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        Electron Microscopy Findings in N-Methyl-N-Nitrosourea-Induced Mammary Tumors
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        Electron Microscopy Findings in N-Methyl-N-Nitrosourea-Induced Mammary Tumors
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        Electron Microscopy Findings in N-Methyl-N-Nitrosourea-Induced Mammary Tumors
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Abstract

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.

Introduction

The rat model of mammary tumors chemically induced by the carcinogen agent N-methyl-N-nitrosourea (MNU) has been extensively used by several research groups in order to give new insights on mammary cancer physiopathology, and to develop new preventive and therapeutic strategies (Faustino-Rocha et al., 2013 a , 2014 a , 2014 b , 2015 b ). Although these tumors have been extensively studied by histopathology, immunohistochemistry, and genetic approaches, there is a lack of ultrastructural studies (Faustino-Rocha et al., 2013 b , 2015 a , 2016). The present work aimed to perform an ultrastructural evaluation of MNU-induced mammary tumors in female rats. Considering that our research team has also focused on the effects of tumor microenvironment on mammary tumors development, particularly in the role of mast cells, we also examined mast cell infiltration in these tumors.

Materials and Methods

Animals

In total, 25 female Sprague-Dawley rats, 4–5 weeks of age, were obtained from Harlan Interfauna Inc. (Barcelona, Spain). Animals were housed at the facilities of the University of Trás-os-Montes and Alto Douro in filter-capped polycarbonate cages with corncob for bedding under controlled temperature conditions (23±2°C), humidity (50±10%), and air filtration system (10–20 ventilations/h) and on a 12 h:12 h light:dark cycle. Tap water and a basic standard laboratory diet (4RF21; Mucedola, Settimo Milanese, Milan,Italy) were supplied ad libitum. Cages were cleaned and water was changed once per week. All procedures were done in accordance with European and National Legislation (European Directive 2010/63/EU and National Decree-law 113/2013). The Portuguese Ethics Committee for Animal Experimentation approved all the experiments and procedures carried out on the animals (Direcção-Geral de Alimentação e Veterinária, Approval no. 008961).

Animal Experiments

Animals were submitted to a period of quarantine for 1 week and acclimated to laboratory conditions for 2 weeks. Then they were randomly divided into two experimental groups: MNU (n=15) and control (n=10). Mammary tumor development was induced in animals from the MNU group by a single intraperitoneal administration of the carcinogen agent MNU (Isopac, lot 100M1436V; Sigma Chemical Co., Madrid, Spain) at a dose of 50 mg/kg at 7 weeks of age. Animals from control groups received a single administration of the vehicle (saline solution 0.9%). All animals were observed daily to monitor their general health status. They were palpated weekly for the detection of mammary tumor development.

Animal Sacrifice and Samples

After 35 weeks of MNU administration, all surviving animals were humanely sacrificed by intraperitoneal administration of ketamine (75 mg/kg; Imalgene 1000, lot LBF133BB; Merial S.A.S., Lyon, France) and xylazine (10 mg/kg; Rompun 2%, lot KPO78X0; Bayer Healthcare S.A., Kiel, Germany) followed by exsanguination by cardiac puncture as indicated by the Federation of European Laboratory Animal Science Associations (Forbes et al., 2007).

All animals were skinned and the skin was carefully observed under a light for the detection of small mammary tumors, and all tumors were removed. Three fragments with <1 mm of diameter were randomly taken from the two malignant histological patterns (papillary carcinoma and cribriform carcinoma) most frequently developed by MNU-exposed animals for transmission electron microscopy (TEM) processing. After this, all mammary tumors were immersed in buffered formalin for 12 h.

Histological Analysis

After fixation in buffered formalin, all mammary tumors were routinely processed, embedded in paraffin, and 2 µm-thick sections were stained with hematoxylin and eosin (H&E). They were histologically classified by a pathologist according to the classification previously established by Russo & Russo, 2000).

TEM

Fragments obtained from three mammary tumors from the MNU group were fixed with 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h at 4°C (Hyttel et al., 1986). After washing in buffer for 2 h at 4°C, they were postfixed in 2% osmium tetroxide (OsO4) in buffer, dehydrated in an ethanol series followed by propylene oxide, and embedded in Epon. Semithin sections 0.5–1 µm-thick were cut with a glass knife in a RMC Power Tome XL ultramicrotome (Boeckeler Instruments, Tucson, Arizona, USA), stained with a mixture of methylene blue and azur II (2:1), and observed by light microscopy. Ultrathin sections from mammary tumors embedded in Epon were cut in a RMC Power Tome XL ultramicrotome with a diamond knife (Diatome, Hatfield, Pennsylvania, USA), collected on copper grids (Taab, Aldermaston, Berks, UK), contrasted with uranyl acetate (20 min) and Reynolds lead citrate (10 min), and observed at 60 kV in a LEO 906E transmission electron microscope (Zeiss, Oberkochen, Germany) (Calado et al., 2011).

Results

Animals

Five animals died during the course of the experiment: four animals from the MNU group and one animal from the control group. Data from these animals were excluded from the study.

Mammary Tumors

As expected, animals from the control group did not develop any mammary tumor. All animals from the MNU group developed mammary tumors (incidence of 100%) and a total of 28 mammary tumors was counted.

Histological Classification

At histological analysis of H&E stained mammary tumors, it was observed that each mammary tumor exhibited more than one histological lesion. In this way, a total of 71 lesions were identified in the MNU group, as previously published by our research team (Faustino-Rocha et al., 2016). The cribriform and papillary carcinomas were the malignant histological patterns most frequently identified (Faustino-Rocha et al., 2016). At analysis of mammary tumor sections embedded in Epon, the three tumors from the MNU group sampled for TEM analysis were histologically classified as follows: two of them were classified as cribriform carcinoma and one was classified as papillary carcinoma (Fig. 1).

Figure 1 Semithin sections of mammary tumors stained with methylene blue and azur II (2:1) by light microscopy. The lesions were classified as cribriform carcinoma (a) and papillary carcinoma (b). Scale bar of 100 µm.

TEM

Similar ultrastructural features were found in the two histological patterns (cribriform and papillary carcinomas) analyzed in the present work. Neoplastic cells usually presented a round or elongated regular shaped nucleus, always accompanying a cuboid or a prismatic cell shape. Perinuclear heterochromatin was evident, and uniformly distributed below the entire inner envelope (Figs. 2a, 2b). Other cells showed an increased nuclear size, with a very irregular shape and also with numerous folds. In these cells the nucleus had clumps of chromatin (Fig. 2c). In the cytoplasm, abundant rough endoplasmic reticulum, free ribosomes and Golgi cisterns were observed. Mitochondria were abundant and presented a pleomorphic shape (Fig. 2d). These cells revealed a clear relationship between Golgi cisterns and mitochondria positions. Some dense vesicles (two or three on each cell section) were also observed. The Golgi apparatus presented a developed lamellar membranous structure with a curved parallel series of flattened saccular vesicles (dictyosomes) expanded at their ends. Vesicles of rough endoplasmic reticulum fused on the cis-part of the Golgi apparatus (Fig. 2d). Cells also presented abundant primary and secondary lysosomes, multivesicular bodies, and residual bodies. Some cells were closely embedded between adjacent ones, and showed numerous interdigitating cytoplasmic processes and desmosomes, with very electron-dense plaques attached to the inner side of the cell membrane. Other cells showed a clear rupture with adjacent cells, and in these regions the plasma membrane developed several long projections (Figs. 2e, 2f). These cells, especially those that had lost their contact with the adjacent ones, lost large portions of cytoplasm that were detached from the rest of the cell (Fig. 2g). The fragmented portions of the cytoplasm were deposited on the extracellular space. These fragments contained several organelles, such as rough endoplasmic reticulum, ribosomes, several cisterns and round mitochondria with visible cristae. Other cells lost large portions of cytoplasm through the formation of large vacuoles just near the nucleus (Figs. 2h, 2i). It was also observed in some neoplastic cells that after losing parts of the cytoplasm, the remaining part became devoid of plasma membrane (Figs. 2j, 2k).

Figure 2 Ultrastructural findings in N-methyl-N-nitrosourea-induced cribriform and papillary carcinomas. a,b: Elongated (a) or round (b) nuclei with perinuclear heterochromatin (black arrows) were observed. Several cells are connected with adjacent ones by several interdigitating cytoplasmic processes and desmosomes (black arrow heads). c: Nuclei with an irregular shape and increased size were observed in some cells. d: Golgi cisterns (white arrows) and mitochondria (white asterisks) were closely positioned in cell cytoplasm. e,f: Abundant lysosomes, multivesicular bodies and residual bodies were observed with different material inside (white arrow heads). Free ribosomes and rough endoplasmic reticulum were abundant. Cells exhibited several long projections of contact with adjacent ones (black arrows). g: Some cells lost large portions of cytoplasm with several organelles by a torsion-like process (white arrows). h,i: Other cells formed vacuoles with variable size and shape. These vacuoles increased in size and fused with each other, forming large vacuoles (black asterisks). These vacuoles initiated the separation of cytoplasm portions. j,k: Cells lost parts of cytoplasm and the remaining part became devoid of plasma membrane (blak arrows). l: Mast cells were frequently observed, especially near cells in fragmentation.

Several mast cells were observed near cells in fragmentation. They appeared as round to elongated cells with a diameter of ~10 µm. A nonsegmented heterochromatic nucleus, abundant granules, elongated mitochondria, free ribosomes, and profiles of endoplasmic reticulum were observed in the mast cell cytoplasm (Fig. 2l).

Discussion

Cancer occurs when a normal cell fails to function properly, leading to abnormalities in cell cycle that induces alterations in a tissue (Kaul-Ghanekar et al., 2009). Some authors consider that several ultrastructural features detected by TEM are useful for the differential diagnosis of breast tumors (benign versus malignant tumors) (Tsuchiya & Li, 2005), and advise the evaluation of breast lesions using this technique. However, the time consumed for fixation and Epon embedding, as well as advanced skills for preparing ultrathin sections, forces TEM use for research purposes only (Winey et al., 2014). Although the rat model of MNU-induced mammary tumors is one of the most frequently used in breast cancer research, little is known about the ultrastructure of these cancer cells (Faustino-Rocha et al., 2015 a ). Once cells undergo transformations during the process of carcinogenesis (Jaafar et al., 2012), we consider it important to analyze their ultrastructure in order to better understand and justify the observable macroscopic alterations. Thus, TEM was carried out in three samples from MNU-induced mammary tumors in female Sprague-Dawley rats.

TEM analysis revealed similar features between the histological patterns (cribriform and papillary carcinomas) identified in this work. Although a low number of samples (three samples) was used due to the cost, laborious sample preparation, and requirement of a specialist to complete the protocol, they were considered representative, given that the cribriform and papillary carcinomas were the most frequent malignant histological patterns identified in the present protocol (Faustino-Rocha et al., 2016).

Neoplastic cell nuclei from MNU-induced rat mammary tumors exhibited a round or elongated shape, which is in accordance with that previously reported in human mammary tumors. Cell nuclei from malignant mammary tumors showed an oval or round shape when compared with the nuclei of cells from normal mammary gland (Tsuchiya & Li, 2005). Nuclei were rich in heterochromatin, as previously observed in the nuclei of normal cells from mammary gland and from carcinoid tumors (Tsuchiya & Li, 2005). Although previous works in human mammary tumors concluded that breast cancer cells from both benign and malignant tumors were characterized by absence of atypia (Sloane, 1985; Scott et al., 1997), some cells from MNU-induced rat mammary tumors exhibited cell atypia, characterized by nuclear enlargement, irregular shape, and an increase in heterochromatin. Similar to normal cells from mammary gland, cytoplasmic organelles appeared to be uniformly distributed throughout the cell (Tsuchiya & Li, 2005). The organelles observed in these cells, namely mitochondria, Golgi complex (composed of a few flat saccules and small vesicles), endoplasmic reticulum, free ribosomes, and lysosomes had been previously observed by Russo et al. (1977) in a work performed in a human breast cancer carcinoma cell line (MCF-7). Mitochondria are essential sources of adenosine triphosphate (ATP) to the cells. Although alterations of mitochondrial morphology were not frequently reported in human mammary cancer cells (Ghadially, 1985), mitochondria with pleomorphic shape were found in cells from MNU-induced mammary tumors. Rough endoplasmic reticulum and its attached polyribosomes are responsible for the production of secretory proteins. Cells that produce a protein-rich secretion, like hepatocytes, pancreatic cells, and fibroblasts, have a well-developed rough endoplasmic reticulum (Ghadially, 1985). The reticulum remains in tumors arising in these organs. An inverse relationship was described between the tumor growth rate and the amount of rough endoplasmic reticulum (Ghadially, 1985). The Golgi complex is well developed in secretory cells. It is responsible for modifying, condensing, and packaging materials to form secretory granules. So, similarly to what occurs with the rough endoplasmic reticulum, the Golgi complex is an indicator of cellular differentiation and functional activity. Immature or undifferentiated cells, like stem cells, have a poorly developed Golgi complex and poor rough endoplasmic reticulum when compared with normal mature cells (Ghadially, 1985). Concerning tumors, the Golgi complex is poorly developed in fast-growing tumors. In addition, the less differentiated tumors have smaller Golgi complexes, in contrast to well-differentiated mammary tumors, which normally present a well-differentiated Golgi complex. In some tumors, it may exhibit hypertrophy, dilation, or distortion (Ghadially, 1985). Secondary lysosomes were observed in most mammary tumors (Ghadially & Parry, 1966).

The neoplastic cells exhibited several projections from their body that allow them to attach to neighboring/adjacent ones. This contact among cells is established in order to enhance their ability to survive and become more resistant to apoptosis induced by therapeutic approaches, namely administration of anticancer drugs (Luparello et al., 1991; Kataoka & Tsuruo, 1996). These projections may also be involved in cell migration. Kramer et al. (1986) verified that the formation of projections maximize the cell membrane surface area and leads to the beginning of tumor cells Invasion. Benbow et al. (1999) described three distinct steps during the process of carcinogenesis: adhesion, invasion, and migration. The process begins with attachment between cancer cells, followed by the formation of protrusions, and ends with the movement of cells along the matrix (Benbow et al., 1999). One of the most interesting phenomena observed in our study was the loss of cytoplasm and the formation of vacuoles. Furthermore, mast cells were also frequently observed near tumor cells in fragmentation, confirming our research team findings in MNU-induced mammary tumors in Sprague-Dawley female rats (Soares-Maia et al., 2013; Faustino-Rocha et al., 2014 a , 2014 b , 2015 b ). The characteristics of mast cells observed in the present work were similar to those previously described in mammary tumors (Blank, 2011, Ribatti & Crivellato, 2011).

Conclusions

Although a low number of samples (three samples) were analyzed by TEM in the present study, we consider that it may contribute to a better understanding of the carcinogenesis process of MNU-induced mammary tumors in a rat model. Ultrastructural characteristics of the two most common mammary carcinomas MNU-induced in female rats described in the present work can be useful to distinguish them from other histological patterns. In addition, in the present work the loss of cytoplasm in neoplastic cells and formation of vacuoles were described.

Acknowledgments

This work was supported by European Investment Funds by FEDER/COMPETE/POCI – Operational Competitiveness and Internationalization Program, under Project POCI-01-0145-FEDER-006958 and Portuguese Foundation for Science and Technology (FCT), under the Project UID/AGR/04033/2013, the Project PTDC/DES/114122/2009, and Postgraduation grant SFRH/BD/102099/2014.

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