Experimental orthotopic breast cancer as a model for investigation of mechanisms in malignancy and metastasis to the lymph nodes

Citation: Soares Sousa CR, Miranda-Vilela AL, de Almeida MC, Soares Fernandes JM, Sebben A, et al. (2019) Experimental Orthotopic Breast Cancer as a Model for Investigation of Mechanisms in Malignancy and Metastasis to the Lymph Nodes. Int J Vet Sci Res 5(2): 046-057. DOI: http://dx.doi.org/10.17352/ijvsr.000041 https://dx.doi.org/10.17352/ijvsr DOI: 2640-7604 ISSN: L IF E S C IE N C E S G R O U P


Introduction
Breast cancer is the second most common type of cancer in all countries and the most common among women, accounting for 25% of all cancers in women and 14% of cancer deaths [1,2].
In spite of the emergence and evolution of new therapeutic approaches over the last half century, such as conservative surgery, chemotherapy, radiotherapy, hormonal therapy and immunotherapy, breast cancer remains the most common cause of cancer deaths among women worldwide [2,3] with an estimated 521,900 cancer deaths and a global increase of 1.7 million new cases diagnosed in 2012 [1]. Thus, early diagnosis of breast cancer is especially important because it enables more effective and less aggressive therapies, may slow disease progression and can lead to a decrease in the mortality rate in breast cancer patients [1,4].
In this regard, axillary lymph node dissection has been accepted for its staging and prognostic value for breast cancer [5]. Further, just as the surgical treatment of breast cancer has evolved from radical mastectomy to breast conserving surgery, surgical treatment of regional lymph nodes has also become less invasive. Lymphatic mapping with sentinel lymph node (SLN) biopsy has contributed to this development, since it has the potential for reducing the morbidity associated with breast carcinoma staging [6][7][8].
Notwithstanding this progress, to understand the fundamental mechanisms behind malignancy and also contribute to the discovery of improved methods for prevention, diagnosis and treatment, animal cancer models remain essential [9]. In this context, mouse cancer models have been for Investigation of Mechanisms in Malignancy and Metastasis to the Lymph Nodes. Int J Vet Sci Res 5(2): 046-057. DOI: http://dx.doi.org/10.17352/ijvsr.000041 used successfully, while mouse cancer cells also represent a good model to evaluate the impact of new developments [10,11].
Previous research in modeling cancer in laboratory animals provided new insights into the biology of cancer. Among the existing models, the orthotopic murine is characterized by the transplantation of tumor cells or fragments in the same anatomical site where the tumor was originally developed, or the primary site. This cancer model has been widely used [12,13] and, although complex, favors interactions between the stromal and vascular cells in order to provide a microenvironment that will play a crucial role in the tumor cells' development [14][15][16].
Currently, the most common animal models used are tumor xenografts in immunodefi cient mice [14,17]. However, they fail to model human populations accurately due to important genetic differences between the two species, such as the polymorphism [18]. Thus, an immunocompetent mouse cancer model could help researchers to approximate the use of a more effective model system, even though it still fails to model the human population accurately in this respect.
A wide variety of experimental tumor models has been established for educational and scientifi c purposes in order to understand the tumor biology better and improve cancer diagnosis and therapeutic strategies [14]. The ascites and solid Ehrlich tumor, originating from a spontaneous breast cancer in female mouse, was used by Apolantem and Ehrlich in 1905 as an experimental tumor model, by transplanting tumor tissues subcutaneously into mice [19,20].
The Ehrlich tumor resembles human tumors; its neoplastic cells are undifferentiated and have a fast growth rate, leading to an increase in chemotherapy sensitivity in mice [19]. This tumor has been used as an experimental model for several different investigations, including antitumor effi ciency of photodynamic therapy and magnetohyperthermia [3,[21][22][23] pharmacokinetic studies of chemotherapeutics [24,25] evaluation of tumor growth under effects of Agaricus blazei, plant extracts and antioxidants [26]. However, most studies have been conducted using ectopic transplantation into the peritoneal cavity in order to acquire the ascitic tumor, or into the head, femoral region, tongue and footpad, in order to obtain solid tumors [3,[21][22][23][24][25][26][27][28]. Therefore, to intentionally develop an optimal animal cancer model either to investigate the fundamental mechanisms in malignancy and metastasis, or to assess the effi cacy of novel agents and procedures, this work aimed to establish an orthotopic model for breast cancer using Swiss mice. To meet this goal, detailed microanatomy of the breast, SLN and lymphatic mapping, histopathological description, characterization of the orthotopic tumor transplantation by computed microtomography (CMT), and interleukin dosage in tumor fragments and samples from the contralateral mammary gland were performed. To improve the inoculation procedure, a needle without bevel was used. The tumor model development also involved the optimization of microdissection and staining techniques. Possible systemic impairment was evaluated through hemogram and histological analysis of the heart, liver, kidneys and brain.

Animals
Non-isogenic female Swiss albino mice, 3-4 months of age and with a body mass of 29.86 ± 0.74 g at the beginning of the experiment, were purchased from the Multidisciplinary Center for Biological Investigation in Laboratory Animal Science (Cemib) of the State University of Campinas (Unicamp, SP/ Brazil). Animals were housed in plastic cages (5,8 or 10/cage, depending on the experiment) at room temperature (20 ± 2 °C) in a 12 h light/dark cycle with lights on at 6 a.m. and free access to food and water.
For the experiments, animals were previously anesthetized by intraperitoneal route using xylazine (10 mg/Kg) and ketamine (80 mg/Kg), in a fi nal dose of 0.1mL/30g. Euthanasia was carried out by cervical dislocation after anesthesia, according to the guidelines on Euthanasia of Federal Council of Veterinary Medicine (CFMV) [29]. All care and procedures were conducted according to the guidelines of the Animal Research Ethics Committee of the University of Brasilia -Institute of Biologic Sciences, Brazil (process nº 44783/2013).

Identifi cation of the ductal network of the mouse breast
The distribution of the ductal network model was observed employing staining techniques previously described by Krause et al., [30] and Assis et al. (2010) [31], with slight alterations in the microdissection technique. Mice were randomly distributed in two groups (N=5): one group was used as control and the other group injected in the primary duct of the fi fth mammary gland with 50 μL of the vital dye Trypan Blue 2% 24 hours before euthanasia.

Ehrlich tumor cells preparation
Ehrlich ascites tumor (EAT) cells were obtained from frozen aliquots maintained in liquid nitrogen and subcultured every week by intraperitoneal (ip) inoculations in mice [27]. EAT cells (1 × 10 6 ) either fresh or frozen for 30 days at −80 o C were used for the orthotopic transplantation. Fresh and frozen EAT cell viabilities were evaluated using Trypan Blue (Sigma) 0.2% staining method followed by counting in a Neubauer chamber [32]. The inoculum concentration (1 × 10 6 ) was adjusted with phosphate-buffered saline (PBS) to 100 μL.

Orthotopic transplantation and tumor growth curve
In order to improve the technique of orthotopic transplantation and minimize the occurrence of metastasis that might occur due to the slightest contact with the needle bevel, or cause transfi xation of adjacent anatomical sites, such as muscle and viscera of the abdominal cavity, as well as to restrict the degree of invasiveness of the procedure, the needle (30 cc short needle 8 mm in length and 0.30 mm caliber short 30-gauge BD®) was shortened, thus removing the bevel.
The orthotopic transplantation of tumor cells was modifi ed from Behbod et al., [33]. Ehrlich-solid-tumor-bearing mice were randomly distributed in 10 experimental groups (N= 5 per group), according to fresh (5 groups) or frozen (5 groups) tumor cell use and the time of the tumor analysis, carried out after 1, 2, 7, 14 and 21 days after the inoculation of tumor cells. The negative control group received fi ltered water and no tumor was implanted.
For the inoculation procedure, anesthetized animals were placed in supine position and observed under stereoscopic microscope. The papilla of the fi fth right inguinal mammary gland was clamped and the inoculation needle inserted until a depth of 1 mm directly into the mammary papilla. Subsequently 100 μL (1 × 10 6 ) of fresh or frozen tumor cells were inoculated into the mammary fat at a 90-degree angle, thus completing the orthotopic transplantation.
Tumors were weighed on an analytical balance (Shimadzu of Brazil, São Paulo) and measured using a digital pachymeter. The tumor volume was calculated according to the formula: length × (width 2 ) × 0.5 mm 3 [34]. Data were used to obtain a tumor growth curve.

Lymphatic mapping and sentinel lymph node (SLN)
Trypan Blue dyeing technique was used to access the lymphatic mapping and thus reveal the SLNs and nodal network, mimicking anatomically and functionally the lymphatic metastatic path of the neoplastic cell from the tumor primary site to the nodal region.
To investigate the occurrence of micrometastasis in SLN, 100 μL (1 × 10 6 ) of fresh Ehrlich tumor cells were inoculated into the fi fth right mammary gland of 34 animals, randomized into 3 groups: control (N = 8) and EAT inoculated animals examined at 7 and 14 days after tumor cell implantation (N = 10/group). Trypan Blue (100 μL) was injected subcutaneously in the subpapillary region on the adipose cushion (subcutaneous mammary papillae), in the 5 th right mammary gland and the 4 th left mammary gland, 24 hours before euthanasia.
After euthanasia, SLN (right subiliac node) [35,36] was surgically removed, fi xed in formaldehyde 3.7% for 8 hours, and processed for histology according to the methodology described above (sections of 2 to 5 m of thickness). Each slide was fi tted with a series of three semi-serial sections (50 μm) and stained with hematoxylin-eosin (HE). Sections were photographed under the light microscope Carl Zeiss Axio Vision 4.8.2 SP2, using an image capture Zen System 2011 (Zen blue edition program; Carl Zeiss).

Histopathological evaluations
Samples from the tumors, the contralateral mammary gland and also from several organs were collected and washed in 0.9% saline solution in order to remove the excess of blood and tissue residues. The liver, kidneys, lung, spleen and brain samples were fi xed in formaldehyde 3.7% for 8 hours; the mammary samples were fi xed in Davidson solution at 4° C for 8-10 hours. All samples were then processed in an automatic tissue processor (OMA ® DM-40, São Paulo, Brazil), cut to 5 μm of thickness with a series of three serial semisections (50 μm) in a Leica RM2235 manual microtome (Leica Microsystems, Nussloch, Germany) and stained with HE. The photomicrography of the slides was analyzed by light microscopy (Carl Zeiss Axio Vision 4.8.2 SP2, image capture Zen System 2011 -Zen blue edition program).

Computed microtomography characterization (CMT)
CMT images of orthotopic tumor were acquired from the anesthetized mouse positioned on a specifi c small animal scanner of a SkyScan Micro CT 1076 (SkyScan, Kontich, Belgium) operating at 50 kV, 141 mA, with a 0.5 mm aluminum fi lter. Vital signs were monitored during all the scanning period. Three-dimensional (3D) image reconstruction was performed by Nrecon software (SkyScan, Kontich, Belgium), adjusting the parameters smoothing, beam-hardening and ring-artifact to the values of 01, 10, 07, respectively. For the 3D image evaluation the CTanalyze software (SkyScan, Kontich, Belgium) was used.

Cytokine profi le of mammary glands after orthotopic ehrlich tumor cells inoculation
To investigate whether the orthotopic tumor cells implantation could trigger an immunomodulatory response, the levels of the anti-infl ammatory cytokine IL-4 (Th2) and pro-infl ammatory cytokines IL-17 (Th1), IL-1 and IL-12p70 (Th1) were evaluated in mammary gland lysates inoculated with fresh and frozen tumor cells using normal mammary glands as control. Because Ehrlich tumor is very aggressive and grows very quickly after the second week, reaching large sizes in short periods [22], cytokines were evaluated at the 14 th day after the tumor cell inoculation. For this evaluation, 100 mg of mammary gland was homogenized in lysis buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1% Triton X-100, 60 mM octyl glucoside) containing protease inhibitors (Boehringer Mannheim). IL-4, IL-17, IL-1 and IL-12p70 levels were measured in mammary gland tissue lysates by enzyme-linked immunosorbent assay (ELISA), in accordance with the manufacturer's instructions (R&D Systems). For each experiment, test and control samples were examined at least in duplicate. Standard curves were prepared with a group of serially diluted standards and used for the calculation of cytokine concentrations.

Hemogram
Mice were anesthetized with ketamine and xylazine according to the method described above. Blood samples (400 μL) collected by cardiac puncture were used to carry out hemogram in a multiple automated hematology analyzer for veterinary use, Sysmex pocH-100iV Diff (Curitiba/Paraná, Brazil) calibrated for mice in microtubes containing EDTA as anticoagulant.

Lymphatic mapping and sentinel lymph node (SLN) of healthy and tumor bearing mouse
The lymphatic and SLN mapping of a healthy control mouse are showed in fi gure 2. The mammary glands of the negative control group showed no changes and the lymph nodes (LN) were free of tumor involvement. Macroscopically, ipsilateral SLN (right subiliac node) [36], could be viewed in the 4 th and 5 th mammary glands, lymph vessels, and afferent and efferent blood vessels in the lymph node hilum.
In the group with tumor inoculation of 7 days, positive lymph node involvement with micrometastases was observed in 85% of the animals in ipsilateral SLN (right subiliac node) [36] and contralateral LN (left subiliac node) [36], however, in the group of 14 days, there were metastatic deposits in the right (SLN) and left (contralateral LN) subiliac nodes in 100% and 50% of animals, respectively.

Characterization of the tumor: Growth curve, histopathology and computed microtomography (CMT)
The inoculation of frozen Ehrlich cells did not induce tumor growth until the 14 th day, with little inter-animal variation. However, the tumor growth curve of fresh tumor cells revealed that the tumor development started as soon as 1 day (24 h) after the inoculation and led to high inter-animal variation ( Figure   3), as also observed by clinical and histological evaluation. The Histopathological analysis revealed undifferentiated tumors with large necrotic areas invading the host mammary gland, including destruction of ductal structures. The adjacent dermis and skeletal muscle were also infi ltrated. Lymphatic vascular invasion was frequently observed inside tumor, peritumoral adjacent mammary tissue, muscle endomysium and dermis after 7 days of tumor inoculation (Figure 4). Micrometastases and overt metastases were identifi ed in the SLN (right subiliac node) and contralateral LN (left subiliac node) [36], after 14 and 21 days ( Figure 5). Neurovascular bundle tumor invasion and focal metastases in mammary adipose tissue were rarely observed (data not shown).
The computed microtomography (CMT) of the abdominal wall in the groups inoculated with fresh cells showed the tumor mass (isolated node) with irregular shape and margin in the right inguinal mammary gland, presenting image with a density that matches with the soft parts and isoechogenicity (same texture) of mammary tissue, confi rming the tumor implantation after 1 day (24 hours) of tumor inoculation, as well as expansion of the lesion and progressive mass effect (lesion pushing against adjacent structures), particularly at 14 days after inoculation ( Figure 6).     (Figure 7c), as compared to normal mammary glands (control). This IL-1 secretion was also higher than that observed for frozen cells. Further, the secretion of IL-17 was not signifi cantly changed after fresh cell implantation ( Figure   7b). However, the implantation of frozen tumor cells triggered signifi cantly higher IL-17 (Figure 7b) secretion levels than both control and fresh tumor cells inoculated in mammary glands.

Cytokine profi le of mammary glands after orthotopic ehrlich tumor cells inoculation
No detectable levels of IL-12p70 were observed for both fresh and frozen tumor cells implantation (data not shown).

Histopathological evaluations of organs
No morphological changes were observed in the liver, kidneys, spleen, lung and brain sections at 1, 2, 7, 14 and 21 days after inoculation of EAT cells, as compared to the negative control organs (data not showed).

Hemogram
Due to the positive lymph node involvement with micrometastases from 7 days after tumor inoculation, results of the hemogram will be presented from this time period (Table 1). Compared to the negative control, fresh and frozen tumor cells at 7 days after inoculation promoted signifi cantly decreased red blood cells (RBC), which were also below the reference values for mice [37,38] with fresh tumor cells. At this time, fresh tumor cells also promoted a signifi cant reduction in hemoglobin (HGB; also below the reference values for mice [37,38]

Discussion
Preclinical cancer studies fall into two main categories: those using tumor cell transplantation, and those in which tumors arise spontaneously or are chemically induced in the host [39]. Although several animal species may be used, the   mouse is the most common animal for cancer models [39][40][41]. In spite of the morphological differences between mouse and human mammary tissue (such as the presence in human mammary tissue of a branching network of ducts ending in clusters of small ductules that constitute the terminal ductal lobular units, and their absence in mouse mammary epithelial tissues, which comprise alveolar buds that are formed during each estrous cycle), signifi cant insights into breast cancer have emphasized functional similarities between them [42].
These similarities, together with easy accessibility, make the mammary gland of mouse the most widely studied organ in improving the understanding of the fundamental cellular and molecular properties of normal and neoplastic development [11,[42][43][44][45].
Classical tumor diagnostics have for decades been based on surgical pathology and histology, using tumor cell differentiation status as one important aspect to score, evaluate, and communicate tumor aggressiveness. This has prognostic implications where, as a rule, a high degree of differentiation purports a better prognosis than a low degree [46]. As Ehrlich tumor is very poorly differentiated and can morphologically resemble a poorly differentiated or undifferentiated ductal human breast cancer, it can be considered a good model for studying the behavior of this type of aggressive solid cancer. Also, it has the advantage of being successfully used in an immunocompetent mouse cancer model, unlike other established mouse-into-mouse metastatic mammary tumor models, such as 4T1 and EMT6 breast tumor cells, which require syngenetic mice [47]. Another advantage of the Ehrlich tumor cancer model is that it does not require growth in culture medium; it is easily cultivated and transferred in vivo [27].
Although experimental tumor biology has over the years not focused on the differentiation processes, but rather studied molecular pathways leading to growth, migration, and cell death [46], our study aimed for a classical diagnostic approach, since establishing an optimal orthotopic cancer model for breast   in which the tumor grows, so that the effect of the tumor on its microenvironment can be modulated [58]. Therefore, the orthotopic tumor transplantation seems to be a better predictor of clinical success than ectopic models, as reported in the literature [59][60][61].
It is known that the invasiveness generated by surgical orthotopic transplantation signifi cantly increases the likelihood of metastases [62][63][64] caused by mechanical injuries that may lead even to an increased expression of genes promoting metastasis of breast cancer to the lungs. Additionally, SLN is defi ned as the fi rst LN to receive lymphatic drainage from breast cancer, due to the ordered progression of cells through the lymphatic system [66][67][68][69][70]. In view of this, we selected the 5 th mammary gland due to the proximity of the right subiliac node [36]. This choice also took into account a reduction in the discomfort of the animals during clinical exams, and facilitated clinical and imaging visualization.
Evaluation of the proliferative profi le of the Ehrlich tumor makes it feasible to determine the evolution of this tumor type in its different forms (fresh and frozen cell aliquots), as well as its growth curve. Isolated tumor cells were found in cervical lymph nodes when the Ehrlich tumor was inoculated in mouse tongue, suggesting a metastatic process [76]. Moreover, many human tumors and those transplanted into nude mice have only metastasized when orthotopically inoculated; if held ectopically they often do not metastasize [59,77]). In this regard, the accurate assessment of the SLN, as perfomed in the present study, is important not only for staging and prognosis, but also to guide the treatment selection [78,79], since metastasis to regional LN is an important step in the dissemination of cancer, and often occurs at a relatively early stage of tumor development compared with distant metastasis, such as that to the liver and lung [78].
SLN is the very fi rst lymph node to be reached by metastasing cancer cells coming from the primary tumor site. Indeed SLN is representative of the whole nodal network. Metastases to the SLN are not random, instead following predictable sequential steps. The axillary lymph nodes are the preferred sites for the spread of breast cancer, and the axillary stadium is considered the main prognostic factor, accurately predicting the identifi cation of micrometastases when these cannot be detected by investigatory images. So, it is an important factor in the assessment of prognosis and in decisions about the treatment of breast cancer [6,79,80], and the lymphatic mapping has a great contribution as a diagnostic tool capable of providing regional lymph node status [6,8]. In this respect, as far as we know, observation of micrometastases in Ehrlich tumor in both the ipsilateral SLN (right subiliac node) and contralateral LN (left subiliac node) [35,36] has not previously been described. Actually, in the literature there are no Ehrlich tumor metastasis reports and lymph node involvement. Moreover, for breast cancer, the majority of occurrences are unilateral, with higher tumor incidence on the left. Left-side predominance also occurs in bilateral cases, in which more tumors develop fi rst in the left breast or are larger than those in the right [44]. So, this could explain the rapid metastatic deposits in contralateral LN (left subiliac node) at 7 days after tumor inoculation. However, there was no evidence of metastasis in the several analyzed organs, which may be due to the fact the metastasis searches were performed only until the 21 th day. Bioluminescence signals of cells metastasized from the subiliac lymph nodes to the axillary lymph nodes themselves has been described in mutant mice, exhibiting remarkable systemic lymphadenopathy (MRL/lpr) after 3 to 9 days, but were not evident in other organs until the 14 th day [78]. Considering our immunocompetent mouse cancer model and the importance, discussed above, of the as-presented nonsurgical orthotopic transplantation, a greater length of time than this could be expected to pass before fi nding metastasis to organs.
It has been reported that several innate cytokines play a crucial role in controlling breast cancer progression. Although much research has focused on the genetic abnormalities that initiate and drive cancer, there is now overwhelming evidence that the behavior of tumorigenic cells is also highly infl uenced by their microenvironment [81]. Thus, even though the inoculated Ehrlich tumor caused very little antitumor reaction of a cellular nature, and lymphocyte reaction was rare, results were consistent with those reported in the literature, in which modifi cations in cytokine profi le mediated both directly and indirectly by the tumor are important parameters that affect the course of the disease [81], as discussed below.
Overall, orthotopic tumor cell implantation triggered a polarization towards a pro-infl ammatory immune response, since Th1 cytokine secretion levels (IL-1 and IL-17) were signifi cantly higher than anti-infl ammatory Th2 (IL-4). IL-4 was originally described as a B cell growth factor, and is now known to provide potent anti-tumor activity against various tumors, including breast cancer [82,83]. Simultaneously, IL-17-and IL-4-producing CD8+ T lymphocytes have been implicated in breast cancer progression, since they have been found within the tumor-draining lymph nodes of breast cancer patients [84]. Likewise, IL-17 secretion has been shown to be up-regulated in breast cancer patients, since it is positively associated with tumor progression aggressiveness [85]. The production of IL-17 in murine models of breast cancer has been described as an important marker of tumor progression [86]. Du and colleagues (2012) [87] showed that injection of 4T1 tumor-bearing mice with recombinant IL-17 resulted in increased tumor volume and microvascular density, as measured by the immunohistochemical detection of CD34 expression in microvessels. IL-1 is another important proinfl ammatory cytokine related to cancer progression. It has been established that IL-1 was predominately responsible for the increased metastatic potential of the tumors [88,89]. In the present study the orthotopic tumor implantation with fresh cancer cells triggered signifi cant amounts of IL-1, and it could be related with metastasis generation in the analyzed mice. As the immune system exerts both inhibitory and stimulatory effects on breast tumors, and the balance of these effects may profoundly infl uence tumor growth [81], the signifi cantly reduced percentage of peripheral lymphocytes at all times (7, 14 and 21 days) with fresh tumor cells, and after 7 and 14 days with frozen tumor cells do corroborate the literature, evidencing that reduced lymphocyte-dependent immunity can favor carcinogenesis [90,91]. Moreover, it has been demonstrated that the development of Ehrlich tumor (both ascites and solid forms) is associated with production of infl ammation [27,92], which is in accordance with our results, where tumor cells induced pro-infl ammatory cytokines and increased neutrophils+monocytes. This could also be explained by tumor necrosis; the histology is concordant.
Anemia is a common complication in patients with breast cancer, either as a consequence of the disease itself (blood loss, bone marrow infi ltration or nutritional defi ciencies), or with reduced tumor control or its treatment [93]. Results of erythrogram corroborate this, mainly for fresh tumor cells at 7 days after inoculation. As anemia is a causative factor in the development of tumor hypoxia, which gives rise to a more aggressive tumor phenotype and increased likelihood of distant metastases [93], the aggressiveness of the undifferentiated tumor cells at 7 days with large necrotic areas invading the host mammary gland, destroying ductal structures and infi ltrating the adjacent dermis and skeletal muscle, besides the lymphatic vascular invasion, could be explained by anemia.
In conclusion, our study indicates that, with orthotopic inoculation of the Ehrlich tumor, there is a marked invasion of mammary host structures, dermis and muscle associated with vascular lymphatic invasion in these areas. Importantly, micrometastases were frequently seen in the SLN and