Rat Mammary Tumors Research Articles

A new class of peroxisome proliferator-activated receptor γ (PPARγ) agonists that inhibit growth of breast cancer cells: 1,1-Bis(3′-indolyl)-1-(p-substituted phenyl)methanes

Abstract 1,1-Bis(3′-indolyl)-1-(p-trifluoromethylphenyl)methane (DIM-C-pPhCF3) and several p-substituted phenyl analogues have been investigated as a new class of peroxisome proliferator-activated receptor γ (PPARγ) agonists. Structure-activity studies in PPARγ-dependent transactivation assays in MCF-7 breast cancer cells show that 5–20 μm concentrations of compounds containing p-trifluoromethyl, t-butyl, cyano, dimethylamino, and phenyl groups were active, whereas p-methyl, hydrogen, methoxy, hydroxyl, or halogen groups were inactive as PPARγ agonists. Induction of PPARγ-dependent transactivation by 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2) and DIM-C-pPhCF3 was inhibited in MCF-7 cells cotreated with the PPARγ-specific antagonist N-(4′-aminopyridyl)-2-chloro-5-nitrobenzamide. In mammalian two-hybrid assays, DIM-C-pPhCF3 and PGJ2 (5–20 μm) induced interactions of PPARγ with steroid receptor coactivator (SRC) 1, SRC2 (TIFII), and thyroid hormone receptor-associated protein 220 but not with SRC3 (AIB1). In contrast, DIM-C-pPhCF3, but not PGJ2, induced interactions of PPARγ with PPARγ coactivator-1. C-substituted diindolylmethanes inhibit carcinogen-induced rat mammary tumor growth, induce differentiation in 3T3-L1 preadipocytes, inhibit MCF-7 cell growth and G0/G1-S phase progression, induce apoptosis, and down-regulate cyclin D1 protein and estrogen receptor α in breast cancer cells. These compounds are a novel class of synthetic PPARγ agonists that induce responses in MCF-7 cells similar to those observed for PGJ2.

Optical coherence tomography: feasibility for basic research and image-guided surgery of breast cancer.

Diagnostic trends in medicine are being directed toward cellular and molecular processes, where treatment regimens are more amenable for cure. Optical imaging is capable of performing cellular and molecular imaging using the short wavelengths and spectroscopic properties of light. Diffuse optical tomography is an optical imaging technique that has been pursued as an alternative to X-ray mammography. While this technique permits non-invasive optical imaging of the whole breast, to date it is incapable of resolving features at the cellular level. Optical coherence tomography (OCT) is an emerging high-resolution biomedical imaging technology that for larger and undifferentiated cells can perform cellular-level imaging at the expense of imaging depth. OCT performs optical ranging in tissue and is analogous to ultrasound except reflections of near-infrared light are detected rather than sound. In this paper, an overview of the OCT technology is provided, followed by images demonstrating the feasibility of using OCT to image cellular features indicative of breast cancer. OCT images of a well-established carcinogen-induced rat mammary tumor model were acquired. Images from this common experimental model show strong correlation with corresponding histopathology. These results illustrate the potential of OCT for a wide range of basic research studies and for intra-operative image-guidance to identify foci of tumor cells within surgical margins during the surgical treatment of breast cancer.

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In this issue

Inhibition of Rat Mammary Gland Carcinogenesis by Simultaneous Targeting of Cyclooxygenase-2 and Peroxisome Proliferator-activated Receptor γ

We examined the effect of celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, and N-(9-fluorenyl-methyloxycarbonyl)-L-leucine (F-L-Leu), a peroxisome proliferator-activated receptor gamma (PPAR gamma) agonist, separately and combined, on the development of methylnitrosourea (MNU)-induced rat mammary gland carcinogenesis. Celecoxib and F-L-Leu significantly reduced tumor incidence and multiplicity (P < 0.05). Combining both agents exerted higher (synergistic) cancer inhibition than separate treatments (P < 0.05). The effects of the test drugs on COX-2 and PPAR gamma expression and on the synthesis of prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) were examined in rat mammary normal (MNU-untreated), uninvolved, and tumor (MNU-treated) tissues. Celecoxib and F-L-Leu, separately, inhibited COX-2 and up-regulated PPAR gamma expression. These effects were paralleled by inhibition of PGE(2) synthesis and up-regulation of 15d-PGJ(2). Combined treatment resulted in higher alterations in COX-2 and PPAR gamma transcripts and PG synthesis compared with separate administrations. The effect of the test agents on Bcl(2), BAX, and protein kinase C alpha expression levels were examined in the rat mammary gland and the pro-(BAX:Bcl(2)) and anti-[PKC alpha*(Bcl(2)/BAX)] apoptotic ratios were evaluated. Each drug increased the proapoptotic ratio by 2- to 7-fold and reduced the antiapoptotic ratio by 2- to >8-fold in all tissues. Combined treatment, however, resulted in >9- to 14-fold up-regulation in the proapoptotic processes and 15- to >30-fold down-regulation in the antiapoptotic ones. Analyses were also carried out on the drug-induced modulation of cell cycle regulators and proliferation markers (cyclin-dependent kinase 1 and proliferating cell nuclear antigen). F-L-Leu and celecoxib each reduced the cyclin-dependent kinase 1 and proliferating cell nuclear antigen expression in the tumor. Higher down-regulation was attained in all tissues by combined treatment where cyclin-dependent kinase 1 and proliferating cell nuclear antigen almost retained the expression levels observed in the normal glands. In conclusion, simultaneous targeting of COX-2 and PPAR gamma may inhibit mammary cancer development more effectively than targeting each molecule alone. COX-2 inhibitors and PPAR gamma agonists coordinately mediate their anticancer effect via both COX-dependent (inhibition of COX-2, activation of PPAR gamma, and modulation PG synthesis) and COX-independent (induction of proapoptotic factors and inhibition of cell proliferation) pathways.

Human soluble p66 and p51 tumor-associated antigens promote the suppression of rat mammary tumors in comparison to commercial human albumin

This study examined whether the soluble 66- and 51 kDa tumor-associated antigens (sTAA), isolated from the serum of breast cancer patients, possess specific suppressive effects on chemically-induced rat mammary tumorigenesis in comparison to commercial human albumin. Dimethylbenzanthracene (DMBA, 10 mg/rat, 2 administrations) was used to induce mammary tumors in 8-week-old Sprague Dawley rats. After the appearance of many large tumors, preparations of sTAA (50-60 micro g/rat in 0.5 ml sterile PBS) or commercial human albumin (HA, in the same doses as sTAA) were administered weekly, for 10-14 more weeks. The following groups of mammary tumor-bearing rats were studied: i) control non-treated rats, ii) rats treated with HA, iii) rats treated with sTAA. The experiment was terminated when tumors in 70% of the rats became ulcerous. The treatment with sTAA significantly decreased, compared to controls, the yield and total area of the tumors. In rats treated with sTAA, the appearance of new tumors stopped at week 5 as compared to week 7 in rats treated with HA and week 10 in control rats. In rats treated with sTAA, the time of appearance of ulcerous tumors increased to 8 weeks, as compared to 6 weeks in controls and in rats treated with HA. Duration of the experiment increased from 11 weeks in controls to 12 weeks in rats treated with HA and to 14 weeks in rats treated with sTAA. We conclude that sTAA have tumor-suppressive properties, which are well-defined if the treatment is begun on small tumors.

Aurora-A Kinase Regulates Telomerase Activity through c-Myc in Human Ovarian and Breast Epithelial Cells

Aurora-A kinase is frequently overexpressed/activated in human ovarian and breast cancers. A rat mammary tumor model study indicates that alterations of Aurora-A are early events during mammary tumor development (T. M. Goepfert et al., Cancer Res., 62: 4115-4122, 2002), suggesting that Aurora-A plays a pivotal role in transformation. However, the molecular mechanism by which Aurora-A induces ovarian and breast cell transformation remains elusive. Here we show that ectopic expression of Aurora-A induces telomerase activity in human ovarian and breast epithelial cell lines HIOSE118 and MCF-10A. The mRNA and promoter activities of human telomerase reverse transcriptase (hTERT) are stimulated by Aurora-A. Furthermore, we have demonstrated that the c-Myc binding sites of hTERT promoter are required for Aurora-A-induced hTERT promoter activity. Ectopic expression of Aurora-A up-regulates c-Myc. Knockdown of c-Myc by RNA interference attenuates Aurora-A-stimulated hTERT expression and telomerase activity. To our knowledge, these findings demonstrate, for the first time, that Aurora-A induces telomerase activity and hTERT by up-regulation of c-Myc and provides an additional mechanism for the role of Aurora-A in malignant transformation in addition to its cell cycle control.

Enhancement of laser cancer treatment by a chitosan-derived immunoadjuvant

A chitosan derivative, glycated chitosan (GC), has been used as an immunostimulant for cancer treatment in laser immunotherapy. The function of GC is to enhance the host immune response after direct cancer cell destruction by a selective laser photothermal interaction. To further test its effects, laser immunotherapy was extended to include several different adjuvants for immunological stimulation and to include photodynamic therapy (PDT) as a different tumor-destruction mechanism. Complete Freund (CF) adjuvant, incomplete Freund (IF) adjuvant and Corynebacterium parvum (CP) were selected for treatment of metastatic mammary tumors in rats, in combination with a selective photothermal interaction. The solution of the immunoadjuvants admixed with indocyanine green (ICG), a light-absorbing dye, was injected directly into the tumors, followed by noninvasive irradiation of an 805 nm laser. Combined with PDT, in the treatment of tumors in mice, GC was administered peritumorally immediately after laser irradiation. The survivals of treated animals were compared with untreated control animals. In the treatment of rat tumors, CF, IF and CP raised the cure rates from 0% to 18%, 7% and 9%, respectively. In comparison, GC resulted in a 29% long-term survival. In the treatment of EMT6 mammary sarcoma in mice, GC of 0.5% and 1.5% concentrations increased the cure rates of Photofrin-based PDT treatment from 38% to 63% and 75%, respectively. In the treatment of Line 1 lung adenocarcinoma in mice, a 1.67% GC solution enabled a noncurative meso-substituted tetra(meta-hydroxy-phenyl)chlorin-based PDT to cure 37% of the tumor-bearing mice. The experimental results of this study confirmed our previous studies, showing that immunoadjuvants played an active role in laser-related cancer treatment and that GC significantly enhanced the efficacy of laser cancer treatment.

Cell proliferation and apoptosis in rat mammary cancer after electrochemical treatment (EChT)

Background: Several authors have recently reported encouraging results from Electrochemical treatment (EChT) in malignant tumours. However, EChT is not established and mechanisms are not completely understood. In vivo studies were conducted to evaluate the toxic changes and effectiveness of EChT on an animal tumour model. Methods: Tumours were induced by injecting cells from the R3230AC rat mammary tumour cell line clone D subcutaneously, in 28 female Fischer 344 rats. EChT was conducted by inserting a platinum electrode into the tumours. The positive and negative control groups were subjected to the same conditions but without current. The rats were kept for 0, 7 or 14 days post-treatment. Three hours prior to euthanasia an i.p. injection of Bromodioxyuridine (BrdU) was given. The rats were euthanized, the lesions extirpated and samples were collected for histopathological, and immunohistochemical examination. Results: Significant changes in cell proliferation rate were seen both in the cathode and anode regions. Apoptosis were induced in the anodic treated area outside the primary necrosis, detected with the TUNEL method. Discussion: The results suggest that secondary cell destruction was caused by necrosis with cathodic EChT and apoptosis or necrosis with anodic EChT.

Id4 Regulates Mammary Epithelial Cell Growth and Differentiation and Is Overexpressed in Rat Mammary Gland Carcinomas

Id4 belongs to a family of helix-loop-helix (HLH) proteins that impact cellular growth and differentiation via regulation of basic HLH transcription factors. Herein the rat Id4 gene was cloned (GenBank Accession No. AF468681). The expression of rat Id4 was examined in rat mammary gland tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen found in the human diet. By real-time polymerase chain reaction analysis, relative expression of Id4 mRNA in carcinomas, adenomas, and normal tissue was 27, 6, and 1, respectively. Immunohistochemical analysis indicated statistically elevated nuclear expression for Id4 protein in carcinomas in comparison to adenomas and normal mammary gland. In carcinomas, Id4 nuclear expression was positively correlated with proliferation, invasiveness, and tumor weight (Fisher Exact Test or Spearman Correlation, P < 0.05). The consequence of enforced expression of Id4 on mammary epithelial cell proliferation, differentiation, and growth in soft agar was examined in HC11 cells, a well-characterized model for studying various aspects of mammary epithelial cell biology. After transient and stable transfection of HC11 cells, Id4 overexpression increased cell proliferation and inhibited lactogenic hormone-mediated differentiation as revealed by inhibition of beta-casein promoter activity and beta-casein expression. In addition, enforced expression of Id4 in HC11 cells induced a statistically significant increase in colony growth in soft agar. The results implicate Id4 in rat mammary gland carcinogenesis and suggest that Id4 may contribute to carcinogenesis by inhibiting mammary epithelial cell differentiation and stimulating mammary epithelial cell growth.

Venezuelan equine encephalitis replicon immunization overcomes intrinsic tolerance and elicits effective anti-tumor immunity to the 'self' tumor-associated antigen, neu in a rat mammary tumor model.

Many tumor-associated antigens (TAAs) represent 'self' antigens and as such, are subject to the constraints of immunologic tolerance. There are significant barriers to eliciting anti-tumor immune responses of sufficient magnitude. We have taken advantage of a Venezuelan equine encephalitis-derived alphavirus replicon vector system with documented in vivo tropism for immune system dendritic cells. We have overcome the intrinsic tolerance to the 'self' TAA rat neu and elicited an effective anti-tumor immune response using this alphavirus replicon vector system and a designed target antigen in a rigorous rat mammary tumor model. We have demonstrated the capacity to generate 50% protection in tumor challenge experiments (p = 0.004) and we have confirmed the establishment of immunologic memory by both second tumor challenge and Winn Assay (p = 0.009). Minor antibody responses were identified and supported the establishment of T helper type 1 (Th1) anti-tumor immune responses by isotype. Animals surviving in excess of 300 days with established effective anti-tumor immunity showed no signs of autoimmune phenomena. Together these experiments support the establishment of T lymphocyte dependent, Th1-biased anti-tumor immune responses to a non-mutated 'self' TAA in an aggressive tumor model. Importantly, this tumor model is subject to the constraints of immunologic tolerance present in animals with normal developmental, temporal, and anatomical expression of a non-mutated TAA. These data support the continued development and potential clinical application of this alphaviral replicon vector system and the use of appropriately designed target antigen sequences for anti-tumor immunotherapy.

Inhibition of rat mammary tumorigenesis by concord grape juice constituents.

The effects of Concord grape juice constituents on the promotion of chemically induced rat mammary tumor development and on the proliferation of a rat mammary adenocarcinoma cell line were studied. Isocaloric grape juice formulations provided in the drinking fluid of rats at concentrations of 489 and 651 mg of phenolics/dL of fluid significantly inhibited mammary adenocarcinoma multiplicity compared to controls. Final tumor mass also was significantly decreased for animals provided these two grape juice concentrations compared to controls. In addition, DNA synthesis of the rat mammary adenocarcinoma RBA cell line was significantly inhibited in a dose-dependent manner for cells treated with a grape extract, with an IC50 dose of approximately 14 micrograms of phenolics/mL. This inhibition of DNA synthesis was not accompanied by changes in 8-oxodeoxyguanosine formation or by substantial cell cycle arrest. These studies thus indicate that Concord grape juice constituents can inhibit the promotion stage of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumorigenesis, in part by suppressing cell proliferation.

Human soluble tumor-associated antigens promote the suppression of rat mammary tumors by 5-fluorouracil and stimulate the functional activity of immune organs: Experimental and morphological studies

This study examined whether the soluble 66 and 51 kDa tumor-associated antigens (sTAA) could promote suppression by the anticancer drug 5-fluorouracil (5-Fu) of chemically induced mammary tumorigenesis, and which, if any, morphological changes in the immune organs accompany this treatment. Dimethylbenzanthracene (DMBA, 8 mg/rat, twice) was used to induce mammary tumors. After the appearance of many large tumors, the preparations of sTAA and 5-Fu, alone or in combination, were administered in weekly doses, for 4 weeks. The following groups of mammary tumor-bearing rats were studied: 1) control non treated rats, 2) rats treated with sTAA, 3) rats treated with 5-Fu, 4) rats treated with 5-Fu and sTAA. The experiment was terminated when tumors in 70% of control rats became ulcerous. Treatment with sTAA alone significantly decreased tumor yield and their total area relative to controls. Both of these parameters showed an even larger significant decrease after treatment with 5-Fu, and the most marked decrease was obtained after the combined treatment with 5-Fu and sTAA. Results demonstrated that not only do sTAA have tumor-suppressive properties, they also enhance the anticancer effects of 5-Fu and prevent its toxic side effects. Morphologically, the treatment with sTAA was manifested in a significant increase in the size of the spleen follicles and mantle layer compared to control rats with large tumors. The treatment with 5-Fu decreased the sizes of almost all areas of the spleen compared to control rats, whereas the combined treatment with 5-Fu and sTAA increased all these parameters to the levels found in rats treated with sTAA alone. The total areas of the cortex and paracortex in the lymph nodes increased after treatment with sTAA. Treatment with 5-Fu alone resulted in a significant decrease of these areas which, as seen in the spleen, increased after combined treatment with 5-Fu and sTAA. Similar changes were seen in the areas of the separate lymph node zones. We concluded that the addition of sTAA to conventional tumor chemotherapy regimens has a remarkable synergistic effect on mammary tumors leading to curative antitumor responses of the host's immune organs.

Sulforaphane: a naturally occurring mammary carcinoma mitotic inhibitor, which disrupts tubulin polymerization.

Sulforaphane (SUL), an isothiocyanate found in broccoli and other cruciferous vegetables, has been shown to induce phase II detoxification enzymes, inhibit chemically induced mammary tumors in rats, and more recently to induce cell cycle arrest and apoptosis in cancer cells of the colon. Here, we provide evidence that SUL also acts as a breast cancer anti-proliferative agent. The BALB/c mouse mammary carcinoma cell line F3II was treated with SUL at concentrations up to 15 microM and examined for markers of cell cycle arrest and apoptosis. Treatment of asynchronous F3II cells with 15 microM SUL resulted in G2/M cell cycle arrest, elevated p34cdc2 (cdc2) kinase activity, Bcl-2 down-regulation, evidence of caspase activation, and aggregation of condensed nuclear chromatin. Subsequent exposure of synchronized cells to 15 microM SUL resulted in elevated numbers of prophase/prometaphase mitotic figures, indicating cell cycle progression beyond G2 and arrest early within mitosis. Moreover, cells treated with 15 microM SUL displayed aberrant mitotic spindles, and higher doses of SUL inhibited tubulin polymerization in vitro. In addition, BALB/c mice injected s.c. with F3II cells and subsequently injected daily i.v. with SUL (15 nmol/day for 13 days) developed significantly smaller tumors (approximately 60% less in mass) than vehicle-treated controls. Western blot analysis of tumor proteins demonstrated significantly (P<0.05) reduced PCNA and elevated PARP fragmentation in samples from animals dosed with SUL. Taken together, these results indicate that SUL has mammary cancer suppressive actions both in cell culture and in the whole animal. Inhibition of mammary carcinogenesis appears in part to involve perturbation of mitotic microtubules and early M-phase block associated with cdc2 kinase activation, indicating that cells arrest prior to metaphase exit.

Glucocorticoids control beta-catenin protein expression and localization through distinct pathways that can be uncoupled by disruption of signaling events required for tight junction formation in rat mammary epithelial tumor cells.

In Con8 rat mammary epithelial tumor cells, the synthetic glucocorticoid dexamethasone stimulates the remodeling of tight junctions and adherens junctions before formation of highly sealed tight junctions. In this study, the expression and localization of key components of the apical junction were examined as potential targets of glucocorticoid signaling. Western blot and RT-PCR demonstrated that dexamethasone up-regulated beta-catenin protein and transcript expression and nearly ablated beta-catenin phosphorylation under conditions that led to a significant increase in monolayer transepithelial resistance. Indirect immunofluorescence revealed that dexamethasone treatment also caused beta-catenin to localize predominantly at the cell membrane rather than the nucleus. The glucocorticoid regulation of beta-catenin expression and localization was not a consequence of dexamethasone inhibition of cell growth, because both responses were unaltered in the presence of hydroxyurea. The steroid induction of beta-catenin expression and localization can be uncoupled by altering the function of signaling pathways needed for tight junction formation. Expression of dominant-negative RasN17 abolished dexamethasone up-regulation of beta-catenin protein expression without affecting its localization at the membrane. In contrast, exogenous treatment or constitutive production of TGFalpha abolished the dexamethasone-induced alteration of beta-catenin localization without affecting the dexamethasone stimulation of beta-catenin expression. Taken together, our results demonstrate that glucocorticoids control beta-catenin at two distinct levels of cellular regulation that differ in their cell signaling requirements for the glucocorticoid regulation of mammary epithelial junctional dynamics.

Comparative action of 1,4-phenylenebis(methylene)selenocyanate and its metabolites against 7,12-dimethylbenz[a]anthracene-DNA adduct formation in the rat and cell proliferation in rat mammary tumor cells

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) inhibits 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis and DMBA–DNA binding in the rat mammary gland. Tetraselenocyclophane (TSC) was identified in rat feces as a metabolite of p-XSC. This led us to postulate the metabolic pathway: p-XSC→glutathione conjugate (p-XSeSG)→aromatic selenol (p-XSeH)→TSC. Whether p-XSC or one of its metabolites is responsible for cancer prevention is the focus of this study. We utilized the DMBA–DNA binding assay with p-XSC as a positive control to evaluate the chemopreventive potential of p-XSC metabolites at dietary selenium levels of 10ppm. Rats were fed AIN-76A diet supplemented with various selenium compounds for 1 week prior to the oral administration of a single dose of [3H]DMBA (5mg per rat, specific activity 51.3mCi/mmol). The rats were sacrificed 24h later and DNA was isolated from the mammary fat pads. Relative levels of total binding were: [pmol/mg DNA, mean±S.D., n=6]; DMBA [7.2±1.6]; DMBA+p-XSC [3.5±2.7]; DMBA+p-XSeSG [2.2±1.1]; DMBA+TSC [5.6±2.9]. All selenium compounds, except TSC, significantly inhibited DMBA–DNA adduct formation; however, the difference between p-XSC and p-XSeSG was not statistically significant. The inhibition of total binding was attributed to a reduction in the formation of the three major adducts derived from bay-region diol epoxides of DMBA. On the basis of their chromatographic characteristics, these were identified as anti-diol-epoxide:deoxyguanosine, syn-diol-epoxide:deoxyadenosine, and anti-diol-epoxide:deoxyadenosine. Our results suggest that p-XSeSG, but not TSC, is the likely inhibitor of mammary cancer. Selenium levels measured by atomic absorption spectroscopy in the target organ (mammary fat pads) and in plasma following the dietary administration of selenium compounds were in the order of p-XSeSG≅p-XSC>TSC. These results appear to be consistent with their order of inhibitory effects on total DMBA–DNA binding. Further in vitro studies of the effect of selenium compounds on cell proliferation suggest that, depending on the dose and time point selected, p-XSC is comparable to or better than p-XSeSG; but both are more effective than TSC. Collectively, our in vivo and in vitro results indicate that p-XSC and its conjugate are better candidates than TSC for future studies on mammary cancer chemoprevention.

Induced antitumor immunity against DMBA-4 metastatic mammary tumors in rats using laser immunotherapy.

Induced antitumor immunity is a highly effective and long-term cure for cancer, particularly for metastatic tumors. Laser immunotherapy was developed to induce such an immunologic response. It involves intratumoral administration of a light-absorbing dye and a specially formulated immunoadjuvant, followed by noninvasive irradiation of a near-infrared laser. Treatment of DMBA-4 metastatic mammary tumors in rats with this approach has resulted in local control of primary tumors and eradication of untreated distant metastases. After laser immunotherapy, rats were resistant to tumor rechallenge and developed immunity, which could be adoptively transferred. To better understand the immunity induced in this tumor model, immunization using freeze-thaw DMBA-4 cell lysates was performed, followed by tumor challenge 21 days later. Tumor cell lysate immunization delayed the emergence of metastases but did not provide immunity against the tumor challenge. Also performed was surgical resection of primary tumors before the observation of metastatic tumors. Removal of primary tumors was unsuccessful at changing the course of tumor progression. Tumors re-emerged at the primary sites, and metastases developed at multiple remote sites. In contrast, tumor-bearing rats successfully treated by laser immunotherapy experienced tumor regression and eradication and developed strong resistance to repeated challenges by tumor cells of the same type. Our results show that laser immunotherapy could have potential for the treatment of metastatic tumors by inducing tumor-specific, long-lasting immunity.

Radiosensitivity of rat mammary tumors correlates with early vessel changes assessed by power Doppler sonography.

To investigate the changes occurring in the vascularization of tumors during irradiation, we used a model of autochthonous mammary tumors in rats and assessed early vascular changes after irradiation by power Doppler sonography. Mammary tumors were induced in 24 female Sprague Dawley rats by a single subcutaneous injection of N-nitroso N-methyl urea. After tumor areas reached 1 cm2, the animals received a single fraction of 18-Gy radiation or intraperitoneal saline injection. Power Doppler sonographic quantification of detected vessels was performed 1 day before irradiation and 7 days after the use of a power Doppler index of 5 different tumor imaging planes. Final tumor shrinkage was compared with early changes in the power Doppler index. Not all tumors regressed in a similar fashion. Radiosensitive tumors were defined as tumors with a greater than 50% decrease in baseline area 28 days after irradiation, whereas radioresistant tumors were tumors with a less than 50% decrease in baseline area. Statistical analysis was performed by the Mann-Whitney U test. Tumor area changes were similar in radioresistant and radiosensitive tumors 7 days after irradiation (-41% and -35%, respectively; P > .05, not significant), whereas reduction in the power Doppler index was significantly greater in radiosensitive tumors (mean value, -63%) than in radioresistant tumors (mean value, -12%) (P = .001). Late tumor regrowth was correlated with day 7 power Doppler index changes (P = .009). A 40% reduction in the power Doppler index at day 7 distinguished 8 of 9 radiosensitive tumors and 8 of 9 radioresistant tumors (P = .003). This study suggests that early changes in tumor perfusion as assessed by power Doppler sonography after tumor irradiation may precede the long-term tumor regression.

Po-topic V-01: Parametric imaging of spontaneous mammary tumors in rats for the purposes of tissue microstructure characterization

The characterization of tissue microstructure using ultrasound backscatter can assist in the detection and classification of diseased tissues. Parametric images can be formed from tissue characterizations that show structure information below that resolved for conventional ultrasound. Eight retired breeders were acquired that had developed spontaneous mammary tumors. 2D B-mode images of the rat tumors were constructed from backscattered echoes. After scanning, tumors were dissected free from each rat, trimmed in the plane of ultrasound exposure, fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. Tumors were diagnosed microscopically as mammary gland fibroadenomas. Regions of interest (ROIs) were selected from the ultrasound B-mode images of the tumors and surrounding tissues. Average scatterer properties (effective scatterer size and acoustic concentration) were estimated from the backscattered RF signal used to make the B-mode images. Scatterer estimates were made by using least squares to fit a line to the measured form factor that was calculated from the backscattered power spectrum. The effect of noise in the signal from increasing attenuation with depth was to reduce the ability to make estimates of scatterer properties at greater depths. A weighting scheme was used to reduce the effects of noise and increase the ability to make accurate estimates to greater depths. Comparison of scatterer estimates between normal tissues (intercostal tissues) and tissues inside the tumors were made. On average, the estimated effective scatterer diameters inside the tumors were 30% larger at 107 μm than estimates of effective scatterer diameters outside the tumors averaging 82 μm. The average acoustic concentration estimated inside the tumor was 3.16 × 10−2/mm−3 as opposed to 0.746 mm−3 for outside the tumor. In all but one of the rats, there was a statistically significant difference (P < .05) between estimates of scatterer properties made inside the tumors and in surrounding healthy tissues. Feature analysis plots clearly showed distinction between the diseased tissues and the normal tissues. Enhanced B-mode images were constructed by superimposing colored pixels corresponding to the estimated average scatterer properties in different ROIs on the gray-scale B-mode images. The enhanced B-mode images highlighted differences between tissues inside and outside the tumors.

Effects of Seocalcitol (EB1089) on nitrosomethyl urea-induced rat mammary tumors.

Although 1,25-dihydroxyvitamin D3 is a potent cell-differentiating agent, its use in cancer prevention or therapy is precluded because it induces hypercalcemia. Synthetic analogs have been developed which inhibit tumor progression in animal models of breast cancer. One analog, Seocalcitol (EB1089) has been shown to be effective in causing regression of N-methyl-nitrosourea-induced rat mammary tumors. However, at the most effective oral dose, a significant increase in serum and urinary calcium levels were observed. In order to compare the efficacy of different dosing schedules of Seocalcitol, rats were treated either 6 times weekly (1 microg/kg) or by intermittent dosing to achieve the same total weekly dose. All dosing schedules of Seocalcitol were effective in inhibiting tumor progression. Once daily dosing was significantly more effective than intermittent dosing but was associated with a greater rise in serum calcium concentration. In order to evaluate alternative treatment strategies to limit calcemic effects, we assessed the efficacy of limiting vitamin D-induced hypercalcemia using bisphosphonates. Seocalcitol (2.5 microg/kg daily p.o. for 4 weeks) alone and in combination with pamidronate (APD 0.4 mg/kg per day s.c.) or the same dose of the bisphosphonate EB 1053 caused substantial tumor regression. No statistically significant difference was seen between combination treatment and Seocalcitol treatment alone. Co-treatment with APD or EB 1053 did not limit the rise in serum calcium induced by Seocalcitol alone. Cessation of treatment or administration of a lower dose (1microg/kg twice weekly) reversed hypercalcemia, hypercalciuria and weight loss induced by high dose Seocalcitol. However, reduction in tumor volume was maintained in the majority of animals.

Serum Pyrrolidone Carboxypeptidase Activity in N-methyl Nitrosourea Induced Rat Breast Cancer

Pyrrolidone carboxypeptidase (Pcp) (E.C. 3.4.19.3) is an omega peptidase widely distributed in animal fluids and tissues and hydrolyses N-terminal pyroglutamic residues from biologically active peptides such as gonadotropin releasing hormone (GnRH). Previous results obtained by us showed a decrease in human breast cancer Pcp activity, suggesting that this enzyme activity or its putative substrates may play a major role in breast cancer pathogenesis. The aim of the present work is to analyse serum Pcp activity in N-methyl-nitrosourea (NMU) induced rat mammary tumours using pyroglutamyl-beta-naphthylamide as substrate. Serum Pcp activity was significantly lower in NMU-treated rats than in controls. Moreover, multiple regression analysis showed a significant correlation between Pcp activity and the number and size of tumours and the body weight of the animals. Since NMU-induced carcinomas are mainly oestrogen-dependent, the decrease observed in Pcp activity may reflect an increase in circulating levels of GnRH that lead to an increase in gonadal steroid hormones production responsible, at least in part, for the initiation and promotion of the disease.