumors were resected, and the mice were treated for an additional 4 weeks (a total of 5 weeks of treatment). The synthetic glycoamines did not have significant antitumor effects, and there was no difference in the tumor incidence or tumor growth rates in mice treated continuously with synthetic glycoamines or PBS. The significant antimetastatic activity of synthetic glycoamines was detected in both experimental treatment protocols. In mice continuously treated with synthetic glycoamines according to protocol A, the incidence of metastasis was decreased 4.6-fold (P = 0.014) and 2.7-fold (P = 0.031) in mice treated with Fru-D-Leu and Lac-L-Leu, respectively. In mice in protocol B, the incidence of pulmonary metastasis was decreased 1.9-fold (P = 0.069) and 2.5-fold (P = 0.042) in mice treated with Fru-D-Leu and Lac-L-Leu, respectively. Correspondingly, the average number of spontaneous pulmonary metastases was reduced from 37 in control mice to 0.2 (P = 0.005) and 0.9 (P < 0.02) in mice treated according to the protocol A with Fru-D-Leu and Lac-L-Leu, respectively. Treatment of mice with N-(1-deoxy-D-fructos-1-yl)-L-leucine did not have significant antimetastatic effects, and no reduction in metastasis incidence or number was noted in mice treated with this synthetic glycoamine analogue. The treated animals had no apparent toxicity from chronic daily injection (up to 17 weeks of treatment) of synthetic glycoamines, and no obvious pathology was noted in the histological slides of the livers, kidneys, or spleens of the treated mice. Therefore, we have identified two synthetic glycoamines (Fru-D-Leu and Lac-L-Leu) that were the effective inhibitors of spontaneous human breast cancer metastasis in nude mice. Potential mechanisms for antimetastatic activity of synthetic glycoamines may include the inhibition of beta-galectin-mediated homotypic cancer cell aggregation and induction of apoptosis in target cells.