Preclinical validation of Aurora kinases-targeting drugs in osteosarcoma.

BACKGROUND: Aurora kinases are key regulators of cell cycle and represent new promising therapeutic targets in several human tumours. METHODS: Biological relevance of Aurora kinase-A and -B was assessed on osteosarcoma clinical samples and by silencing these genes with specific siRNA in three human osteosarcoma cell lines. In vitro efficacy of two Aurora kinases-targeting drugs (VX-680 and ZM447439) was evaluated on a panel of four drug-sensitive and six drug-resistant human osteosarcoma cell lines. RESULTS: Human osteosarcoma cell lines proved to be highly sensitive to both drugs. A decreased drug sensitivity was observed in doxorubicin-resistant cell lines, most probably related to ABCB1/MDR1 overexpression. Both drugs variably induced hyperploidy and apoptosis in the majority of cell lines. VX-680 also reduced in vitro cell motility and soft-agar cloning efficiency. Drug association experiments showed that VX-680 positively interacts with all conventional drugs used in osteosarcoma chemotherapy, overcoming the cross-resistance observed in the single-drug treatments. CONCLUSION: Aurora kinase-A and -B represent new candidate therapeutic targets for osteosarcoma. In vitro analysis of the Aurora kinases inhibitors VX-680 and ZM447439 indicated in VX-680 a new promising drug of potential clinical usefulness in association with conventional osteosarcoma chemotherapeutic agents.

Human responses against HER-2-positive cancer cells in human immune system-engrafted mice.

BACKGROUND: Human immune system (HIS)-engrafted mice are new tools to investigate human immune responses. Here, we used HIS mice to study human immune responses against human HER-2-positive cancer cells and their ability to control tumour growth and metastasis. METHODS: BALB/c Rag2(-/-), Il2rg(-/-) mice were engrafted with CD34(+) or CD133(+) human cord blood hematopoietic stem cells (HSC) and vaccinated with human HER-2-positive cancer cells SK-OV-3 combined to human IL-12. RESULTS: Both CD34(+) or CD133(+) human HSC gave long-term engraftment and differentiation, both in peripheral blood and in lymphoid organs, and production of human antibodies. Vaccinated mice produced specific anti-HER-2 human IgG. An s.c. SK-OV-3 challenge was significantly inhibited (but not abolished) in both vaccinated and non-vaccinated HIS mice. Tumours were heavily infiltrated with human and murine cells, mice showed NK cells and production of human interferon-γ, that could contribute to tumour growth inhibition. Vaccinated HIS mice showed significantly inhibited lung metastases when compared with non-vaccinated HIS mice and to non-HIS mice, along with higher levels of tumour-infiltrating human dendritic cells. CONCLUSION: Anti-HER-2 responses were elicited through an adjuvanted allogeneic cancer cell vaccine in HIS mice. Human immune responses elicited in HIS mice effectively inhibited lung metastases.

Advances in preclinical therapeutics development using small animal imaging and molecular analyses: the gastrointestinal stromal tumors model.

The large use of target therapies in the treatment of gastrointestinal stromal tumors (GISTs) highlighted the urgency to integrate new molecular imaging technologies, to develop new criteria for tumor response evaluation and to reach a more comprehensive definition of the molecular target. These aspects, which come from clinical experiences, are not considered enough in preclinical research studies which aim to evaluate the efficacy of new drugs or new combination of drugs with molecular target. We developed a xenograft animal model GIST882 using nude mice. We evaluated both the molecular and functional characterization of the tumor mass. The mutational analysis of KIT receptor of the GIST882 cell lines and tumor mass showed a mutation on exon 13 that was still present after in vivo cell growth. The glucose metabolism and cell proliferation was evaluated with a small animal PET using both FDG and FLT. The experimental development of new therapies for GIST treatment requires sophisticated animal models in order to represent the tumor molecular heterogeneity already demonstrated in the clinical setting and in order to evaluate the efficacy of the treatment also considering the inhibition of tumor metabolism, and not only considering the change in size of tumors. This approach of cancer research on GISTs is crucial and essential for innovative perspectives that could cross over to other types of cancer.

A multi-DNA preventive vaccine for p53/Neu-driven cancer syndrome.

The highly aggressive cancer syndrome of female mice carrying a p53 knockout allele and a rat HER-2/neu (Neu) transgene (BALB-p53Neu) can be prevented by a cell vaccine presenting three components: Neu, interleukin (IL)-12 production, and allogeneic major histocompatibility complex (MHC) alleles (Triplex cell vaccine). Here we tested a second-generation Triplex DNA-based vaccine (Tri-DNA), consisting of the combination of three gene components (a transmembrane-extracellular domain fragment of the Neu gene, IL-12 genes, and the H-2D(q) allogeneic MHC gene), carried by separate plasmids. The Tri-DNA vaccine was at least as effective as the Triplex cell vaccine for cancer immunoprevention, giving a similar delay in the onset of mammary cancer and complete protection from salivary cancer. Both vaccines induced anti-Neu antibodies of the murine IgG2a isotype at similar levels. The Tri-DNA vaccine gave more restricted immunostimulation, consisting of a fully helper T cell type 1 (Th1)-polarized response, with effective production of interferon (IFN)-gamma in response to the vaccine but no spontaneous production, and no induction of anti-Neu IgG3 antibodies. On the other hand, the Triplex cell vaccine induced both Th1 and Th2 cytokines, a strong increase in spontaneous IFN-gamma production, and high levels of IgG3 antibodies recognizing Neu-positive syngeneic cells. In conclusion, the Tri-DNA vaccine is as effective as Triplex cell vaccine, exploiting a more restricted immune stimulation.

Experimental results and related clinical implications of PET detection of epidermal growth factor receptor (EGFr) in cancer.

The epidermal growth factor receptor (EGFr) is one of the most studied molecules as a target for cancer therapy. Over these last few years, several studies attempting to identify predictive biomarkers of treatment response, such as the receptor status or other molecules related to the downstream signalling pathway, have been conducted. However, from a clinical point of view, the information obtained from ex vivo analyses still has various limitations that may be overcome by the combination with molecular imaging technologies which may provide a noninvasive, global, in vivo evaluation of the molecular tumour background. The aim of this review is to report the preclinical results of all positron emission tomography (PET) tracers synthesized until now for in vivo detection of EGFr in cancer. Two classes of PET compounds have been developed: labelled small molecules such as tyrosine kinase inhibitors and labelled monoclonal antibodies. The in vitro and in vivo results of these PET tracers are very different depending on the chemical properties, positron emission radionuclide, or animal models. As a consequence, various critical questions are still open, and the implications of a translation in the clinical setting for EGFr imaging in cancer patients is discussed.

Toward the definition of immunosuppressive regimens with antitumor activity.

Immunosuppressive therapies associated with organ transplantation produce an increased risk of cancer development. Malignancies are increased in transplant recipients because of the impaired immune system. Moreover, experimental data point to a tumor-promoting activity of various immunosuppressive agents. In this study, we compared the effects of 4 immunosuppressive agents with different mechanisms of action (cyclosporine, rapamycin, mycophenolic acid, and leflunomide) on the in vitro growth of various tumor cell lines and umbilical vein endothelial cells. To varying degrees rapamycin (10 ng/mL), mycophenolic acid (300 nmol/L), and leflunomide (30 micromol/L) highly inhibited the growth of human rhabdomyosarcoma, hepatocellular carcinoma, colorectal carcinoma, and endothelial cells. In contrast, cyclosporine (100 ng/mL) did not affect their growth. Our data suggest that regimens containing rapamycin, mycophenolic acid, or leflunomide, which have both immunosuppressive and antitumor activities, should be preferred in transplant recipients to minimize the risk of tumors.

New target antigens for cancer immunoprevention.

Prevention of cancer through the activation of the immune system has been explored in recent years in preclinical systems thanks to the availability of several new transgenic mouse models that closely mimic the natural history of human tumors. The most thoroughly investigated model of cancer immunoprevention is the mammary carcinoma of HER-2/neu transgenic mouse. In this system it has clearly been shown that the activation of immune defences in healthy individuals can effectively prevent the subsequent onset of highly aggressive mammary carcinomas. A complete prevention was obtained using a combination of three signals (the so called "triplex" vaccine) that included the specific antigen (p185, the product of HER-2/neu) and nonspecific signals like allogeneic histocompatibility antigens and interleukin 12. The analysis of protective immune responses in models of cancer immunoprevention revealed some unexpected features, in particular the central role of antibodies in immunoprevention, at variance with conventional immuno-therapy which is firmly based on cytotoxic T cells. In the HER-2/neu system anti-p185 antibodies, in addition to immunological functions leading to tumor cell lysis, inhibit p185 dimerization and induce its internalization, resulting in the inhibition of mitogenic signaling. Most current tumor antigens appear to be unsuitable targets for cancer immunoprevention. An ideal antigen should have a crucial pathogenetic role in tumor growth to avoid the selection of antigen loss variants. Downregulation of major histocompatibility complex (MHC) expression during tumor progression frequently limits antigen recognition by MHC-restricted T cells. Thus an ideal antigen for cancer immunoprevention should be recognized both by T cells and by antibodies. Antibody binding to cell surface oncogenic determinants, in addition to complement- and cell-mediated tumor cell lysis, can block mitogenic signaling and induce internalization, resulting in tumor growth arrest. A search for new tumor antigens should be conducted among molecules that are directly involved in neoplastic transformation and are recognizable by the immune response also in MHC loss variants. Novel tumor antigens fulfilling both conditions will be crucial for the development of cancer immunoprevention and will provide new targets also for cancer immunotherapy.

Combined allogeneic tumor cell vaccination and systemic interleukin 12 prevents mammary carcinogenesis in HER-2/neu transgenic mice.

Transgenic Balb/c mice expressing the transforming rat HER-2/neu oncogene develop early and multifocal mammary carcinomas. Within the first 5 months of life the tissue-specific expression of HER-2/neu causes a progression in all their 10 mammary glands from atypical hyperplasia to invasive carcinoma. It was previously observed that chronic administration of interleukin (IL)-12 increased tumor latency, but every mouse eventually succumbed to multiple carcinomas. A significant improvement in tumor prevention was sought by administering allogeneic mammary carcinoma cells expressing HER-2/neu combined with systemic IL-12. This treatment reduced tumor incidence by 90% and more than doubled mouse lifetime. For the maximum prevention p185(neu) antigen must be expressed by allogeneic cells. IL-12 treatment strongly increased the cell vaccine efficacy. The mammary glands of mice receiving the combined treatment displayed a markedly reduced epithelial cell proliferation, angiogenesis, and HER-2/neu expression, while the few hyperplastic foci were heavily infiltrated by granulocytes, macrophages, and CD8(+) lymphocytes. Specific anti-HER-2/neu antibodies were produced and a nonpolarized activation of CD4(+) and CD8(+) cells secreting IL-4 and interferon (IFN)-gamma were evident. A central role for IFN-gamma in the preventive effect was proven by the lack of efficacy of vaccination in IFN-gamma gene knockout HER-2/neu transgenic Balb/c mice. A possible requirement for IFN-gamma is related to its effect on antibody production, in particular on IgG2a and IgG2b subclasses, that were not induced in IFN-gamma knockout HER-2/neu mice. In conclusion, our data show that an allogeneic HER-2/neu-expressing cell vaccine combined with IL-12 systemic treatment can prevent the onset of genetically determined tumors.

Identification of new genes related to the myogenic differentiation arrest of human rhabdomyosarcoma cells.

Rhabdomyosarcoma is a soft tissue tumor committed to the myogenic lineage but arrested prior to terminal differentiation. To identify new genes implicated in the block in myogenic differentiation of rhabdomyosarcoma cells and those responsible for their proceeding along the myogenic pathway we used cDNA microarrays to compare gene expression profiles of two clones of the human embryonal rhabdomyosarcoma cell line RD with different myogenic potentials: RD/12, which is unable to differentiate, and RD/18, which shows elements able to terminally differentiate, as defined by the expression of myosin heavy chain (up to 50% of the population) and the formation of multinucleated myotube-like structures. We identified 80 genes differentially expressed by the two clones. Differentiating RD/18 cells overexpressed the myogenic transcription factor myogenin along with known myogenic markers; myogenin transfection into undifferentiated RD/12 cells was able to revert the phenotype giving rise to 94% of clones displaying a differentiated morphology. RD/18 cells also expressed several genes not known to be expressed in rhabdomyosarcoma or muscle cells, such as pigment-epithelium derived factor and endothelin-3. Poorly differentiated RD/12 cells, along with genes related to mesenchymal lineage or early myogenic commitment, also expressed genes not previously known to be related to the differentiation block of human rhabdomyosarcoma, such as monocyte chemotactic protein-1, connective tissue growth factor and insulin-like growth factor binding protein-5. Differential expression of these genes in a time course of differentiation suggested their potential roles as either new myogenic markers or repressors of differentiation. These results identify a cluster of new genes related to the aberrant myogenic differentiation program of human rhabdomyosarcoma cells.

An anti-apoptotic role for NGF receptors in human rhabdomyosarcoma.

The expression and biological function of Nerve Growth Factor (NGF) receptors was studied in a panel of rhabdomyosarcoma cell lines derived from embryonal and alveolar histotype. All the cell lines expressed both the high affinity receptor TrkA and the low affinity receptor p75(NTR). Treatment with exogenous NGF did not considerably alter rhabdomyosarcoma cell growth or differentiation, but significantly inhibited spontaneous apoptosis as well as apoptosis, and induced by serum starvation or apoptosis induced by treatment with cycloheximide (CHX). Rhabdomyosarcoma cell lines expressed NGF and other neurotrophins and trace amounts of NGF protein were found in the supernatants of rhabdomyosarcoma cell cultures. Blocking the putative autocrine loop with an anti-NGF antibody resulted in an increase in apoptosis compared with control cultures. These data suggest that the simultaneous presence of both high and low affinity NGF receptors engaged by endogenous or exogenous NGF might contribute to the escape from apoptosis exhibited by the rhabdomyosarcoma cells.

Therapy of lung metastases through combined vaccination with carcinoma cells engineered to release IL-13 and IFN-gamma.

TS/A spontaneous mouse mammary adenocarcinoma cells were engineered to release interferon-gamma (IFN-gamma), a Th1 cytokine (TS/A-IFNgamma) and interleukin-13 (IL-13), a Th2 cytokine (TS/A-IL13). Mice bearing lung micrometastases induced by parental TS/A cells received repeated subcutaneous vaccinations with TS/A-IFN-gamma admixed with TS/A-IL13 engineered cells. This combined treatment cured up to 75% of mice, whereas vaccinations with either TS/A-IFNgamma or TS/A-IL13 alone cured only 20-40% of mice. Combined TS/A-IL13 and TS/A-IFNgamma therapeutic vaccinations elicited a reactive infiltrate of CD4+ and CD8+ lymphocytes in lung metastases and an increased production of IFN-gamma in the spleen and lung, suggesting a shift of the immune response toward the Th1 type. The type of infiltrating cells along with the lack of efficacy in T cell-deficient mice point to a major role of T cells. In conclusion, no antagonism but a synergistic and effective definitive cure stems from the combined vaccination with tumor cells engineered to release a Th1 and a Th2 cytokine.

The metastatic ability of Ewing's sarcoma cells is modulated by stem cell factor and by its receptor c-kit.

Ewing's sarcoma is a primitive highly malignant tumor of bone and soft tissues usually metastasizing to bone, bone marrow, and lung. Growth factor receptors and their ligands may be involved in its growth and dissemination. We analyzed the expression of c-kit and its ligand stem cell factor (SCF) in a panel of six Ewing's sarcoma cell lines. All cell lines exhibited substantial levels of surface c-kit expression, and five of six displayed transmembrane SCF on the cell surface. Expression of c-kit was down-modulated in all lines by exposure to exogenous SCF. The SCF treatment was able to confer to cells a growth advantage in vitro, due both to an increase in cell proliferation and to a reduction in the apoptotic rate. When used in the lower compartment of a migration chamber, SCF acted as a strong chemoattractant for Ewing's sarcoma cells. The pretreatment of cells with SCF reduced their chemotactic response to SCF. In athymic nude mice, Ewing's sarcoma cells injected intravenously metastasized to the lung and to a variety of extrapulmonary sites, including bone and bone marrow. Metastatic sites resembled those observed in Ewing's sarcoma patients and corresponded to SCF-rich microenvironments. The in vitro pretreatment of cells with SCF strongly reduced the metastatic ability of Ewing's sarcoma cells, both to the lung and to extrapulmonary sites. This could be dependent on the down-modulation of c-kit expression observed in SCF-pretreated cells, leading to a reduced sensitivity to the chemotactic and proliferative actions of SCF. Our results indicate that the response to SCF mediated by c-kit may be involved in growth, migration, and metastatic ability of Ewing's sarcoma cells.

DNA vaccination against rat her-2/Neu p185 more effectively inhibits carcinogenesis than transplantable carcinomas in transgenic BALB/c mice.

The ability of vaccination with plasmids coding for the extracellular and the transmembrane domain of the product of transforming rat Her-2/neu oncogene (r-p185) to protect against r-p185(+) transplantable carcinoma (TUBO) cells and mammary carcinogenesis was evaluated. In normal BALB/c mice, DNA vaccination elicits anti-r-p185 Ab, but only a marginal CTL reactivity, and protects against a TUBO cell challenge. Massive reactive infiltration is associated with TUBO cell rejection. In BALB/c mice transgenic for the rat Her-2/neu gene (BALB-neuT), DNA vaccination elicits a lower anti-r-p185 Ab response, no CTL activity and only incompletely protects against TUBO cells, but markedly hampers the progression of carcinogenesis. At 33 wk of age, when control BALB-neuT mice display palpable tumors in all mammary glands, about 60% of immunized mice are tumor free, and tumor multiplicity is markedly reduced. Tumor-free mammary glands still display the atypical hyperplasia of the early stages of carcinogenesis, and a marked down-modulation of r-p185, along with a massive reactive infiltrate. However, BALB-neuT mice protected against mammary carcinogenesis fail to efficiently reject a TUBO cell challenge. This suggests that the mechanisms required for the rejection of transplantable tumors may not coincide with those that inhibit the slow progression of carcinogenesis.

Expression of HER/erbB family of receptor tyrosine kinases and induction of differentiation by glial growth factor 2 in human rhabdomyosarcoma cells.

Five human rhabdomyosarcoma cell lines were used to investigate the surface expression of HER/erbB receptors, particularly of HER-2, HER-3 and HER-4, by flow cytometry. HER-2 was expressed in 3/5 rhabdomyosarcoma cell lines. HER-3 was expressed in all rhabdomyosarcoma cell lines. None of the rhabdomyosarcoma cell lines investigated showed HER-4 surface expression. To study the biological activity of HER/erbB receptors in rhabdomyosarcomas, cells were cultured in the presence of glial growth factor 2 (GGF-2), a specific ligand of HER-3 that is able to stimulate myogenesis in normal myoblasts. In 3 of 3 human rhabdomyosarcoma cell lines positive for both HER-2 and HER-3 receptors, the treatment with GGF-2 induced a significant increase in myogenic differentiation.

p185(neu) protein is required for tumor and anchorage-independent growth, not for cell proliferation of transgenic mammary carcinoma.

Transgenic FVB-NeuN mice (N202) bearing the rat neu protooncogene driven by the mouse mammary tumor virus promoter/enhancer develop focal mammary carcinomas overexpressing the neu-encoded p185(neu) protein. In vitro expression of p185(neu) among mammary carcinoma cultures was heterogeneous, and we could establish some cell lines and clones displaying a complete loss of p185(neu) expression, along with others with very high p185(neu) protein level. Upon in vivo injection, p185(neu)-positive cells gave rise to fast-growing tumors with a short latency, while p185(neu)-negative cells required a very long latency time, and the resulting tumors were invariably p185(neu)-positive. The lower growth ability of p185(neu)-negative cells in vivo was also confirmed in athymic nude mice. In vitro, analysis of anchorage-independent growth in soft agar revealed colony formation from p185(neu)-positive but not p185(neu)-negative cells. The direct involvement of p185(neu) in clonogenicity was demonstrated by the inhibition of p185(neu)-positive colony growth in soft agar in the presence of an anti-p185(neu) monoclonal antibody. By contrast, a higher level of anchorage-dependent clonogenic growth and proliferation was observed in p185(neu)-negative cells as compared to p185(neu)-positive cells, thus explaining the relative ease with which p185(neu)-negative cell lines and clones were established in vitro. Together, the results indicate that p185(neu) expression can lead to tumor formation and metastasis through the modification of intrinsic properties of cells related to anchorage-independent growth ability rather than to proliferation or host-dependent mechanisms.

Murine model for skeletal metastases of Ewing's sarcoma.

Ewing's sarcoma shows a strong tendency to metastasize to the lungs or the skeleton, or both. A peculiar feature of the secondary involvement of bone with this tumor is that it may also appear in the absence of clinically evident lung metastases, both at clinical presentation and during the course of the disease. Although osseous metastases are critically relevant for prognosis, the pathogenesis of this peculiar feature of Ewing's sarcoma is poorly understood, partly due to the lack of appropriate experimental in vivo models. We show that the intravenous injection of TC-71 Ewing's sarcoma cells into athymic 4-5-week-old Crl/nu/nu (CD1) BR mice reproducibly colonizes specific sites of the skeleton in addition to the lungs and lymph nodes. The distribution and the morphologic appearance of these experimental bone metastases mimic the pattern of skeletal involvement observed in humans. This experimental model of bone metastasis of Ewing's sarcoma may be the basis for future studies aimed at understanding the pathophysiology and treatment of Ewing's sarcoma.

Wild-type p53-mediated down-modulation of interleukin 15 and interleukin 15 receptors in human rhabdomyosarcoma cells.

We recently reported that rhabdomyosarcoma cell lines express and secrete interleukin 15 (IL-15), a tightly regulated cytokine with IL-2-like activity. To test whether the p53-impaired function that is frequently found in this tumour type could play a role in the IL-15 production, wild-type p53 gene was transduced in the human rhabdomyosarcoma cell line RD (which harbours a mutated p53 gene), and its effect on proliferation and expression of IL-15 was studied. Arrest of proliferation was induced by wild-type p53; increased proportions of G1-arrested cells and of apoptotic cells were observed. A marked down-modulation of IL-15 expression, at both the mRNA and protein level, was found in p53-transduced cells. Because a direct effect of IL-15 on normal muscle cells has been reported, the presence of IL-15 membrane receptors was studied by cytofluorometric analysis. Rhabdomyosarcoma cells showed IL-15 membrane receptors, which are down-modulated by wild-type p53 transfected gene. In conclusion, wild-type p53 transduction in human rhabdomyosarcoma cells induces the down-modulation of both IL-15 production and IL-15 receptor expression.

Production of stem cell factor and expression of c-kit in human rhabdomyosarcoma cells: lack of autocrine growth modulation.

Human rhabdomyosarcoma cells produce autocrine and paracrine growth factors that can sustain their growth and malignancy. Here we report constitutive production of stem cell factor (SCF) by 5 of 5 human rhabdomyosarcoma cell lines both of alveolar and embryonal histotype. SCF production, ranging from 30 to 162 pg/ml, was independent from the degree of myogenic differentiation and was not modulated by exogenous addition of retinoic acid (RA) or tumor necrosis factor-alpha (TNF-alpha). Four of 5 rhabdomyosarcoma cell lines expressed the mRNA for SCF receptor c-kit, while the 5th cell line became weakly positive for c-kit mRNA only after stimulation with retinoic acid. On the cell surface, c-kit protein was detectable at very low levels in only 1 of 5 rhabdomyosarcoma cell lines and was not up-regulated by RA or TNF-alpha. Addition of anti-c-kit and anti-SCF blocking antibodies, or of exogenous SCF did not alter the in vitro growth ability of rhabdomyosarcoma cells. In conclusion, our data show that rhabdomyosarcoma cells produce consistent amounts of SCF but did not demonstrate autocrine growth modulation. SCF secretion may thus have a paracrine, rather than an autocrine activity in this tumor.

Blockage of insulin-like growth factor-I receptor inhibits the growth of Ewing's sarcoma in athymic mice.

Innovative, more effective treatment modalities are needed for Ewing's sarcoma (ES), a neoplasm with a disappointingly low survival rate despite the use of aggressive multimodal therapeutic approaches. We have previously shown (K. Scotlandi et al, Cancer Res., 56: 4570-4574, 1996) the existence and the pathogenetic relevance of an autocrine loop that is mediated by the insulin-like growth factor-I receptor (IGF-IR) and is crucial for the survival and proliferation of ES cells in vitro. In this study, we report that the IGF-IR-blocking monoclonal antibody alphaIR3 may also significantly inhibit ES cell growth in vivo. In particular, in almost one-half of the animals tested, after s.c. inoculation with TC-71 ES cells, the blockage of IGF-IR by alphaIR3 induced a complete regression of tumors that developed, which suggests that IGF-IR is valuable as a specific target for novel therapeutic strategies. In addition, suramin, a drug that can interfere with growth factor binding with their receptors, inhibited the tumorigenic and the metastatic ability of TC-71 cells and, therefore, is a promising agent to be combined with conventional cytotoxic drugs for the design of more effective therapeutic regimens.

Interleukin 12-mediated prevention of spontaneous mammary adenocarcinomas in two lines of Her-2/neu transgenic mice.

The ability of interleukin (IL)-12 to prevent tumors when administered to individuals with a genetic risk of cancer was studied in two lines of transgenic mice expressing rat HER-2/neu oncogene in the mammary gland. Female BALB/c (H-2(d)) mice carrying the activated HER-2/ neu oncogene show no morphological abnormalities of the mammary gland until 3 wk of age. They then progress through atypical hyperplasia to in situ lobular carcinoma and at 33 wk of age all 10 mammary glands display invasive carcinomas. Adult FVB mice (H-2(q)) carrying the HER-2/neu protooncogene develop mammary carcinomas with a longer latency (38-49 wk) and a lower multiplicity (mean of 2.6 tumors/mice). Treatment with IL-12 (5 daily intraperitoneal injections, 1 wk on, 3 wk off; the first course with 50 ng IL-12/day, the second with 100 ng IL-12/day) begun at 2 wk of age in BALB/c mice and at 21 wk of age in FVB mice markedly delayed tumor onset and reduced tumor multiplicity. Analogous results were obtained in immunocompetent and permanently CD8(+) T lymphocyte-depleted mice. In both transgenic lines, tumor inhibition was associated with mammary infiltration of reactive cells, production of cytokines and inducible nitric oxide synthase, and reduction in microvessel number, in combination with a high degree of hemorrhagic necrosis.