Transfection of the genes for interleukin-12 into the K1735 melanoma and the EMT6 mammary sarcoma murine cell lines reveals distinct mechanisms of antitumor activity

Interleukin 12 (IL-12) is a pleiotropic cytokine with multiple effects on the immune system. The antitumor effects of locally produced IL-12 were examined in 2 tumor model systems. IL-12 expressing EMT6 mammary sarcomas (EMT6/IL-12) grew temporarily and then regressed resulting in mice that were immune to a further challenge of EMT6 cells. Interestingly, the IL-12 expressing K1735 melanomas (K1735/IL-12) maintained a lag phase of nonmeasurable growth for several weeks, followed by tumor outgrowth that was associated with a loss of IL-12 production. Tumor-infiltrating lymphocytes (TILs) isolated from EMT6/IL-12 tumors effectively lysed EMT6 target cells, whereas K1735/IL-12 TILs lacked lytic activity. Both IL-12 expressing tumors, however, grew progressively in nude mice indicating an important role for T cells in each case. Recombinant murine interferon gamma (rmIFN-gamma) inhibited the growth of EMT6 cells, but not K1735 cells in vitro, and strongly induced the expression of the antiangiogenic chemokine interferon-inducible protein 10 (IP-10) by both cell lines. Of interest, only the EMT6 cell line was able to secrete the proangiogenic molecule, vascular endothelial growth factor (VEGF), in response to low oxygen conditions. Fluorescent staining of the vascular endothelium at the tumor injection site provided images depicting early stages of angiogenesis prior to K1735/IL-12 tumor outgrowth. These results indicate that locally produced IL-12 likely mediates the rejection of EMT6 tumors through tumor cell lysis by host immune cells, whereas its antiangiogenic potential may be counterbalanced by the strong induction of VEGF by hypoxic tumor cells. In contrast, IL-12 does not induce protective immunity to K1735 tumors. However, an antiangiogenic mechanism may be responsible for controlling tumor growth.     

Disparate effects of endostatin on tumor vascular perfusion and hypoxia in two murine mammary carcinomas

PURPOSE: Recent results in the literature have demonstrated that the antiangiogenic agent endostatin can enhance antitumor effects when administered before or during radiotherapy. To better understand the underlying pathophysiologic basis for this radiosensitization, the current study investigated whether short-term endostatin administration is linked to alterations in tumor vascular perfusion and oxygen delivery. METHODS AND MATERIALS: Three daily doses of recombinant endostatin (20 mg/kg) were administered to two murine mammary carcinomas, the highly vascularized MCa-35 and the less vascularized MCa-4. Image analysis techniques were used to quantify (1) total and perfused vascular spacing, and (2) changes in tumor hypoxia as a function of distance from the nearest blood vessel. RESULTS: In MCa-35 tumors, endostatin had no effect on vessel spacing, tumor hypoxia, or tumor growth. In MCa-4 tumors, total and perfused vessel spacings were also unchanged, but tumor growth was inhibited, and tumor hypoxia significantly decreased. These tumors demonstrated an increased vascular functionality suggestive of an increase in the number of intermittently perfused vessels, without corresponding alterations in tumor oxygen consumption rate. CONCLUSIONS: Poorly vascularized, hypoxic mammary carcinomas were much more responsive to short-term endostatin treatment than well-vascularized, more homogeneously oxygenated tumors. Oxygen levels in the responsive tumors were transiently improved after treatment, which could have substantial implications with respect to the therapeutic effectiveness of combining antiangiogenic agents with conventional therapies.     

Nonspecific T-cell reactivity in mice bearing autochthonous tumors or early-generation transplanted spontaneous mammary tumors.

The mitogenic response to phytohemagglutinin (PHA) by spleen cells from C3H/HeJ mice bearing either autochthonous tumors or early-generation transplanted spontaneous mammary tumors was depressed in some, but not all, tumor-bearing animals. T-cell hyporesponsiveness in both groups was associated with the larger (more rapidly growing) tumor burdens. The growth patterns of the transplanted tumors and the associated PHA-induced responses were not affected by the initial tumor cell dose or by the number of in vivo passages of the tumor cell lines. Suppressor cell activity was detected in the hyporesponsive spleens of mice bearing transplanted tumors. Depletion of phagocytic macrophages, rayon wool-adherent cells, or theta-positive lymphocytes did not remove the suppressor cell activity. The mitogenic response of some but not all hyporesponsive spleens from autochthonous tumor bearers was restored after removal of phagocytic macrophages. The results demonstrated the heterogeneity of the factor(s) influencing non-specific T-cell reactivity in animals bearing spontaneous mammary tumors. Furthermore, our data suggest that nonspecific immunosuppression does not precede spontaneous tumor appearance but is probably a late result of rapid, extensive tumor growth.     

Endothelial Akt signaling is rate-limiting for rapamycin inhibition of mouse mammary tumor progression

Chronic activation of Akt signaling in the endothelium recapitulates the salient features of a tumor vasculature and can be inhibited by rapamycin, an inhibitor of mammalian target of rapamycin. This led to the hypothesis that the antitumor efficacy of rapamycin may be partially dependent on its ability to inhibit endothelial Akt signaling, making rapamycin an antiangiogenic agent and endothelial Akt pathway inhibitor. Dose-response studies with rapamycin showed that primary human endothelial cells and fibroblasts had a bimodal Akt response with effective reductions in phosphorylated Akt (pAkt) achieved at 10 ng/mL. In contrast, rapamycin increased pAkt levels in tumor cell lines. When tumor-bearing mice were treated with rapamycin doses comparable to those used clinically in transplant patients, we observed strong inhibition of mammary tumor growth. To test whether Akt activation in the endothelium was rate-limiting for this antitumor response, we engineered mouse mammary tumor virus-polyoma virus middle T antigen mice with endothelial cell-specific expression of constitutively activated Akt. We observed that the antitumor efficacy of rapamycin was reduced in the presence of elevated endothelial Akt activation. Just as we observed in MCF7 cells in vitro, rapamycin doses that were antiangiogenic resulted in increased pAkt levels in total mouse mammary tumor virus-polyoma virus middle T antigen tumor lysates, suggesting that tumor cells had an opposite Akt response following mammalian target of rapamycin inhibition compared with tumor endothelial cells. Together, these data support the hypothesis that endothelial Akt signaling in the tumor vasculature is an important target of the novel anticancer drug rapamycin.     

In vitro demonstration of tumor-specific antigens in spontaneous mammary tumors of mice

The colony-inhibition test has been used to demonstrate cellular immunity against spontaneous murine mammary tumors, both in autochthonous, mammary tumor virus (MTV)-carrying BALB/cfC3H and in immunized, MTV-free, BALB/c mice. Immunity could be demonstrated in nearly all cases against a mammary tumor with lymph-node cells from mice which had experienced that tumor. One of eight tumors tested with lymph-node cells from BALB/cfC3H mice which had spontaneously developed mammary tumors showed evidence of immunological cross-reactivity, while seven of thirteen tumors did so when tested with immune cells from MTV-free BALB/c mice. The data, therefore, indicate the presence of at least two types of tumor-specific antigen (s) in mouse mammary tumors — a common one and an antigen specific for each neoplasm.     

Effect of resveratrol on the development of spontaneous mammary tumors in HER-2/neu transgenic mice

Resveratrol (Res) has been reported to possess cancer chemopreventive activity on the basis of its in vitro effects on tumor cells and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of Res on the development of mammary tumors appearing spontaneously in HER-2/neu transgenic mice at an early age. The mechanisms involved in the Res antitumor effect were evaluated by studying the immune effectiveness, tumor apoptosis and expression of mRNA and protein for HER-2/neu in tumoral mammary glands from Res-treated mice and in tumor cell lines. Res supplementation delayed the development of spontaneous mammary tumors (p < 0.001), reduced the mean number and size of mammary tumors (p < 0.0001) and diminished the number of lung metastases in HER-2/neu transgenic mice. The effects of Res were associated with downregulation of HER-2/neu gene expression and increased apoptosis both in tumoral mammary glands and in murine (N202) and human (SKBr3) tumor cell lines. Neither the basal, the IL-2-induced NK activity nor the lymphocyte number and proliferation was modified in Res-supplemented compared to control mice. Our results demonstrate that Res supplementation delays the development and reduces the metastasizing capacity of spontaneous mammary tumors in HER-2/neu transgenic mice. The antitumor effect of Res might be related to the downregulation of HER-2/neu expression and the induction of apoptosis in tumor cells.     

Antitumor activity of endostatin against carcinogen-induced rat primary mammary tumors

Endostatin, a Mr 20,000 fragment of collagen XVIII, potently inhibits the growth of experimental tumors implanted in mice. Here we report the cloning, expression, and antitumor activity of the rat form of endostatin. When tested on breast carcinomas arising in female virgin rats after intragastric administration of 9,10-dimethyl-1,2-benzanthracene (DMBA), endostatin induced significant inhibition of mammary tumor growth in all of the treated rats during a 4-week treatment period without signs of systemic toxicity. Interestingly, this arrest of tumor growth persisted throughout a four-week off-therapy period. Moreover, endostatin was effective in counteracting the development of multiple primary tumors. These results confirm that rat endostatin is a potent anticancer agent in a carcinogen-induced, spontaneously arising rat breast cancer model. It not only stops the growth of existing tumors but also decreases the incidence of the development of multiple neoplastic lesions.     

The extracellular region of heregulin is sufficient to promote mammary gland proliferation and tumorigenesis but not apoptosis

Heregulin (HRG) is a member of the neuregulin family of ligands that have been shown to interact with and activate erbB receptors. A transgenic mouse model in which full-length HRG is overexpressed has proven that this protein can induce carcinomas in the murine mammary gland. These tumors display a high level of apoptosis, which appears to be mediated by the cytoplasmic domain of HRG. Because both proliferation and apoptosis play a role in tumor formation, we wished to separately view those perturbations by removing the suspected apoptosis-inducing cytoplasmic domain of HRG. We thereby sought to determine whether overexpression of the extracellular region of HRG would be sufficient to induce mammary gland carcinomas. A HRG construct lacking the cytoplasmic domain was targeted to the mammary gland using the murine mammary tumor virus promoter. Multiple lines of transgenic mice carrying the transgene developed mammary gland tumors at approximately 15 months of age. These tumors did not display high levels of apoptosis as compared with tumors from murine mammary tumor virus/full-length HRG transgenic animals. In addition, virgin transgenic mice show a persistence of terminal end bud structures, which normally disappear at the onset of puberty in wild-type mice. To examine the signal transduction pathway activated by extracellular HRG in tumors, we investigated the phosphorylation status of the epidermal growth factor receptor family members. Western blot analysis showed activation of ErbB2 and ErbB3, suggesting a possible mode of action of extracellular HRG in mammary gland carcinomas. We conclude that the extracellular and transmembrane domains of HRG are sufficient for the induction of tumorigenesis but that induction of apoptosis requires the cytoplasmic tail.     

Long-term exposure to elevated levels of circulating TIMP-1 but not mammary TIMP-1 suppresses growth of mammary carcinomas in transgenic mice

Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates matrix metalloproteinase activity, acts as a growth stimulator and inhibits apoptosis. We developed transgenic mice to evaluate the relevance of circulating versus mammary TIMP-1 in mammary carcinogenesis. The transgene was placed under the control of the albumin (Alb) promoter for the production of large amounts of TIMP-1 in the liver and release into the systemic circulation to achieve chronically elevated blood levels. The initial 7,12-dimethylbenz[a]anthracene (DMBA) mammary carcinogenesis study showed greatly decreased tumor incidence in heterozygous Alb-TIMP-1 mice (25%), compared with their wild-type (wt) littermates (83.3%). Metastatic mammary carcinomas were induced in the Alb-TIMP-1 mice through breeding with mice expressing the polyomavirus Middle T antigen (MT) under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Both the mammary tumor burden and the incidence of lung metastases were lower in the Alb-TIMP-1/MMTV-MT mice than their MMTV-MT littermates. Analysis of the Alb-TIMP-1/MMTV-MT tumors showed evidence of decreased proliferative activity and inhibition of apoptosis, whereas microvascular density was not affected. Transgenic expression of TIMP-1 in mammary epithelial cells was accomplished by using MMTV-LTR. In contrast to the Alb-TIMP-1 mice, there was insignificant difference in the growth of both DMBA- and MT-induced mammary tumors between heterozygous MMTV-TIMP-1 mice and their wt littermates. The MT-induced mammary tumors of the MMTV-TIMP-1 mice were separated into 'low' and 'high' TIMP-1 expressing groups. The 'high' TIMP-1 expressing tumors exhibited significantly higher proliferative activity than the tumors of the MMTV-MT only mice, whereas the number of apoptotic cells and microvascular density were not different. The findings of this study show that circulating TIMP-1, but not mammary-derived TIMP-1, has growth suppressive effects on DMBA and MT-induced mammary carcinomas.     

Matrix metalloproteinases play an active role in Wnt1-induced mammary tumorigenesis

The Wnt signaling transduction pathway plays a critical role in the pathogenesis of several murine and human epithelial cancers. Here, we have used mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice, which develop spontaneous mammary adenocarcinoma, to examine whether matrix metalloproteinases (MMPs)--a family of extracellular proteases implicated in multiple steps of cancer progression--contributed to Wnt1-induced tumorigenesis. An analysis of the expression of several MMPs by RT-PCR and in situ hybridization revealed an increase in the expression of MMP-2, MMP-3, MMP-9, MMP-13, and MT1-MMP (MMP-14) in hyperplastic glands and in mammary tumors of MMTV-Wnt1 transgenic mice. Interestingly, whereas MMP-2, MMP-3, and MMP-9 were exclusively expressed by stromal cells in mammary tumors, MMP-13 and MT1-MMP were expressed by transformed epithelial cells in addition to the tumor stroma. To determine whether these MMPs contributed to tumorigenesis, MMTV-Wnt1 mice were crossed with transgenic mice overexpressing tissue inhibitor of metalloproteinase-2-a natural MMP inhibitor-in the mammary gland. In the double MMTV-Wnt1/tissue inhibitor of metalloproteinases-2 transgenic mice, we observed an increase in tumor latency and a 26.3% reduction in tumor formation. Furthermore, these tumors grew at a slower rate, exhibited an 18% decrease in proliferative rate, and a 12.2% increase in apoptotic rate of the tumor cells in association with a deficit in angiogenesis when compared with tumors from MMTV-Wnt1 mice. Thus, for the first time, the data provides evidence for the active role of MMPs in Wnt1-induced mammary tumorigenesis.     

Wwox inactivation enhances mammary tumorigenesis

Breast cancer is the leading cause of cancer-related death in women worldwide. Expression of the WWOX tumor suppressor is absent or reduced in a large proportion of breast tumors suggesting that loss of WWOX may contribute to breast tumorigenesis. Wwox-deficient mice die by 3-4 weeks of age precluding adult tumor analysis. To evaluate the effect of WWOX-altered expression on mammary tumor formation, the Wwox-heterozygous allele was back crossed onto the C3H mammary tumor-susceptible genetic background (Wwox(C3H)+/-) and incidence of mammary tumor formation was evaluated. Although 50% of the female Wwox(C3H)+/- mice developed mammary carcinomas, only 7% of Wwox(C3H)+/+ mice did. Intriguingly, mammary tumors in Wwox(C3H)+/- mice frequently lost WWOX protein expression suggesting a genetic predisposition toward mammary tumorigenesis. Immunohistochemical staining of hormone receptors revealed loss of estrogen receptor-α (ER) and progesterone receptor in the majority of these tumors. In vitro, depletion of WWOX in MCF7 ER-positive cells led to reduced ER expression and reduced sensitivity to tamoxifen and estrogen treatment and was associated with enhanced survival and anchorage-independent growth. Finally, cDNA array analyses of murine normal mammary epithelial cells and mammary tumors identified 163 significantly downreguated and 129 upregulated genes in the tumors. The majority of differentially expressed genes were part of pathways involved in cellular movement, cell-to-cell signaling and interaction, cellular development, cellular growth and proliferation and cell death. These changes in gene expression of mouse mammary tumors in Wwox(C3H)+/- mice resemble, at least in part, human breast cancer development. Our findings demonstrate the critical role that the WWOX tumor suppressor gene has in preventing tumorigenesis in breast cancer.     

Comparative responsiveness and pharmacokinetics of doxorubicin in human tumor xenografts

The antineoplastic activity of the anthracyclin antibiotic doxorubicin (Adriamycin®) differs in its cytotoxic effectiveness against different types of human tumors. In the present study the effect of doxorubicin on the growth of two human lung carcinomas and one human mammary carcinoma transplanted into athymic mice was correlated with the pharmacokinetics of doxorubicin in the same tumors after intraperitoneal administration. Doxorubicin produced a greater inhibition of tumor growth in the lung carcinomas than in the mammary carcinoma. Furthermore, the pharmacokinetic characteristics of doxorubicin differed widely within the three human solid tumors. No apparent correlation was found to exist between the different tumor growth sensitivities to doxorubicin and the pharmacokinetic parameters of doxorubicin within the tumor tissue. It is suggested that the differences in the demontrated antitumor effectiveness of doxorubicin may be due to differences in the “intrinsic sensitivity” of the three human solid tumors.     

Comparison of biologic behavior and histology of transplanted mammary tumors in GR mice

Mammary tumors that arose spontaneously in inbred GR mice were transplanted into syngeneic castrated males. The hormone responsiveness of the transplants was studied in mice treated with estrone and progesterone and was compared with the hormone responsiveness in mice that received no hormone treatment. Microscopic examination of hormone-responsive and hormone-independent tumors revealed similar histologic patterns in both groups. It was evident that pale cells, which are classically associated with hormone-responsive tumors, may also be present in transplanted hormone-independent tumors in this strain. No correlation was found between the histologic pattern of these transplanted tumors and the biologic behavior, hormonal status, or presence of a specific murine mammary tumor virus (MuMTV) proviral fragment. Mammary tumors also appeared capable of undergoing differentiation into more than one morphologic type. Two cotransplanted tumors (derived from the same parental tumor) had markedly different histologic patterns; however, analysis of MuMTV proviral fragments indicated that the MuMTV-infected cells were of the same parentage.     

Potentiation of cytotoxic therapies by TNP-470 and minocycline in mice bearing EMT-6 mammary carcinoma

Summary The ability of the antiangiogenic agents TNP-470 and minocycline, singly or in combination, to potentiate the antitumor effects of several cytotoxic therapies was assessed in the murine EMT-6 mammary carcinoma as well as in two drug resistant sublines of that tumor designated EMT-6/CTX and EMT-6/CDDP.The antiangiogenic agents alone or in combination did not alter the growth of the tumors. However, their administration along with cyclophosphamide, CDDP, or thiotepa substantially increased the tumor growth delay produced by these cytotoxic therapies in tumors responsive to the drugs — the increase was about 2-fold for TNP-470 and minocycline together. In drug resistant tumors, treatment with the antiangiogenic agents did not reverse drug resistance but did increase the effect of the cytotoxic drugs.Treatment with TNP-470/minocycline also increased the oxygenation of each of the three tumors. Thus, TNP-470/minocycline administration increased the efficacy of fractionated radiation therapy, especially when used along with a perflubron emulsion oxygen delivery agent/carbogen.These results indicate that treatment regimens including therapies directed toward the proliferating normal cells within a tumor mass as well as therapies directed toward the malignant cells can produce improved outcomes.     

Detection of different tumor growth kinetics in single transgenic mice with oncogene‐induced mammary carcinomas by flat‐panel volume computed tomography

Transgenic mouse models offer an excellent opportunity for studying the molecular basis of cancer development and progression. Here we applied flat‐panel volume computed tomography (fpVCT) to monitor tumor progression as well as the development of tumor vasculature in vivo in a transgenic mouse model for oncogene‐induced mammary carcinogenesis (WAP‐T mice). WAP‐T mice develop multiple mammary carcinomas on oncogene induction within 3 to 5 months. Following induction, 3‐dimensional fpVCT data sets were obtained by serial single scans of entire mice in combination with iodine containing contrast agents and served as basis for precise measurements of tumor volumes. Thereby, we were able to depict tumors within the mammary glands at a very early stage of the development. Tumors of small sizes (0.001 cm³) were detected by fpVCT before being palpable or visible by inspection. The capability to determine early tumor onset combined with longitudinal noninvasive imaging identified diverse time points of tumor onset for each mammary carcinoma and different tumor growth kinetics for multiple breast carcinomas that developed in single mice. Furthermore, blood supply to the breast tumors, as well as blood vessels around and within the tumors, were clearly visible over time by fpVCT. Three‐dimensional visualization of tumor vessels in high resolution was enhanced by the use of a novel blood pool contrast agent. Here, we demonstrate by longitudinal fpVCT imaging that mammary carcinomas develop at different time points in each WAP‐T mouse, and thereafter show divergent growth rates and distinct vascularization patterns. © 2009 UICC     

Hypoxia-mediated apoptosis from angiogenesis inhibition underlies tumor control by recombinant interleukin 12.

The role of angiogenesis inhibition in the antitumor activity of recombinant murine interleukin 12 (rmIL-12) was studied in K1735 murine melanomas, the growth of which is rapidly and markedly suppressed by rmIL-12 treatment. On the basis of the prediction that tumor ischemia should result from therapeutic angiogenesis inhibition, tumor cell hypoxia was evaluated as a marker of ischemia using the EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide] approach. This method measures intracellular binding of the nitroimidazole EF5, which covalently binds to cellular macromolecules selectively under hypoxic conditions. Whereas 1 week of rmIL-12 treatment effectively inhibited K1735 cell-induced angiogenesis in Matrigel neovascularization assays, 2 weeks of treatment were needed before severe tumor cell hypoxia was detected in K1735 tumors. The hypoxia that developed was regional and localized to tumor areas distant from blood vessels. The great majority of severely hypoxic tumor cells were apoptotic, and in vitro studies indicated that the degree of hypoxia present within treated tumors was sufficient to trigger K1735 apoptosis. Tumor cell apoptosis was also prevalent in the first week of rmIL-12 treatment when few cells were hypoxic. In vitro studies indicated that this non-hypoxia-related apoptosis was induced directly by IFN-gamma produced in response to rmIL-12 administration. These studies reveal that rmIL-12 controls K1735 tumors initially by IFN-gamma-induced apoptosis and later by hypoxia-induced apoptosis. They also establish hypoxia as an expected result of tumor angiogenesis inhibition and a mediator of its therapeutic effect.     

Characterization of two human mammary carcinomas,MT-1 and MT-3, suitable forin vivo testing of ether lipids and their derivatives

Summary The human mammary carcinomas MT-1 and MT-3 originate from surgical material and were transplanted in nude mice. Both tumors have been classified as estradiol- and progesterone receptor-negative. Therapeutic doses of hormones and anti-hormones remained without growth inhibitory effect. MT-1 and MT-3 proved to be sensitive to conventional cytostatic drugs used for treatment of mammary carcinomas; striking is their sensitivity to ether lipids. Therefore, they are considered suitable tumor models for this class of substances.