A Novel Animal Model of Induced Breast Precancerous Lesion in Tree Shrew

Abstract

Chinese tree shrew, an animal exhibited closer evolutionary relationship with humans compared to rodents, is getting increasingly attentions as an appealing experimental animal model for human diseases. However, a high-efficiency and stable method to establish tree shrew breast precancerous lesions model has not been clearly elucidated. Thus, the current study aimed to explore the way of establishing breast precancerous model in tree shrew and investigate the pathologic characteristics of induced breast precancerous lesions. The results indicated that 7,12-dimethylbenz(a)anthracene (DMBA) could induce breast lesions in tree shrews. However, comparing to DMBA alone, an addition of medroxyprogesterone acetate (MPA) to DMBA critically increased the rate of induced breast lesion in tree shrews. Half of induced breast lesions were intraductal papilloma and the others were atypical ductal hyperplasia. Induced lesions showed positive expression of estrogen receptor α (ERα), progesterone receptor (PR) and cytokeratin 5/6 (CK5/6), but negative expression of human epidermal growth factor receptor-2 (Her-2). The expression of B cell lymphoma-extra large (Bcl-xl) was significantly higher and the expression of B cell lymphoma 2 associated X protein (Bax) was significantly lower in the precancerous lesions (atypical ductal hyperplasia) compared to benign tumor (intraductal papilloma). These results suggest that DMBA is able to induce breast lesions in tree shrews. Combination of DMBA and MPA may be more effective to establish breast precancerous lesion tree shrew models. Tree shrew might be a promising animal model for studying the tumorogenesis of breast cancer.

INTRODUCTION

Global incidence of breast cancer has been gradually increasing. Worldwide, breast cancer is the predominant cancer and possesses the highest cancer-related mortality in women.¹⁾ So, adequate surveillance and interventions to the process from the initial occurrence of breast precancerous lesions to ultimate breast cancer are crucial to reduced incidence and mortality of breast cancer. Given no mature means of monitor and prevention of conversion from normal tissue to precancerous lesions, and to breast cancer ultimately, there is unmet need to establish animal model in every stage of the breast tumor development, particularly precancerous model of breast.

Rodent models have been widely utilized in cancer research. There are many useful features of rodent for cancer research, such as rapid reproduction, sufficient supply, low cost, convenient for breeding, clear genetic background and mature transgenic techniques.²⁾ However, referring to either basic biologic characteristics or tumor biologic activity, rodent species are diverging markedly from human being, which is taken into account as the limitation for studying human disease. So, novel and more proximate to human animal models are definitely needed for more effective breast tumor research.

Tree shrew (Tupaia belangeri chinensisis) classified as a primitive prosimian and belongs to the separate order Scandentia.²⁾ As well, tree shrew is small sized, economic, and highly reproductive. However, as a primate-like animal, tree shrew exhibits closer phylogenetic relationship with humans compared to rodent and become an increasingly popular alternative animal model.³⁾ In 1966, Elliot et al. reported the spontaneous breast tumor of tree shrew for the first time.⁴⁾ Recently, various tumor models in tree shrews were developed, including lung cancer,⁵⁾ hepatocellular carcinoma,⁶⁾ and glioblastoma.⁷⁾ The database has been built based on the genome sequence data of tree shrews.⁸⁾ Based on the above descriptions, it is reasonable to generate an ideal breast tumor model in tree shrew.

As a polycyclic aromatic hydrocarbon, 7,12-dimethylbenz(a)anthracene (DMBA) is identified as a powerful organ-specific chemical carcinogen. The oxidation of DMBA produces metabolites that form covalent adducts with DNA in mammalian cells, leading ultimately to particular tumor induction.^9,10) Given its mammary-specific characteristic, DMBA is most commonly used to selectively induce breast tumor in animals.¹¹⁾ The DMBA-induced rat mammary carcinoma model is extensively investigated in breast tumor research. Medroxyprogesterone acetate (MPA) is an artificial progestogen. It has been confirmed that the development of breast tumor is highly correlated to the expressions of hormones like estrogen and progestogen, so MPA was proven to accelerate tumourigenesis initiated by DMBA.¹²⁾

Here, we performed this study to explore an ideal method to generate breast precancerous lesions in tree shrew by DMBA and MPA injection.

MATERIALS AND METHODS

Materials

Animals

Female tree shrews of 10–12 months-old are purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences and Kunming Medical University. All tree shrews were artificially propagated and kept in a room maintained at constant temperature of 20–25°C and humidity of 30–60%. The tree shrews received water and food ad libitum. All procedures were approved by the Guangxi Medical University Experimental Animal Committee.

Reagents and Antibodies

DMBA (Purity ≥ 95%) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). MPA (Purity ≥ 99%) and dimethyl sulfoxide (DMSO) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Primary monoclonal antibodies to progesterone receptor (PR), estrogen receptor α (ERα), cytokeratin 5/6 (CK5/6) and human epidermal growth factor receptor-2 (HER-2) were purchased from Maxim Biotech Company (Fuzhou, China). Primary monoclonal antibodies against B cell lymphoma-extra large (Bcl-xl), B cell lymphoma 2 associated X protein (Bax) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.).

Methods

Tree Shrew Model and Experimental Design

Ninety tree shrews were randomly averaged to 3 groups, with 30 tree shrews in each group: DMBA group were injected of DMBA (10 mg/kg) dissolved in DMSO into the one side of lumbar mammary fatty pad for 4 times, administrated once per week for 2 weeks and suspended for one week; DMBA + MPA group were received DMBA as mentioned above and intramuscularly injected MPA (100 mg/kg) once per 2 weeks for 5 times from the first DMBA injection; Control group were injected equal volume of DMSO in corresponding time point.

Physical Examination

Physical examination of breast was performed weekly. When lesions of breast could be palpated, the latency period was recorded and volume (volume = length × width² × π/6)¹³⁾ of lesions was measured and calculated weekly.

Ultrasound Examination

In addition to ultrasound examination, contrast-enhanced ultrasound was also conducted to examine the breast lesion by Aplio 500 color Doppler imaging after intravenous injection of Sonovue (5 mL/kg).

Histological Examination

Histological examination was performed on breast lesions of tree shrews. The breast lesion samples were fixed in 10% formalin, embedded in paraffin, cut into 5 µm sections, and then stained with hematoxylin and eosin. The pathological features were observed under a light microscope by an experienced pathologist.

Immunohistochemistry Staining

Slides were first deparaffinized and rehydrated. Following that, endogenous peroxidase was blocked with hydrogen peroxide. Then, antigen was retrieved by citrate buffer and washed with phosphate-buffered saline (PBS). For blocking the non-specific reaction, PBS with 2% bovine serum albumin (BSA) was adopted. After three washes with PBS, the slides were incubated at 4°C overnight with the primary antibodies (anti-PR (1 : 100), anti-ERα (1 : 100), anti-CK5/6 (1 : 100), and anti-HER-2 (1 : 100). Subsequently, the slides were incubated with the secondary antibody at room temperature for 15 min. Diaminobenzidine (DAB) was used as the chromogen and the slides were counterstained with hematoxylin buffer. Finally, after rehydration, the slides were cleaned and mounted with Permount TM clwfchbyhjg. The immunohistochemical characters were observed under a light microscope.

Western Blot

Tumor samples were quick frozen in liquid nitrogen and then stored at −80°C in order to further study. To extract the total protein, the tissues were suspended in RIPA lysis buffer (Solarbio, Beijing, China) containing a protease inhibitor cocktail (Solarbio). The expression level of protein was examined via the bicinchoninic acid method. An aliquot of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Solarbio, Beijing, China). Blocked by 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against Bcl-xl (1 : 1000), Bax (1 : 1000), and GAPDH (1 : 1000), followed by horseradish peroxidase-conjugated secondary antibodies. The protein expression was detected using enhanced chemiluminescence system ChemiDoc MP (BioRad, Hercules, CA, U.S.A.).

Statistical Analysis

To compare the effects of breast precancerous induction between DMBA group, DMBA + MPA group and control group, Chi-square test was conducted. Western blot results were analyzed using Student’s t test or one-way ANOVA. with p < 0.05 considered statistically significant.

RESULTS

DMBA Combined with MPA Induced Breast Lesions in Tree Shrews

As tree shrews are timid, each group appeared accidently died tree shrews after reagent injection. When the experiments endpoint was reached at last, there are 25 tree shrews remained alive in control group, 21 in DMBA group, and 20 in DMBA + MPA group. The characteristics of tree shrews succeed in breast lesion induction were demonstrated in Table 1. Only 2 tree shrews (2/21) from DMBA group generated breast lesion; whereas 10 tree shrews (10/20) from DMBA + MPA group were detected breast lesion. No lesion was found in control group. The breast lesion induced rate was noted significantly increased of DMBA + MPA group compared to DMBA group (50 vs. 10.5%, p < 0.05) and control group (50 vs. 0%, p < 0.05), whereas no significance was found between DMBA group and control group (10.5 vs. 0%, p > 0.05). The median latency of breast lesions was 56 d and the earliest tree shrew generated breast lesion was found on the 42nd day after the first chemical injection. The gross view of breast lesions in No. TS01 and No. TS03 tree shrews was exhibited in Fig. 1.

Table 1. The Characteristics of Induced Breast Lesions in Tree Shrews

Group	Case number	Latency of lesion induction (d)	Survival time (d)	Volume induced lesion (mm3)
DMBA	TS01	47	121	628
	TS02	60	245	754
DMBA + MPA	TS03	42	245	4710
	TS04	56	85	132
	TS05	63	92	113
	TS06	45	203	3211
	TS07	68	245	3087
	TS08	56	79	125
	TS09	51	245	4250
	TS10	65	200	1766
	TS11	76	100	904
	TS12	47	235	4187

Fig. 1. Gross View of Progressively Growing Breast Lesions in Tree Shrews

A1. 60 d after first injection (No. TS01); A2. 121 d after first injection (No. TS01). B1. 60 d after first injection (No. TS03); B2. 140 d after first injection (No. TS03); B3. 196 d after first injection (No. TS03); B4. 245 d after first injection (No. TS03).

Ultrasound Examination

Ultrasound examination detected all the breast lesions with regular morphology, clear margin and heterogeneous enhancement. Ultrasound contrast image was revealed that the induced lesions were highly vascular. Ultrasound image of breast lesions in No. TS01 and No. TS03 tree shrews was exhibited in Fig. 2.

Fig. 2. Ultrasound Image of Induced Breast Lesions in Tree Shrews

A. 121 d after first injection (No. TS01). B. 196 d after first injection (No. TS03).

Pathological Characteristics

Histology analysis based on the 12 tree shrews generated breast lesions indicated two type of breast lesion: intraductal papilloma (TS01, TS02, TS04, TS05, TS08, TS10) and atypical ductal hyperplasia (TS03, TS06, TS07, TS09, TS11, TS12). The basic microscopic structure of induced intraductal papilloma consisted of proliferation of gland alveolus and mammary duct. Breast atypical ductal hyperplasia is generally considered a precancerous condition based on WHO Classification of Tumor of the Breast developed by American Joint Committee on Cancer (AJCC), with bigger ductal epithelial cells, increased cytoplasm-nucleus ratio, and enhanced basophilicity of nucleus. Hematoxylin-eosin (H&E) staining results of breast lesions in No. TS01 and No. TS03 tree shrews were exhibited in Fig. 3.

Fig. 3. H&E Staining Results of Induced Breast Lesions in Tree Shrews (×100)

A. Intraductal papilloma (No. TS01); B. Atypical ductal hyperplasia (No. TS03).

Immunohistochemistry Analysis

Immunohistochemistry analysis based on the samples from all of the 12 tree shrews succeed in breast lesions identified that all induced lesions demonstrated positive expression of ERα, PR and CK5/6, but negative expression of HER-2. Immunostaining results of breast lesions in No. TS01 and No. TS03 tree shrews were exhibited in Fig. 4.

Fig. 4. Immunostaining Results of Induced Breast Lesions in Tree Shrews

ER × 200, PR × 200, CK5/6 × 100, Her-2 × 100.

The Protein Expressions of Bcl-xl and Bax in Induced Breast Lesions

As shown in Fig. 5, the results of Western blot based on the samples from all of the 12 tree shrews generated breast lesions indicated that the Bcl-xl protein expression in breast lesion of atypical ductal hyperplasia (precancerous lesion) was higher than intraductal papilloma (benign tumor) (p < 0.05), while the Bax protein expression of atypical ductal hyperplasia was lower than that of intraductal papilloma (p < 0.05).

Fig. 5. The Protein Expressions of Bcl-xl and Bax in Induced Breast Lesions of Intraductal Papilloma and Atypical Ductal Hyperplasia Were Measured by Western Blot Analysis

* p < 0.05. Intraductal papilloma (TS01, TS04, TS05) vs. atypical ductal hyperplasia (TS03, TS06, TS07).

DISCUSSION

A series of progressive pathological characteristics involved hyperplasia, atypical hyperplasia, carcinoma in situ and invasive carcinoma is taken part in the breast tumorigenesis. Some patients have developed benign breast tumor, whereas others have experienced further progression to ultimate carcinoma. It is of great importance to generate animal models in every stage from benign hyperplasia to carcinoma in studies referring to pathogenesis and prevention of breast tumor. As primate-like animals, tree shrews are deemed closely related to humans. Xiao et al. demonstrated that the whole genome sequence of tree shrews were very close to humans based on immune, nervous, and metabolic systems.¹⁴⁾ Compared to rodent, tree shrew is able to share more similarity with human regarding either tumor characteristics or tumor development. So, it is no doubt that tree shrew shows more superiority over rodent as an ideal tumor model.

Xia et al. was succeeded in generating breast tumor in tree shrew by orally DMBA administration and MPA sustained-release tablets implant.¹⁵⁾ However, current methods reported to induce breast tumors in tree shrew were demonstrated a low frequency and long latency. Then, the induction method needs further improvement and optimization. In our study, we established a more stable and efficient breast precancerous tree shrew model. Breast atypical ductal hyperplasia is deemed to be one type of precancerous lesions.¹⁶⁾ By DMBA plus MPA injection, the induced rate of breast lesions in tree shrew reached 50% (intraductal papilloma 20% and atypical ductal hyperplasia 30%), which was comparable to reported results previously.¹⁵⁾ However, the latency of tumourigenesis was from 25 to 33 weeks by intragastric administration of DMBA and implanted MPA sustained-release tablets.¹⁵⁾ Whereas in our study, the median latency of breast lesions was 56 d and the earliest tree shrew generated breast lesion was found on the 42nd days after the first chemical injection. With shorter latency and more safety for avoiding diarrhea resulting from oral administration, our tree shrew model demonstrated superiority over previous model.

In the procedure of tree shrew model establishment, after breast lesions were found by physical examination, we conducted ultrasound examination to the breast lesion. Meanwhile, contrast-ultrasound was performed and exhibited highly vascular lesions of breast. Then, H&E staining revealed that half of induced lesions were intraductal papilloma and the others were atypical ductal hyperplasia. Nevertheless, none of induced breast lesions developed to invasive carcinoma. Finally, we succeeded in establishment of stable breast precancerous lesion on tree shrew model. In the future study, we will further develop invasive carcinoma on tree shrew model by altering dosage and action time of carcinogen, and prolonging observation time.

Immunohistochemistry analysis revealed that all of induced breast lesions positively expressed of ERα, PR and CK5/6, whereas negatively expressed of HER-2, which may be attributed to the development of breast lesion correlated to MPA. Then, the tree shrew model of breast precancerous lesion through our method may just represent one of molecular subtypes. Even the induced lesions further develops to invasive carcinoma, the induced breast cancer may be just hormone-dependent subtype. So, the combination of DMBA and MPA to induce breast precancerous lesion in our study may represent an ideal method to develop hormone-dependent breast tumor.

Loss of apoptotic signaling is of great relationship with cancer development and progression.¹⁷⁾ Based on the inhibition of apoptosis-promoting gene and the activation of inhibitor of apoptosis protein gene, cancer cells develop to immortal cells with unrestricted proliferation.^18,19) Bcl-xl is a member of the Bcl-2 family of proteins, and acts as an anti-apoptosis protein to down-regulate the mitochondrial pathway of apoptosis.²⁰⁾ Bcl-xl is widely accepted its role of apoptosis inhibition by binding to pro-apoptosis protein from Bcl-2 family to form the heterodimer, interacting with apoptotic protease activating factor-1 (Apaf-1), or inhibiting the release of cytochrome c activated by caspase.^17,21,22) Bax is a member of the Bcl-2 family, acting as a pro-apoptotic role. Bax protein could interact with and increase the opening of mitochondrial permeability transition pore (MPTP), leading to the loss of membrane potential, the increase of mitochondrial permeability and the release of cytochrome c, which ultimately activate apoptosis.^23,24) The results of Western blot in our study demonstrated that, compared to benign tumor (intraductal papilloma), the expression of Bcl-xl was significantly higher and the expression of Bax was significantly lower in the precancerous lesions (atypical ductal hyperplasia). Based on these results, we may attribute the breast tumor development in tree shrew to the imbalance of cell apoptosis.

However, there were several limitations in our tree shrew model yet. First, results analysis should take account of bias derive from limited tree shrews of each study group; second, more related genes research during breast tumorogenesis that need further exploration; last, it is of contradiction regarding diagnostic criterion to distinguish high grade atypical ductal hyperplasia and low grade ductal carcinoma in situ.

In conclusion, DMBA is able to induce breast lesions in tree shrews. Combination of DMBA and MPA may be more effective to establish breast precancerous lesion in tree shrew models, and atypical ductal hyperplasia is the predominant breast precancerous lesion type. The induced breast lesions positively expressed of ERα, PR and CK5/6, whereas negatively expressed of HER-2. These findings suggest that tree shrew is a promising animal model for studying the tumorogenesis of breast cancer.

Acknowledgments

Thanks are due to Kezhi Li, Yanping Tang, Qi Zeng and Yawen Chen for assistance with the experiments. This work was supported by the foundation of Guangxi Science and Technology Infrastructure Project (No. 15-235-05), the Natural Science Foundation of China (No. 81360396, No. 81860341), the Natural Science Foundation of Guangxi (No. 2015GXNSFAA139204, No. 2017GXNSFAA198103), the Medication and Health Care Research Program of Guangxi (No. S201418-03) and the Innovation Project of Guangxi Graduate Education (No. YCSW2017108, No. YCBZ2018041).

Conflict of Interest

The authors declare no conflict of interest.

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