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The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The extracellular matrix (ECM) is a master regulator of cellular phenotype and behavior. It has a crucial role in both normal tissue homeostasis and disease pathology. Here we present a fast and efficient approach to enhance the study of ECM composition and structure. Termed in situ decellularization of tissues (ISDoT), it allows whole organs to be decellularized, leaving native ECM architecture intact. These three-dimensional decellularized tissues can be studied using high-resolution fluorescence and second harmonic imaging, and can be used for quantitative proteomic interrogation of the ECM. Our method is superior to other methods tested in its ability to preserve the structural integrity of the ECM, facilitate high-resolution imaging and quantitatively detect ECM proteins. In particular, we performed high-resolution sub-micron imaging of matrix topography in normal tissue and over the course of primary tumor development and progression to metastasis in mice, providing the first detailed imaging of the metastatic niche. These data show that cancer-driven ECM remodeling is organ specific, and that it is accompanied by comprehensive changes in ECM composition and topological structure. We also describe differing patterns of basement-membrane organization surrounding different types of blood vessels in healthy and diseased tissues. The ISDoT procedure allows for the study of native ECM structure under normal and pathological conditions in unprecedented detail.

Although the rat model of mammary tumors chemically induced by N-methyl-N-nitrosourea (MNU) has been frequently used by several research teams, there is a lack of ultrastructural studies in this field. The main aim of this work was to perform an ultrastructural characterization of MNU-induced mammary tumors in female rats. Some alterations previously reported in human mammary tumors, such as nucleus size and shape, accumulation of heterochromatin in the perinuclear region, and interdigitating cytoplasmic processes between cancer cells were also observed in MNU-induced mammary tumors. Although a low number of samples were analyzed by transmission electron microscopy in the present study, we consider that it may contribute to a better understanding of MNU-induced mammary carcinogenesis in a rat model. The ultrastructural characteristics of the two most frequently diagnosed mammary carcinomas described in the present work can be useful to differentiate them from other histological patterns. In addition, the loss of cytoplasm in neoplastic cells and formation of vacuoles were described.

Several studies have assumed that myoepithelial cells (MECs) loss may contribute to epithelial tumor induction and/or progression. We adopted an in vitro assay and a syngeneic mice breast cancer model with histological and molecular characteristics resembling human lesions to evaluate tumor suppression effects of MECs. Flow cytometric, cell viability, blood chemistry, transmission electron microscope, immunohistochemistry and qRT-PCR assays were performed at the end of the study. We demonstrated that MECs could significantly suppress the viability of cancer cells at different time points (P<0.05). At the end of the fourth and fifth weeks, treated mice had smaller tumor volume compared with control animals. Average tumor volume was significantly less in treated groups than control group at days 21 (0.38±0.19 vs. 1.99±0.13 cm3), 28 (0.57±0.3 vs. 2.5±0.37 cm3) and 35 (0.7±0.35 vs. 2.65±0.4 cm3) after tumor cell injection (P<0.05). No hematological, hepatocellular, and renal toxicities were seen in MECs treated groups. Ultrastructural features revealed severe relationship between adjacent tumoral cells and loose interconnections of neoplastic cells in treated group. Immunohistochemical examinations of breast tumors showed high p63 and low alpha-smooth muscle actin protein expression in treated mice compared to control (P<0.05). MRNA expressions of TNF-α, smooth muscle-myosin heavy chain, connexin 43, and maspin were significantly up-regulated in breast tumor tissues in treated group compared to control (P<0.05). VEGF and alpha-smooth muscle actin mRNA expression were reduced in treated animals (P<0.05). The present study highlighted the potential tumor suppression effects of MECs on breast cancer in a typical animal model., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The ability to modulate the fluorescence of optical probes can be used to enhance signal-to-noise ratios for imaging within highly autofluorescent environments, such as intact tissues and living organisms. Here, we report two bacteriophytochrome-based photoactivatable near-infrared fluorescent proteins, named PAiRFP1 and PAiRFP2. PAiRFPs utilize haem-derived biliverdin, ubiquitous in mammalian tissues, as the chromophore. Initially weakly fluorescent PAiRFPs undergo photoconversion into a highly fluorescent state with excitation/emission at 690/717 nm following a brief irradiation with far-red light. After photoactivation, PAiRFPs slowly revert back to initial state, enabling multiple photoactivation-relaxation cycles. Low-temperature optical spectroscopy reveals several intermediates involved in PAiRFP photocycles, which all differ from that of the bacteriophytochrome precursor. PAiRFPs can be photoactivated in a spatially selective manner in mouse tissues, and optical modulation of their fluorescence allows for substantial contrast enhancement, making PAiRFPs advantageous over permanently fluorescent probes for in vivo imaging conditions of high autofluorescence and low signal levels.

Background: ARHI is a Ras-related imprinted gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of breast cancers, and loss of its expression is associated with its progression from ductal carcinoma in situ (DCIS) to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic death in cell culture; however, ARHI re-expression enables ovarian cancer cells to remain dormant when they are grown in mice as xenografts. The purpose of this study is to examine whether ARHI induces autophagy in breast cancer cells and to evaluate the effects of ARHI gene re-expression in combination with paclitaxel., Methods: Re-expression of ARHI was achieved by transfection, by treatment with trichostatin A (TSA) or by a combination of TSA and 5-aza-2'-deoxycytidine (DAC) in breast cancer cell cultures and by liposomal delivery of ARHI in breast tumor xenografts., Results: ARHI re-expression induces autophagy in breast cancer cells, and ARHI is essential for the induction of autophagy. When ARHI was re-expressed in breast cancer cells treated with paclitaxel, the growth inhibitory effect of paclitaxel was enhanced in both the cell culture and the xenografts. Although paclitaxel alone did not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-induced apoptosis and G2/M cell cycle arrest., Conclusions: ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophagy, apoptosis, and G2/M cell cycle arrest.

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved alpha-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated alpha-smooth muscle actin-expression during recovery reflected the recruitment of alpha-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1alpha expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.

The purpose of this study was to examine the localization of macrophages, B-lymphocytes and osteoclasts in tumoral lesions of mammary carcinoma metastasized to bone of non-immunocompromised mice. Mouse mammary carcinoma cells (BALB/c-MC) were injected through the left cardiac ventricle into 5-week-old female wild-type Balb/c mice. The femora and tibiae of mice with metastasized cancer were extracted, and thereafter processed for histochemical analyses. The foci of metastasized tumor cells occupied the metaphyseal area, and the cell death zones could be identified within the tumor mass. Abundant tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were found among the alkaline phosphatase (ALP)-reactive osteoblastic cell layer that covered the bone surface neighboring the metastatic lesion. In contrast, F4/80-positive macrophages/monocytes were localized adjacent to, or invading the metastatic tissue. In addition, some F4/80-positive cells were found in the aforementioned cell death zones. Unlike F4/80-positive cells, CD45R-positive B-lymphocytes did not accumulate at the surfaces of the tumor lesions, nor infiltrate into them, but were found scattered over bone marrow. Interestingly, some CD45R-positive cells were observed close to TRAP-positive osteoclasts in the stromal tissue surrounding the tumor lesion. Our findings suggest that, in the bone metastatic lesions of non-immunocompromised mice, F4/80-positive macrophages/monocytes accumulated on and/or infiltrated into the tumor nests, while CD45R-positive B-lymphocytes were associated with osteoclasts, rather than attacking metastatic tumor cells.

Background: Stromal-epithelial interactions are of particular significance in breast tissue as misregulation of these interactions can promote tumorigenesis and invasion. Moreover, collagen-dense breast tissue increases the risk of breast carcinoma, although the relationship between collagen density and tumorigenesis is not well understood. As little is known about epithelial-stromal interactions in vivo, it is necessary to visualize the stroma surrounding normal epithelium and mammary tumors in intact tissues to better understand how matrix organization, density, and composition affect tumor formation and progression., Methods: Epithelial-stromal interactions in normal mammary glands, mammary tumors, and tumor explants in three-dimensional culture were studied with histology, electron microscopy, and nonlinear optical imaging methodologies. Imaging of the tumor-stromal interface in live tumor tissue ex vivo was performed with multiphoton laser-scanning microscopy (MPLSM) to generate multiphoton excitation (MPE) of endogenous fluorophores and second harmonic generation (SHG) to image stromal collagen., Results: We used both laser-scanning multiphoton and second harmonic generation microscopy to determine the organization of specific collagen structures around ducts and tumors in intact, unfixed and unsectioned mammary glands. Local alterations in collagen density were clearly seen, allowing us to obtain three-dimensional information regarding the organization of the mammary stroma, such as radiating collagen fibers that could not have been obtained using classical histological techniques. Moreover, we observed and defined three tumor-associated collagen signatures (TACS) that provide novel markers to locate and characterize tumors. In particular, local cell invasion was found predominantly to be oriented along certain aligned collagen fibers, suggesting that radial alignment of collagen fibers relative to tumors facilitates invasion. Consistent with this observation, primary tumor explants cultured in a randomly organized collagen matrix realigned the collagen fibers, allowing individual tumor cells to migrate out along radially aligned fibers., Conclusion: The presentation of these tumor-associated collagen signatures allowed us to identify pre-palpable tumors and see cells at the tumor-stromal boundary invading into the stroma along radially aligned collagen fibers. As such, TACS should provide indications that a tumor is, or could become, invasive, and may serve as part of a strategy to help identify and characterize breast tumors in animal and human tissues.

This study was to demonstrate by histological grading whether soy protein protects against dimethylbenz[a]anthracene (DMBA) -induced breast tumors in female rats. At 25 days of age, rats were fed diets containing either casein or soy protein. After 25 days on diets, a single dose of DMBA in sesame oil (80 mg/kg) was administered by gavage. All tumors were detected by palpation. The number of tumors per rat was less in soy group than that in casein group at any time point up to 122 days after DMBA administration. Incidence of tumors was less in soy protein group than that in casein group. Casein group had 20% grade I, 60% grade II, and 20% grade III adenocarcinoma. However, the soy group had 100% grade I adenocarcinoma and no aggressive grade II or grade III tumor. There was a delay in the development of tumor in the soy protein group in comparison to the casein group. Again, unlike casein, the soy group had cessation of angiogenesis at several sites of tumor, and reduced levels of angiogenic markers, VEGF and bFGF. Immunohistochemical analysis of the breast tissues did not show any CD-31 positive stain in soy protein group, whereas some CD-31 positive stain was revealed in casein group, which further suggests that soy protein controls angiogenesis. Furthermore, proliferative index as assessed by Ki-67 staining was less in soy protein group than that in casein group. These findings suggest that the soy protein may protect against the development of a more aggressive breast carcinoma.

We recently reported that uPARAP/Endo180 can mediate the cellular uptake and lysosomal degradation of collagen by cultured fibroblasts. Here, we show that uPARAP/Endo180 has a key role in the degradation of collagen during mammary carcinoma progression. In the normal murine mammary gland, uPARAP/Endo180 is widely expressed in periductal fibroblast-like mesenchymal cells that line mammary epithelial cells. This pattern of uPARAP/Endo180 expression is preserved during polyomavirus middle T-induced mammary carcinogenesis, with strong uPARAP/Endo180 expression by mesenchymal cells embedded within the collagenous stroma surrounding nests of uPARAP/Endo180-negative tumor cells. Genetic ablation of uPARAP/Endo180 impaired collagen turnover that is critical to tumor expansion, as evidenced by the abrogation of cellular collagen uptake, tumor fibrosis, and blunted tumor growth. These studies identify uPARAP/Endo180 as a key mediator of collagen turnover in a pathophysiological context.

I have used the paradigm of mammary cancer induction by the mouse mammary tumor virus (MMTV) to illustrate the body of evidence that supports the hypothesis that mammary epithelial stem/progenitor cells represent targets for oncogenic transformation. Further, it is argued that this is not a special case applicable only to MMTV-induced mammary cancer, because MMTV acts as an environmental mutagen producing random interruptions in the somatic DNA of infected cells by insertion of proviral DNA copies. In addition to disrupting the host genome, the proviral DNA also influences gene expression through its associated enhancer sequences over significant inter-genome distances. Genes commonly affected by MMTV insertion in multiple individual tumors include the Wnt genes, the fibroblast growth factor (FGF) gene family, and the Notch gene family. All of these gene families are known to play essential roles in stem cell maintenance and behavior in a variety of organs. The MMTV-induced mutations accumulate in cells that are long-lived and possess the properties of stem cells, namely, self-renewal and the capacity to produce divergent epithelial progeny through asymmetric division. The evidence shows that epithelial cells with these properties are present in normal mammary glands, may be infected with MMTV, and become transformed to produce epithelial hyperplasia through MMTV-induced mutagenesis and progress to frank mammary malignancy. Retroviral marking via MMTV proviral insertion demonstrates that this process progresses from a single mammary epithelial cell that possesses all the features ascribed to tissue-specific stem cells.

An experimental model developed to investigate premalignant stages of breast cancer was used to establish a rationale for designing experiments that target angiogenesis for cancer prevention. Blood vessels were identified via CD31 immunostaining, and all vessels that occurred in a 50 microm wide region circumscribing each pathology were counted using a digital imaging technique. The blood vessel density associated with terminal end buds was unaffected by carcinogen treatment, whereas vessel density was higher in intraductal proliferations and ductal carcinoma in situ than in terminal end buds (P < 0.001) and total vascularity increased with morphologic progression. In comparison with intraductal proliferation or ductal carcinoma in situ, mammary carcinomas had higher vascular density in the tissue surrounding the cancer with a marked increase in the number of blood vessels <25 microm(2). These data suggest that antiangiogenic chemopreventive agents would inhibit cancer occurrence if initiated at any premalignant stage of the carcinogenic process. Because increased vascular density observed during premalignancy could be explained by the size expansion of the lesion and its encroachment on a preexisting blood supply, by pathology-associated vessel expansion, and/or by angiogenesis, it remains to be determined if antiangiogenic agents will reduce the prevalence of premalignant lesions or cause their accumulation by blocking conversion to carcinomas. Failure to recognize the patterns of vascularization that accompany morphologic progression could limit the success of efforts to target angiogenesis for cancer prevention and lead to misinformation about how agents that affect blood vessel formation or growth inhibit the carcinogenic process.

A novel injectable thermosensitive in situ gelling hydrogel has been developed. The system, which falls under the BST-Gel platform technology developed at Biosyntech Inc. (Laval, QC, Canada), consists of a chitosan solution (C) neutralized with beta-glycerophosphate (GP) that is liquid at room temperature but gels when heated to body temperature. We propose to use this thermosensitive hydrogel for the sustained release of paclitaxel at tumor resection sites in order to prevent local tumor recurrence. The in vitro release profiles demonstrated controlled delivery over 1 month. The initial drug loading substantially affected the release. Local delivery of paclitaxel from the formulation injected intratumorally was investigated using EMT-6 tumors implanted subcutaneously on Balb/c mice. These experiments showed that one intratumoral injection of the thermosensitive hydrogel containing paclitaxel was as efficacious as four intravenous injections of Taxol in inhibiting the growth of EMT-6 cancer cells in mice, but in a less toxic manner. Further histological analysis revealed that while the proportion of necrotic areas was similar for the C/GP/paclitaxel and the Taxol-treated tumors, a disparity between tumor-associated inflammatory cell populations may suggest differing anti-tumor mechanisms.

FTY720 is a unique immunosuppressive agent that exerts its activity by inducing apoptosis in lymphocytes. We conducted the present study to investigate the effects of FTY720 on cancer growth and metastasis, as well as its mechanism of action. In vitro treatment with FTY720 induced dramatic cancer cell apoptosis in a mouse breast cancer cell line, JygMC(A). Electron microscopy revealed distinct changes on the cell surface with decreased filopodias and microvilli in cancer cells treated with FTY720 at 2 microM and clear evidence of apoptosis at 10 microM. Interestingly, the effect of FTY720 was significantly less in the normal fibroblasts than in the cancer cells, indicating greater susceptibility of cancer cells to the agent. We then tested the in vivo effect of FTY720 in a mouse breast cancer model created by inoculating JygMC(A) cells (s.c.) in the flank region of BALB/c-nu/nu mice at three different dosages (2, 5, and 10 mg/kg/day; n = 30/group). Tumor growth was markedly suppressed at a dosage of 5 mg/kg or more without notable side effects. In addition, tumor metastasis, which was dramatically evident in control mice, was significantly prevented even at a low dose (2 mg/kg/day), resulting in a significant prolongation of animal survival. These data led us to additionally investigate the mechanism of action, especially the prevention of metastasis at a low dose. FTY720 treatment at 2 microM caused a remarkable cytoskeletal change with deformed and decreased filopodias in cancer cells. In addition, it significantly decreased the ability of cancer cells to adhere and migrate to extracellular matrix components, and markedly reduced the expression of integrins on the cancer cell surface. These results indicate that FTY720 is a potent anticancer agent that induces cancer cell apoptosis and is markedly effective for prevention of metastasis. The changes of cellular structure with reduction of integrin expression may be one of its underlying mechanisms of action.

The initiation of T-cell-mediated antitumor immune responses requires the uptake and processing of tumor antigens by dendritic cells and their presentation on MHC-I molecules. Here we show in a human in vitro model system that exosomes, a population of small membrane vesicles secreted by living tumor cells, contain and transfer tumor antigens to dendritic cells. After mouse tumor exosome uptake, dendritic cells induce potent CD8+ T-cell-dependent antitumor effects on syngeneic and allogeneic established mouse tumors. Therefore, exosomes represent a novel source of tumor-rejection antigens for T-cell cross priming, relevant for immunointerventions.

Previous studies showed that the hormone relaxin acts on human breast cancer MCF-7 cells in vitro by modulating cell proliferation and promoting cell differentiation toward a duct epithelial phenotype. The present study was designed to investigate whether relaxin retains these properties when acting in vivo on MCF-7 cell tumors developed in athymic nude mice. Mice bearing MCF-7 cell tumors transplanted under the mammary fat pad and estrogenized to sustain tumor growth were treated systemically with relaxin (10 microg/day) for 19 days. Vehicle-treated mice were used as controls. Thirty days later, the mice were sacrificed and tumor fragments were analyzed by light and electron microscopy and immunocytochemistry. Measurements of tumor volume were recorded weekly for the overall experimental period. The results obtained indicate that relaxin treatment promotes differentiation of tumor cells towards both myoepithelial-like and epithelial-like cells, as judged by the ultrastructural features of the cells and by the increased expression of smooth muscle actin and cadherins. Measurements of tumor size and of the number of cycling cells show that relaxin, at the doses and times of exposure used in this study, does not significantly influence tumor growth and cell proliferation.

Using both light and high resolution electron microscopy, we analyzed the spatial and temporal relationships between the Arp2/3 complex and the nucleation activity that is required for lamellipod extension in mammary carcinoma cells after epidermal growth factor stimulation. A rapid two- to fourfold increase in filament barbed end number occurs transiently after stimulation and remains confined almost exclusively to the extreme outer edge of the extending lamellipod (within 100-200 nm of the plasma membrane). This is accompanied by an increase in filament density at the leading edge and a general decrease in filament length, with a specific loss of long filaments. Concomitantly, the Arp2/3 complex is recruited with a 1.5-fold increase throughout the entire cortical filament network extending 1-1.5 microm in depth from the membrane at the leading edge. The recruitment of the Arp2/3 complex at the membrane of the extending lamellipod indicates that Arp2/3 may be involved in initial generation of growing filaments. However, only a small subset of the complex present in the cortical network colocalizes near free barbed ends. This suggests that the 100-200-nm submembraneous compartment at the leading edge of the extending lamellipod constitutes a special biochemical microenvironment that favors the generation and maintenance of free barbed ends, possibly through the locally active Arp2/3 complex, severing or decreasing the on-rate of capping protein. Our results are inconsistent with the hypothesis suggesting uncapping is the dominant mechanism responsible for the generation of nucleation activity. However, they support the hypothesis of an Arp2/3-mediated capture of actin oligomers that formed close to the membrane by other mechanisms such as severing. They also support pointed-end capping by the Arp2/3 complex, accounting for its wide distribution at the leading edge.

[meso-1,2-Bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]- dichloroplatinum(II) (meso-1-PtCl2), an estrogenic and cytotoxic platinum complex, shows activity against ER+ but not against ER- breast cancers in vivo (ER: estrogen receptor; ER+ and ER- indicate the presence or absence of the ER). To clarify whether its estrogenic or its cytotoxic potency or both properties are the cause of this specific inhibitory effect, we tested meso-1-PtCl2 comparatively in vivo on an ER+ and an ER- murine breast cancer (MXT-M-3.2 MC and MXT-M-3.2(ovex) MC, respectively), and in vitro on two cell lines derived from the former in vivo models (MXT+ and MXT-, respectively). The estrogens diethylstilbestrol (DES) and the ligand of meso-1-PtCl2 (meso-1), responsible for the hormonal effect of meso-1-PtCl2, and the cytotoxic drug cisplatin (cDDP) were used as comparative substances. Meso-1-PtCl2. DES and cDDP showed a strong and comparable activity on the ER+ MXT-M-3.2 MC in vivo, meso-1 being somewhat less inhibitory. In experiments on the murine, ER- MXT-M-3.2(ovex) MC only cDDP caused a marked inhibitory effect. The other compounds were inactive or only marginally active. In accordance with the in vivo results cDDP was also very active on the MXT+ and MXT- breast cancer cell line. In contrast to this meso-1-PtCl2, meso-1, and DES proved to be only weakly active or inactive on both cell lines. From these results it can be concluded that there is only little if any contribution of the cytotoxic PtCl2 moiety of meso-1-PtCl2 to the anti-breast cancer activity in vivo. On the ER+ MXT-M-3.2 MC transplanted into ovariectomized mice meso-1-PtCl2 yielded a biphasic dose activity curve, i.e. an increase of the tumor growth at low doses followed by a decrease at high doses, identical with those of the estrogens DES and meso-1. These results indicate that meso-1-PtCl2 inhibits ER+ breast cancers by its estrogenicity in the same manner as meso-1 and DES. The complex mechanism of anti-breast cancer active estrogens involves presumably the endocrine and/or the immune system. Its investigation is the subject of further studies.

Injection of interleukin-4 (IL4) gene-transduced tumour cells into syngeneic immunocompetent mice resulted in tumour rejection in which a key role for eosinophils was suggested. To evaluate whether IL5 inhibits tumour growth by selectively inducing eosinophil recruitment and activation, a poorly differentiated mammary adenocarcinoma cell line (TSA) was transfected with the IL5 gene and the cells secreting IL5 (TSA-IL5) were injected subcutaneously (s.c.) in syngeneic mice. The oncogenicity of TSA-IL5 was compared with that exhibited by TSA cells transfected with the IL4 gene (TSA-IL4) and with the neomycin resistance gene only (TSA-neo). At progressive times after subcutaneous challenge, tumour growth areas were studied histologically, ultrastructurally, and immunohistochemically to identify the reactive cells, visualize tumour vessels, and detect the cytokines and chemokines involved in the anti-tumour reaction. Both the morphological and the functional data showed that TSA-IL5, despite the large eosinophil infiltrate, grew progressively like TSA-neo, suggesting that eosinophils per se do not play a crucial role in TSA tumour rejection. Furthermore, our data indicate that the rejection of TSA-IL4 depends on the IL4-induced expression of VCAM-1 and MCP-1 by endothelial cells. MCP-1 together with VCAM-1 results in recruitment and activation of basophils, mast cells, and macrophages, and hence a pro-inflammatory cytokine cascade that initially favours the influx and activation of neutrophils and finally tumour rejection. In this context, the rejection of TSA-IL4 seems to involve a variety of reactive cells and rests on a continuous cross-talk between basophils, mast cells, macrophages, CD8-positive lymphocytes, and granulocyte subsets, mostly neutrophils.

Both endogenous and exogenous estrogen exposure is associated with an increased breast cancer risk. In some studies, elevated serum testosterone levels have also been linked to an increased breast cancer risk. Estrogen alone or combined with progesterone induces high mammary tumor incidences in various strains of both male and female rats. Mammary gland ductal adenocarcinomas were induced after 17beta-estradiol (E2) and testosterone propionate (TP) treatment in male Noble rats. Tumor incidence was 100% after 8-9 months of treatment. Such neoplasms were not detected after either estrogen or androgen exposure alone within this time period. TP alone caused disruption of mammary gland ducts and proliferation of stromal tissue, while E2 treatment alone induced both ductal epithelial growth and nodular atypical hyperplasia. To study the interaction of these hormones in mammary tumorigenesis, sex hormone receptors were characterized in mammary glands of Noble rats. Estrogen receptor-alpha (ER) was detected in age-matched, untreated mammary gland epithelium; in most early atypical hyperplastic lesions appearing after E2 and E2 + TP treatment and in E2 + TP-induced mammary tumors. Two major ER putative isoforms, 116 and 120 kDa, were detected in E2- and E2 + TP-treated mammary glands, and in the induced tumors. A 54 kDa ER protein was found in untreated and TP-treated mammary glands, and in the induced tumors. Both progesterone receptor-B (PR-B) and PR-A2, as well as androgen receptor-B (AR-B) and AR-A isoforms were markedly elevated in all E2 + TP-induced mammary tumors. However, the levels of both PR and AR were very low in mammary glands of E2- and E2 + TP-treated male rats. Low and moderate levels of AR and PR, respectively, were detected in most atypical hyperplastic lesions induced by E2- and E2 + TP-treated mammary glands. These results suggest that androgens may interact with either AR or PR, and perhaps both receptors, in E2 + TP-induced mammary glands and the induced tumors to effect the reduction in latency period, enhance tumor size, and increase incidence to 100%.

To investigate the roles of telomere length (mean length of the terminal restriction fragments; TRFs), telomerase activity (TA) and telomerase RNA (mTR) expression in relation to mouse mammary tumor progression, we examined a pregnancy-dependent mouse mammary tumor line (TPDMT-4) and its four autonomous sublines (T4-OI320: non-metastatic; and T4-OI165, -OI96, and -OI145: artificial metastatic) of DDD/1 mouse origin, and an autonomous growing mammary tumor (JYG-MC) showing spontaneous lung metastasis developed in BALB/c mice infected with a Chinese feral mice (Sub-Jyg)-derived mouse mammary tumor virus (JYG-MTV). Compared with normal (pregnant) mammary tissue, the TA was elevated in the TPDMT-4 tumor and in the non-metastatic subline tumor (T4-OI320) (x10 fold, respectively), and was further increased (x13-15 fold) in parallel with the acquisition of metastatic potential (T4-OI165, -OI96, and -OI145). The mTR level was upregulated (x2.7-2.8 fold) in all autonomous growing tumors compared to the normal counter-part, but not in TPDMT-4. The TRF was shorter in accord with tumor progression (normal mammary tissue, 48 kb; TPDMT-4, 45 kb; T4-OI320, 37 kb; T4-OI165, -OI96 and -OI145, mean 37.7 kb; and JYG-MC, 21 kb). These results suggest that the activation of TA occurs as an early event at the stage of hormone-dependent tumorigenesis, followed by the up-regulation of mTR expression in accordance with the acquisition of autonomous growth, and then further activation of TA occurs when the tumor acquires metastatic potential. The TRF shortening was in parallel with the tumor progression.

We have established a newly derived breast cell line, called G8. This line was obtained by means of the neoplastic transformation of murine breast cells through exposure to the carcinogen NMU and its subsequent inoculation in athymic nude mice. This cell line is highly tumorigenic in nude mice. The tumors that developed in the mice were mammary adenocarcinomas. The morphological and ultrastructural pattern suggested that these cells are of epithelial origin. Immunocytochemical studies revealed that the cells express desmoplakin, vimentin, epithelial membrane antigen, fibronectin and actin. The possibility of maintaining this cell line in vivo by means of xenografts provides very valuable material for breast cancer research, as not only does tumor growth in vivo provide the ideal material for the testing of new therapies, but it also enables the study of various important interactions between the tumor and the host tissues including tumor-stroma interactions such as angiogenesis.

Loss of heterozygosity (LOH) at the mannose 6-phosphate/insulin-like growth factor 2 receptor gene locus (M6P/IGF2R) on 6q26-27 has recently been demonstrated in approximately 30% of both invasive and in situ breast cancers. LOH was coupled with somatic point mutations in the remaining allele in several instances, leading to the proposition that M6P/IGF2R is a tumor suppressor gene. Somatic mutations in M6P/IGF2R have also been described in hepatoma and gastrointestinal cancers with the replication error positive (RER+) phenotype. These data indicate that M6P/IGF2R loss of function mutations may be involved in the pathogenesis of a wide spectrum of malignancies. Extensive data on the normal function of the M6P/IGF2R suggest that loss of M6P/IGF2R activity may contribute to multiple aspects of tumor pathophysiology, including deregulated growth, apoptosis, angiogenesis and invasion.

To test the hypothesis that the transforming growth factor-beta (TGF-beta) system has tumor suppressor activity in the mammary gland, we have generated transgenic mice overexpressing a dominant-negative mutant form of the type II TGF-beta receptor, under the control of the mouse mammary tumor virus-long terminal repeat. High-level expression of the transgene was observed in the mammary and salivary glands, with lower expression in the lung, spleen, and testis. Older nulliparous transgenic mice (9-17 months) showed a marked increase in the incidence and degree of lobulo-alveolar side-branching in the mammary glands when compared to wild-type littermates (24.8% of glands examined histologically versus 14.4%; P = 0.004), suggesting a role for endogenous TGF-betas in regulating development or maintenance of mammary alveoli. Spontaneous tumorigenesis was unchanged in the transgenic mice. However, following initiation with the carcinogen 7,12-dimethylbenz[a]anthracene, the transgenic group showed a significant increase in the incidence and multiplicity of mammary tumors when compared with wild-type littermates (40% incidence in transgenic mice versus 22% for wild-type, with 4 of 25 transgenics developing multiple mammary tumors versus 0 of 27 wild-type; P = 0.03). An early increase in the incidence of lung tumors was also observed in transgenic mice, but no difference between genotype groups was seen in the incidence of tumors in tissues in which the transgene is not expressed. The data show that the endogenous TGF-beta system has tumor suppressor activity in the mammary gland and lung.

Purpose: To study the cell kinetics and chromatin modifications occurring in function of the fractionated irradiation administered to the MXT mouse mammary adenocarcinoma., Methods and Materials: The MXT tumor cells were submitted to three fractions of a 4.8 Gy dose delivered at 24-h intervals. MXT tumor cells were collected by means of fine needle aspirations (between 5 and 10 samples were obtained after each irradiation) during treatment and submitted to the computer-assisted microscope analysis of Feulgen-stained specimens. Three groups of parameters has been described: i.e., the geometry of the nucleus, the nuclear DNA content, and the chromatin texture. Furthermore, cell cycle parameters were studied in the aim to know the distribution of the cells within the cell cycle., Results: The mean values relating to geometric parameters (i.e., the nuclear area and its standard deviation) decreased during treatment. Variations in the nuclear DNA content appeared as being cyclical and could be explained in terms of the modifications in the distribution of the cells within the cell cycle. The quantitative analysis of the cell cycle parameters revealed that the percentage of S cells increased regularly after each irradiation. In contrast, the percentage of G2 cells decreased between each irradiation. The parameters describing nuclear texture showed regular variations between each irradiation. These variations consisted in two cycles constituted by a decrease in chromatin condensation, followed by an increase., Conclusions: The development of the geometric parameters indicates that fractionated radiotherapy leads to the emergence of a more homogeneous population. The effects of the radiotherapy on the distribution of the cells within the cell cycle could be explained through the phenomenon of repopulation and by the high degree of radiosensitivity of the G2 cells (decrease in the percentage of G2 cells). Last, the variations observed at chromatin pattern level could be explained through DNA repair processes.

Breast tumors from postmenopausal women contain levels of estradiol similar to those in premenopausal patients even though serum estradiol levels fall by an order of magnitude upon cessation of ovarian function. The present study sought to examine enhanced uptake from plasma as one potential mechanism for maintenance of high tissue estradiol levels in postmenopausal patients. Accordingly, we used osmotic minipumps to continuously infuse estradiol (E2) at rates producing serum concentrations ranging from pre- to postmenopausal levels for two weeks to oophorectomized Sprague-Dawley rats bearing nitrosomethylurea-induced mammary tumors. We then measured E2 concentrations in various tissues and sera and reasoned that tissue affinities for estradiol could be directly calculated from in vivo measurements by adapting Scatchard analysis to steroid infusion data. Using this method, we demonstrated a very high affinity estradiol binding component with a Kd two orders of magnitude higher (i.e., 0.35 x 10(-12) M) than determined with standard in vitro techniques. A second estradiol binding component with the expected Kd of 1 x 10(-10) M was also present. Estradiol bound to both classes of binding sites could be 98% displaced with diethylstilbestrol within a 6-hr period. In vivo steroid binding off-times calculated from log-linear slopes averaged approximately 60 min. These data demonstrated that the actual E2 binding affinity in target tissues in vivo, especially at low estrogen concentrations, is much higher than usually estimated from standard, in vitro estrogen receptor assays. These observations provide one mechanism to explain why estradiol concentrations remain high in breast cancer tissue from postmenopausal women and consequently can stimulate tumor proliferation.

We studied the effects of different dietary fats on experimental rat mammary lumorigenesis induced by 9,10-dimethyl-1, 2-benzanthracene (DMBA). Rats were randomly placed into four groups fed different diets: a chow diet, and high-fat (15%) diets derived from avocado, soybean or olive oils. The rats were killed 12 weeks after treatment with DMBA (a single dvse of 10 mg/rat) and maintenance on these diets. The olive diet was associated with a significant reduction in the tumorigenic effect of DMBA: tumor incidence decreased to 30%, as compared to 44%-55% in the other dietary groups studied (p < 0.05). The protective antitumor effect of the olive diet was found to be connected to its dietary content of monounsaturated fatty acids such as oleic and palmitic acids and with serum concentrations of stearic acid. The promotive tumorigenic effects of the other high-fat diets were associated with their high levels of some polyunsaturated fatty acids (linoleic and alinolenic). Malignant mammary tissue exhibited higher values than benign tissue for all the argyrophilic-nucleolar-organizer region parameters measured. The tumor-associated protein p53 was accumulated to high levels in the blood of tumor-bearing rats, but not in that of the non tumor-bearing rats.

The rejection of interferon alpha 1 gene-transfected mammary adenocarcinoma cells (TSA-IFN alpha) injected into syngeneic BALB/c mice was accompanied by an unusual stromal reaction and marked CD8-positive T-lymphocyte involvement. To investigate the biological background of this reaction, the possibility was evaluated that an interaction between TSA-IFN alpha and stromal cells might remodel the extracellular matrix (EM). When fibroblasts were co-cultured with TSA-IFN alpha or treated with exogenous IFN alpha, there was no change in their replication rate or collagen synthesis. By contrast, their fibronectin (FN) production and release were increased, resulting in enhanced fibroblast chemotaxis. These findings were mirrored by increased FN staining in the peritumoural and tumoural areas in vivo. IFN alpha thus determines increased FN production and hence massive local recruitment and activation of fibroblasts, with a modification of the EM. The several activities of IFN alpha should thus be considered prior to its employment in clinical trials.

We have examined the role of urokinase receptor (uPAR) in tumor invasion and metastasis by developing a homologous model of uPAR overexpression in a rat breast cancer cell line (Mat B III) using gene transfer technique. Control (pRc-CMV) and experimental plasmid (pRc-uPAR-S) were transfected into Mat B III cells by using Lipofectin reagent. Levels of uPAR production were accessed by Northern blotting, immunofluorescence, receptor binding and ELISA. At least 3 experimental clones (pRc-uPAR-S), expressing 3- to 5-fold higher levels of uPAR than control (pRc-CMV), were selected for further analysis. Experimental cells overexpressing uPAR showed a 4-to 5-fold higher invasive capacity compared with control cells in a Boyden chamber invasion assay. Both control and experimental cells (1 x 10(6) cells) were injected into the mammary fat pad of syngeneic female Fischer rats. Animals were sacrificed at timed intervals and evaluated for the development of tumor growth and metastasis. Animals receiving cells overexpressing uPAR had significantly larger tumor volume and weight throughout our study. Furthermore, due to increased uPAR expression, experimental animals developed large metastatic lesions in liver, spleen and lymph nodes. Our results therefore demonstrate the role of uPAR in tumor progression, due to its ability to localize uPA within the tumor cell milieu.

This study was aimed at developing a hydrophilic diethylenetriaminepentaacetic acid-tamoxifen (DTPA-Tam) analogue for use in imaging estrogen receptor positive (ER+) lesions. In rat uterine cytosol, the IC50 of DTPA-Tam conjugate was 1 microM and of tamoxifen, 2 microM. Biodistribution, autoradiography, and radionuclide imaging of 111In-DTPA-Tam in breast-tumor-bearing rats showed that tumor-to-tissue ratios increased steadily between 30 min and 48 h. The in vivo response of MCF-7 breast cancer xenografts to tamoxifen and DTPA-Tam in nude mice demonstrated that DTPA-Tam could reduce tumor growth rate. These results indicate that DTPA-Tam, a new hydrophilic ER+ ligand, might be useful in diagnosing ER+ lesions.

The effect of tamoxifen (TAM) was evaluated on a mammary tumor model induced in Sprague-Dawley rats by intraperitoneal administration of three N-nitroso-N-methylurea (NMU) doses. Animals received TAM (1 mg/kg per day) from 10 days before the first NMU dose up to 140 days later. Thereafter, treatment was discontinued and the observation period was extended 60 days longer. Mean overall latency period, tumor number per rat and tumor incidence were recorded. Significant differences between treated and control batches were observed in tumor number per rat (1.8 +/- 1.1 versus 5.2 +/- 1.6; P < 0.05) and in tumor incidence (50% versus 100%; P < 0.05), respectively. No significant difference in latency period between both batches was recorded. All lesions induced in the control batch were malignant, whereas only 45% of those induced in TAM-treated animals were malignant and the remaining 55% were preneoplastic. At 60 days after treatment discontinuance, tumor incidence increased to 90% and also tumor number per rat increased to 4.6 +/- 1.5. TAM effect was also evaluated in rats with NMU-induced tumors by treatment with 1 mg/kg per day during 60 days starting when tumors reached a 1.5-cm diameter. Regression to less than 80% of initial size in 49% of the tumors was observed, while in ovariectomized rats, 33% of tumors regressed. Estrogen receptor content, ER (fmol/mg protein) and Kd (nM) in control tumors were: 56 +/- 10 and 0.5 +/- 0.1. In tumors of TAM-treated animals, ER was less than 5 fmol/mg protein. Findings demonstrate that TAM significantly decreased the appearance of tumors induced in rats by i.p. injection of NMU and when TAM treatment was initiated after tumor induction, some tumors failed to respond to hormonal manipulation. Differential tumor growth response after TAM or oophorectomy in each tumor indicates that in the same rat it is possible to distinguish hormone-dependent and hormone-autonomous tumor populations. Hormonal regulation of tumor growth can be under intrinsic control, regardless of the hormonal status of the whole organism.

An N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin conjugate is currently under clinical evaluation as a new antitumour agent. It has been shown previously that such conjugates exhibit selective tumour accumulation. In this study HPMA copolymer doxorubicin conjugates of low (LMW) or high (HMW) molecular weight were synthesised (which had a weight average molecular weight (Mw) of 25,000 and 94,000 respectively) and additionally contained a small amount (1 mol%) of the comonomer methacryloyltyrosinamide to permit labelling with [123I or 125I]iodide. Gamma camera imaging using the 123I-labelled probes was used to follow time-dependent biodistribution after intraperitoneal (i.p.) or intravenous (i.v.) administration to mice bearing subcutaneously either B16F10 melanoma or a mammary carcinoma. Imaging showed more rapid clearance of LMW conjugate from the peritoneal cavity than HMW conjugate. The images of mice given the LMW conjugate revealed rapid urinary excretion of radioactivity after both i.p. and i.v. injection with an early high concentration of tracer in the bladder, and subsequently a very high concentration in the kidneys, which came to dominate the views. Dissection analysis 2 days after administration of the LMW conjugate revealed a kidney level of radioactivity corresponding to 25-40% dose/g tissue in mice bearing the two tumour models. Following administration of the HMW conjugate kidney accumulation at 2 days was less due to retention of the higher molecular weight polymer molecules in the circulation, and spleen and liver displayed the highest concentrations of radioactivity. The tumour accumulation of LMW and HMW conjugates was; mammary carcinoma 3.18 and 5.29% dose/g respectively; B16F10 melanoma 3.23 and 8.82 %dose/g although these levels of tracer enabled visualisation in the images of the mammary carcinoma with HMW conjugate at later time points. The smaller size of the B16F10 tumour masses did not permit clear visualisation.

A bioluminescence technique involving single photon imaging was used to quantify the spatial distribution of the metabolites ATP, glucose and lactate in cryosections of various solid tumours and normal tissue. Each section was covered with an enzyme cocktail linking the metabolite in question to luciferase with light emission proportional to the metabolite concentration. The photons emitted are imaged directly through a microscope and an imaging photon counting system. In some cases, good agreement was observed between the distribution of relatively high concentrations of ATP and glucose in viable cell regions of the periphery, while the reverse was seen in more necrotic tumour centres with comparatively high lactate levels. In general, lactate was distributed more diffusely over the sections while ATP was more highly localised and glucose assumed an intermediate pattern. In contrast to the large degree of heterogeneity seen in tumours, distribution patterns of metabolites were much more homogeneous in normal tissue, such as heart muscle. Mean values for metabolite levels in cryosections using bioluminescence are in good agreement with those obtained from the same tumour by conventional methods.

The MCM-B2 canine mammary cell line was serially transplanted into nude mice. The tumour masses consisted of elongated pleomorphic cells of varying size in the first to third passages; oval cells, becoming rounder, in the sixth to eighth passages; and cord-like, glandular and duct-like structures with compact radiating projections in the ninth and tenth passages. Ultrastructural and immunohistochemical examination of round cells confirmed their epithelial cell nature, but the morphology of the elongated and oval cells was identical with that of the original cell line. The findings suggest that the MCM-B2 cell line is a multipotential stem cell or is derived from glandular differentiation of mammary gland.

The effects of four Vinca alkaloids (vinblastine, vincristine, vindesine and vinorelbine) on three neoplastic cell lines (the MXT mouse mammary cell line and the T24 and J82 bladder cell lines) were studied at three biological levels, i.e. cell proliferation, cell cycle kinetics and morphonuclear characteristics. These effects were studied by means of digital cell image analysis on Feulgen-stained nuclei. The aim of the present work was to characterize the effects specifically induced by Vinca alkaloids as compared with those obtained previously with other pharmacological classes of anticancer drugs. The results show that Vinca alkaloids inhibit the cell proliferation of neoplastic cell lines at a concentration of 10(-8) M except in the case of the J82 cell line, for which only a slowing down of cell proliferation was observed. Concerning the cell cycle kinetics, the results show that the Vinca alkaloids induce an accumulation of cells in the mitosis phase. This accumulation of mitotic cells was maximal after 15 h incubation in the presence of the drugs. A study of the morphonuclear-induced effects of Vinca alkaloids showed that the variance of the optical density (VOD) is strongly influenced by these Vinca alkaloids. The development of the VOD was parallel with the development of the percentage of mitosis; thus, the VOD enabled the Vinca alkaloid-induced effects to be specifically characterized from a morphonuclear point of view. On the other hand, the results show that the mean value of the variance of the optical density was very highly correlated (P < 0.001) with the efficiency of the Vinca alkaloids in terms of cytotoxicity. In clinical studies, the analysis of the development of this parameter would make it possible to assess the response to chemotherapy in the case of patients treated with Vinca alkaloids.

The cDNA coding for mouse IL-10 (mIL-10) was transduced into the parental cells of a spontaneous adenocarcinoma of BALB/c mice (TSA-pc), and clones secreting small, medium, and large quantities of IL-10 were selected. In vivo, both low and high producer clones do not display an enhanced ability to grow in H-2 and non-H-2 incompatible mice. Instead, the intensity of their rejection increases in function of the amount of mIL-10 released. After an initial growth period in syngeneic mice, high producer clones undergo complete rejection due to the combined action of CD8+ lymphocytes, NK cells, and neutrophils. After this rejection, mice are immune to a subsequent challenge with TSA-pc. This memory rests on a strong lytic activity of CD8+ CTL and granulocytes. Following the rejection, mice also develop anti-TSA Ab that guide the granulocytes in TSA-pc memory reaction. A direct comparison shows that although TSA clones engineered to release IL-2 activate CTL and no anti-TSA Ab, those engineered to release IL-4 activate a strong Ab response but not CTL. The kind of cytokine released by the tumors appears to determine the type of response. However, IL-10 high producer cells do not deviate the immune memory, neither toward a Th1 nor a Th2. Both the CTL activity and the Ab responses induced by IL-10 high producer cells are the strongest so far observed in the TSA system.

By using a chemical extraction assay and confocal laser scanning fluorescence microscopy, the kinetic patterns of uptake, elimination, and localization of meso-tetra(hydroxyphenyl)porphine (m-THPP) and its corresponding chlorin (m-THPC) in tumors and various normal tissues of female C3D2/F1 mice bearing CaD2 mammary carcinoma were studied after an i.p. injection of either 5 mg/kg body weight of m-THPP or 1 mg/kg body weight of m-THPC. Moreover, the histological and ultrastructural alterations of the tumors were evaluated after photodynamic therapy (PDT) with m-THPP or m-THPC. The PDT efficacy with m-THPP and m-THPC was also compared. Both m-THPP and m-THPC had a similar kinetic pattern of distribution in the tumors and most normal tissues examined. The concentrations of the dyes in the tissues peaked at 24-48 h after injection. The peak values of the uptake of m-THPP by the tissues were found to decrease in the following order: spleen > urinary tract > kidney > liver > lung > tumor > heart > skin > muscle > brain. However, higher concentrations of m-THPC were taken up by the tumors than by most of the normal tissues studied except for the liver, urinary tract, and skin. m-THPP was mainly localized in the stroma of the tumors, whereas m-THPC was distributed in both vascular interstitium and neoplastic cells of the tumors. Morphological studies showed that PDT with m-THPP resulted in destructive changes in the microvasculature of the tumors, whereas m-THPC-based PDT destroyed both vascular walls and tumor cells of the tumors. The m-THPP-PDT of the tumors was much less efficient than m-THPC-PDT of the tumors, although the dose of m-THPP used was five times higher than that of m-THPC. m-THPP and m-THPC have different efficiency of sensitizing tumors to photodestruction, although they are similar with respect to hydrophobicity. This is likely due to the differences in their intratumoral localization patterns and in their absorption spectra.

Purpose: To present the effects of different radiotherapeutic treatments on the morphonuclear characteristics and growth of the MXT mouse mammary adenocarcinoma., Methods and Materials: We collected MXT tumor cells by means of fine-needle aspirations during various radiotherapeutic treatments and analyzed the morphological aspects of the cell nuclei by means of the digital cell image analysis of Feulgen-stained nuclei. In addition, we studied the morphonuclear aspects of cells from MXT tumors that had been radioresistant cell enriched. These radioresistant cell-enriched tumors involved MXT tumors that had survived one or two previous radiotherapies. The radiotherapy-induced effects on the morphonuclear characteristics were monitored by means of both monovariate (one-way variance) and multivariate (principal components and step-wise linear discriminant) analyses., Results: The monovariate analyses showed that radiotherapy significantly influenced the values of the parameters relating to nuclear size (nuclear area--NA), the frequency of small dense chromatin clumps (short run length emphasis--SRL) in the nuclei, and the overall chromatin condensation level (local mean--LM). The global effect corresponded to a decrease in the overall chromatin condensation level in the radioresistant cell-enriched MXT tumors. This decrease occurred concomitantly with an increase in the frequency of the small dense chromatin clumps in the nuclei and a decrease in the nuclear area. The multivariate analyses showed that it was possible to quantitate the proportion of "radiosensitive-like" and "radioresistant-like" cell nuclei in the various MXT tumor types under study., Conclusions: The development of certain morphonuclear parameters, that is, the NA, the SRL, and the LM, could be proposed to predict the response of human tumors to radiotherapy as, indeed, could the quantitation of the proportion of radioresistant cells.

To elucidate the relationship of estrogen-depleted condition to apoptosis and tumor regression, 7,12-dimethylbenz[a]anthracene-induced mammary cancers of Sprague-Dawley rats were ovariectomized or treated with the anti-estrogenic agent epitiostanol after which proliferative activity and the incidence of apoptosis were investigated using the nick end labeling method, agarose gel electrophoresis of DNA, electron microscopy, the BrdU-labeling method and mitotic count. Tumor regression was found after 7-day treatment, and apoptosis induced by the agent on the 3rd day was clearly shown in both agarose gel electrophoresis of DNA and electron microscopy, which are two major methods used to judge apoptosis. The incidence of apoptosis revealed by the nick end labeling method reached its maximum, about threefold the control level, on the 3rd day of epitiostanol treatment compared with control tumors (P < 0.01). The incidence of the cells incorporating BrdU reached its maximum of 9.7% on the 2nd day of the treatment, while the incidence in tumors without treatment was 7.5% (P < 0.05). Subsequently, the incidence of apoptosis was reduced after 7-day treatment, and the incidence of BrdU-positive cells was significantly reduced to about 3% after 5-day treatment. The incidence of mitosis did not change until the 3rd day of the treatment and was reduced after 5-day treatment. Similarly, chronological changes of the incidences of BrdU-labeled cells, apoptotic cells and mitosis were observed in the tumors after ovariectomy. BrdU-labeled apoptotic bodies were detected in the tumors on the 3rd day in epitiostanol-treated rats that received a 6-hr bolus of BrdU before sacrifice.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the biological effects of ablation-induced stress waves in vitro. Mouse breast sarcoma cells (EMT-6) were exposed to stress waves that differed only in rise time. Two assays were used to determine cell injury: incorporation of tritiated thymidine (viability assay), and transmission electron microscopy (morphology assay). We present evidence that the rise time of stress waves can significantly modify cell viability and that cell injury correlates better with the stress gradient than peak stress.

A retroviral infection was used to introduce the cDNA coding for mouse IL-4 into the parental cells of a spontaneous adenocarcinoma of BALB/c mice (TS/A-pc). Four clones releasing between 5 to 40 U of IL-4 (10(5) cells) in 48 h culture were selected. The secretion of IL-4 does not affect their in vitro growth, whereas their ability to form tumor in vivo inversely correlates with the amount of IL-4 secreted. Although morphologic observation suggested that the rejection of clone D5.40 cells (releasing 40 U of IL-4) depends on eosinophil cytolysis, lymphocyte depletion experiments showed that this required CD8+ lymphocyte guidance. Mice that had rejected D5.40 cells were immune to a subsequent challenge with TS/A-pc. This memory rests on the interaction between noncytotoxic lymphocytes, eosinophils, and IgG1 and IgE anti-TS/A Abs. Comparison of these memory mechanisms with those elicited by IL-2 gene-transduced TS/A cells shows that the kind of cytokine released by the tumor cells determines the type of response. This Th2 memory seems to be more efficient in protecting against a subsequent challenge of TS/A-pc than the Th1-type memory elicited by IL-2 gene-transduced TS/A cells.

Chlorotriazines are widely used in agriculture as broadleaf herbicides. The compounds specifically inhibit photosynthesis, and, as such, display little interaction with animal systems. However, a 24-month feeding study with atrazine (ATR) revealed a significant dose-related increase of mammary tumors in female Sprague-Dawley (SD) rats. Because numerous studies indicated that ATR had a low mutagenic and oncogenic potential, it was decided to test a hypothesis that the herbicide possessed endocrine activity. Among tests for estrogenic action, oral dosing of ATR up to 300 mg/kg did not stimulate uterine weight of ovariectomized rats. However, ATR administration did reduce estrogen-stimulated uterine weight gain. Further evidence of inhibition came from measures of [3H]-thymidine incorporation into uterine DNA of ATR-treated immature rats. Again, no intrinsic estrogenic activity was observed up to a 300-mg/kg dose. In vitro, ATR competed poorly against estradiol binding to cytosolic receptors, with an approximate IC50 of 10(-5) M. Atrazine administration to SD and Fischer-344 (F-344) rats for 12 months, up to 400 ppm in food, was correlated with significant alterations of estrous cycling activity; but there was a divergent strain response. SD rats showed an increased number of days in vaginal estrus, increased plasma estradiol, and decreased plasma progesterone by 9 to 12 months of treatment. F-344 rats did not demonstrate treatment-related affects. A study of ultrastructure in the hypothalamic arcuate nucleus of female SD rats that were fed diaminochlorotriazine (DACT), an ATR metabolite, suggested that age-associated glial pathology was enhanced by treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-to-cell communication via gap junctions has played a fundamental role in the orderly development of multicellular organisms. Current methods for measuring this function apply mostly to homotypic cell populations. The newly introduced Fluorescence Activated Cell Sorting (FACS) method, albeit with some limitations, is simple, reliable, and quantitative in measuring the dye transfer via gap junctions in both homotypic and heterotypic cell populations. In the homotypic setting, the result in dye transfer from the FACS method is comparable to the scrape-loading and microinjection methods. Using this FACS method, we observed a decline of cell-to-cell communication in transformed and cancer cells. We also observed a differential degree of communication between two heterotypic cell populations depending on the direction of dye transfer.

To evaluate the possible involvement of the salivary glands in the modulation of medroxyprogesterone (MPA)-induced mammary tumorigenesis, 48 sialoadenectomized virgin BALB/c female mice and 47 controls were treated with 40mg MPA depot s.c. every 3 months for 1 year. Mammary tumors developed in 11 sialoadenectomized and in 34 control mice with similar latencies. In both groups, 75% of the tumors were ductal and progestin-dependent (PD) while the remainder were lobular and progestin-independent (PI). Epidermal growth factor (EGF) levels were measured in salivary glands (SG-EGF) and serum (S-EGF) in both groups. MPA induced a significant increase in SG-EGF and in S-EGF that became evident only after 1 month of MPA treatment. No increase in S-EGF was detected in MPA-treated sialoadenectomized mice, indicating that salivary glands are the major source of S-EGF. The presence of EGF receptors (EGF-R) was investigated in ductal PD and PI tumor lines and compared with 8 PI tumor lines of lobular origin. A significant difference in EGF-R content was found between lobular and ductal tumors. No increase in EGF-R was noted when ductal tumors became autonomous. EGF-R did not correlate with tumor growth rate and there was an inverse correlation between EGF-R and steroid receptors. When the effect of sialoadenectomy on tumor growth was tested in vivo in syngeneic transplants of 2 ductal PD, 1 ductal PI and 2 lobular PI mammary adenocarcinomas, it was not found to be significant when compared with the controls. It may be concluded that SG-EGF plays an important role in the induction of mammary adenocarcinomas by MPA, while it has no significant effect on the growth of established tumors.

Scanning force microscopy (SFM) was used for imaging subcellular structures of cultured rat mammary carcinoma cells dried in air. Identification of cellular substructures was achieved by immunofluorescence and specific fluorescence probes. Cells grown attached to a glass support exhibited submicrometer thickness in the dried state. Inside the nuclear domain the nucleoli appeared as prominent conical protrusions. Membrane extensions, microspikes and microvilli were well preserved at the cell periphery after fixation in glutaraldehyde vapor and air-drying and were distinguishable either as isolated elements or intercellular communications. The plasma membrane and soluble proteins were selectively removed with nonionic detergent in a buffer system. The mitochondria were concentrated primarily in the perinuclear space and exhibited a well defined filamentous shape. Their identity was confirmed by specific fluorescence staining with rhodamine 123. In the membrane-free system achieved by dry-cleaving of the sample surface, the cytoskeletal network was resolved as a complex mesh of actin-containing fiber bundles interwoven with a filigree arrangement of thinner filaments. The smallest fibrous substructures revealed by SFM with the scanning tips used to date were approximately 8 to 10 nm in height and 80 nm in width.

Purpose: Gliomas, squamous carcinomas and different adenocarcinomas from breast, colon and prostate might have an increased number of epidermal growth factor (EGF) receptors. The receptors are, in these cases, candidates for binding of receptor specific toxic conjugates that might inactivate cellular proliferation. The purpose of this study was to evaluate whether it is reasonable to try ligand-dextran based conjugates for therapy., Methods and Materials: EGF or TGF alpha were conjugated to dextran and binding, internalization, retention and degradation of eight types of such conjugates were analyzed in EGF-receptor amplified glioma cells. The conjugates were labelled with radioactive nuclides to allow detection and two of the conjugates were carrying boron in the form of carboranyl amino acids or aminoalkyl-carboranes. Comparative binding tests, applying 125I-EGF, were made with cultured breast, colon and prostate adenocarcinoma, glioma and squamous carcinoma cells. Some introductory tests to label with 76Br for positron emission tomography and with 131I for radionuclide therapy were also made., Results: The dextran part of the conjugates did not prevent receptor specific binding. The amount of receptor specific binding varied between the different types of conjugates and between the tested cell types. The dextran part improved intracellular retention and radioactive nuclides were retained for at least 20-24 h. The therapeutical effect improved when 131I was attached to EGF-dextran instead of native EGF., Conclusion: The improved cellular retention of the ligand-dextran conjugates is an important property since it gives extended exposure time when radionuclides are applied and flexibility in the choice of time for application of neutrons in boron neutron capture therapy (BNCT). It is possible that ligand-dextran mediated BNCT might allow, if the applied neutron fields covers rather wide areas around the primary tumor, locally spread cells that otherwise would escape treatment to be inactivated.

Mammary tumour induced by pituitary grafting in SHN male mice is promising as an animal model for male breast cancer. In this study, these mice (group MC) were found to differ little from the intact female SHN (group FC) in cytosolic oestrogen receptor (OR) level in either normal or neoplastic mammary glands. On the other hand, Progesterone receptor (PR) level in normal glands tended to be lower in group MC than in group FC, which was recovered by chronic treatment with oestradiol benzoate (group OB). Little difference in PR level in mammary tumours was seen between groups MC and FC. Mammary tumorigenesis was significantly inhibited by chronic treatment with dihydrotestosterone to male mice with pituitary grafts (group DHT). This is associated with the significant decline in PR level in normal mammary glands, while not in mammary tumours. There were no significant correlations between OR and PR levels in normal or neoplastic mammary glands, or between normal and neoplastic mammary glands in OR or PR in all groups except group MC, in which the correlation between OR and PR in mammary tumour was significant. All findings indicate that OR and PR states in mammary tumours of male mice induced by pituitary grafting are essentially similar to the spontaneous mammary tumours of females as postulated in the human.

So far, no combination of endocrine treatments has been routinely used in the therapy of breast Cancer. It was, therefore, our interest to determine whether the combination of the antiprogestin, onapristone (ON), and the pure antiestrogen, ICI 164384 (ICI) might provide a more effective therapy than either monotherapy in experimental mammary tumors containing both estrogen and progesterone receptors. In the MXT-mammary tumor of the mouse, ON (5 mg/kg) administered for 3 weeks exerted an ovariectomy-like antitumor effect (56% inhibition), whereas ICI (30 mg/kg) was weakly effective (28% inhibition). The combination of ON and ICI was, however, distinctly more effective than the monotherapies or ovariectomy, causing 78% inhibition. A similar potentiation of antitumor effect by the combination was manifested in the dimethylbenzanthracene-induced mammary tumor of the rat when ON (5 mg/kg) and ICI (30 mg/kg) were administered once daily for 4 weeks (s.c.). The remission rates of tumors found after treatment with ICI, ON, the combination and ovariectomy (complete and partial remission) were 15%, 46%, 71% and 100% respectively. In the animals bearing DMBA-induced tumors, treatment with ON alone significantly increased the serum levels of luteinizing hormone and prolactin, but caused only a slight increase in the peripheral levels of estradiol and progesterone. ON had no appreciable effect on the uterine and ovarian weights. ICI reduced the uterine weight and the serum progesterone level. In the combination with ON, ICI reversed the effect of ON on the progesterone level without influencing the luteinizing hormone and prolactin levels. These findings suggest that the augmentation of antitumor effectiveness by the combination of two antihormones can be ascribed not only to their effects at estrogen- and progesterone-receptor-binding sites, but also to the decrease in the peripheral level of progesterone. Thus, an appropriate combination of antiprogestin and pure antiestrogen may be useful in the management of breast cancer.

The antiestrogen toremifene treatment results in regression or stabilization of dimethylbenz(a)anthracene(DMBA)-induced rat mammary tumors by reducing the mitotic activity and possibly by modifying the abundant spontaneous apoptosis seen in these tumors. A comparative study on the modes of cell loss was performed in untreated DMBA tumors and in tumors treated with toremifene and ovariectomy. Ovariectomy results in massive apoptotic cell death throughout the tumors with apoptotic cells exfoliating into the glandular lumina. Apoptosis accounts for most of the cell death also in the untreated tumors. The distinctly different morphology of the apoptotic cells in the untreated tumors (condensed apoptosis) and tumors treated with ovariectomy (blebbing apoptosis) may reflect the different type or schedule of the apoptotic process. Both morphologies of apoptotic cells were seen in the toremifene-treated tumors. Blebbing apoptosis is not easily examined in paraffin-embedded material but is best detectable in plastic sections and by electron microscopy.