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Development of Murine Model for Breast Cancer Metastasis to Bone

Komal Talreja, Archana Moon*

University Department of Biochemistry, Rashtrasant Tukadoji Maharaj Nagpur University Nagpur

Abstract: Breast cancer is the second most common diagnosed cancer. 70% of breast cancers metastasize to the bone. This metastasis is more serious than the original tumor. The present study deals with the generation of animal (rat) model for breast cancer metastasis to bone by insertion of human breast cancer cell line MDA-MB 231. The cell line MDA-MB 231 was administered in the immunosupressed rat femur bone and tumor formation was observed for two months. Blood parameters viz; SGOT, SGPT, ALP, TRAP and calcium and antioxidant enzymes (catalase, SOD, GSH, GSSG, GST, MDA and GPX) in liver tissue were studied after one month and two months of insertion. In our study we observed increase in the antioxidant liver enzyme levels i.e. Catalase, SOD, GSH, GSH and MDA, and SGOT, ALP, TRAP and calcium levels also were increased in serum. Increased levels of antioxidant enzymes are indicative of increases Reactive oxygen species (oxidative stress) are involved in initiation, promotion and progression of carcinogenesis. Further, bone histopathology results indicate necrotic bone in experimental rats, which suggests osteolytic bone metastasis. This developed model for breast cancer metastasisto bone is cost effective and gives insight into the metastatic colonization, in which initially formed micrometastasis succeed in colonizing the marrow and generating osteolytic metastasis.

Keywords: breast cancer, metastasis, cancer cell line, tumor

Citation: Komal Talreja, Archana Moon, Development of Murine Model for Breast Cancer Metastasis to Bone. Journal of Cancer Research and Treatment, 5 (1). 20-27. January 2017. doi:10.12691/jcrt-5-1-4

1. Introduction

Breast cancer metastasis to bone is a multistep process [1]. Although, much progress has been made in understanding primary malignancy, metastasis has been less studied [1]. One of the major obstacles in understanding the complex process of metastasis is lack of an animal model. The existing models do not depict the bone metastasis perfectly. The most common model generated by intravenous injection, leads to the loss of cancerous cells in the route followed [1]. For metastasis, the cancer cells should disseminate from the site of origin and invade the adjacent stroma and enter into the blood circulation, reach the distant site and colonize. In the present study, MDA-MB 231 cancer cells were directly inserted into the femur bone by performing bone ablation surgery.

1.1. Animal Model Generation

Animals for the study underwent bone ablation surgery after approval from the Institutional Animal Ethical Committee, following CPCSEA (Committee for Purpose of Control and Supervision of Experiments on Animals) norms. The breast cancer cells MDA-MB 231 was directly inserted in the femur bone of immunocompromised rats [2].

Cell Lines and Culture Conditions:

MDA-MB231 cell line was procured from National Centre for Cellular Sciences (NCCS), Pune. The cell line was maintained in DMEM (Dulbecco’s Modified Essential Medium) supplemented with 10% FBS (Foetal Bovine Serum), 0.1% antibiotic solution (actinomycin/streptomycin) and 5% CO₂. The cells were trypsinised after every 2-3 days [2].

1.2. Cells Insertion in Rats by Bone Ablation Surgery

Rats were obtained from NIN (National Institute of Nutrition) and maintained in the animal house of Department of Biochemistry, RTMNU, Nagpur with 27⁰C and 60% humidity. All rats were maintained with 12 hour day and night cycle. Food and water were provided ad libitum. The rats were housed 3 per cage. They were divided into 5 groups, viz: Control group, SHAM group, Group I, Group II and Group III. Each group consisted of 6 animals (3 males and 3 females) [2].

1.3. Immunosuppression

Leflunomide was administered to generate immunocompromised rats. Leflunomide is a drug which blocks de- novo pyrimidine synthesis. The active component of leflunomide is A77 1726 [3]. Group I, Group II and Group III rats were given doses of leflunomide orally by gavage. Leading dose of 100 mg/kg BW followed by continuous doses of 10 mg/kg BW for 3 days were administered. After the final dose of leflunomide, different concentration of cells were inserted in Group I (10⁵ cells), Group II (10⁶ cells), Group III (10⁷ cells) in the femur bone of rats.

1.4. Administration of Cancerous Cells into Femur Bone of Rats

The cells were washed with PBS and then trypsinized by adding 2 ml trypsin/EDTA. Then, the cells were incubated for 4 minutes in a CO₂ incubator. When the cells started leaving the surface of the flask (substratum), complete media (DMEM with 10% FBS and 0.1% antibiotic solution) (2 ml) was added to neutralize trypsin/EDTA. Further, the tube was centrifuged and the cells were suspended in 1 ml complete media. Cell counting was done with trypan blue dye to differentiate between viable and dead cells. Required number of cells were suspended in 200 µl complete media and taken in 1 ml syringe [4]. Surgical procedure was performed under general anaesthesia using thiopentane (45 mg/kg body weight). Anaesthesia was administered intraperitoneally. The knee and leg area of the rat was prepared and wiped with 70% alcohol. A longitudinal medial and parapatellar incision was made. Knee was held in 90° flexion, and a hole was drilled into the femur with a syringe, and the cell suspension (200 µl) was released into the bone [5]. After surgery, the drilled hole was sutured using catgut. After one month and two months, serum parameters (SGOT, SGPT, ALP, protein, TRAP and calcium), liver parameters (SGOT, SGPT and ALP) and antioxidant enzymes (Catalase, SOD, GST, GSSG, GSH, GPX and MDA) were investigated. To obtain serum, the blood was withdrawn from retro orbit plexus.

• Serum Glutamate oxaloacetate Transaminase (SGOT)

SGOT was estimated by Reitmann and Frankle method. In this method, the amount of oxaloacetate released, is measured colorimetrically; by the formation of hydrazones with DNPH reagent which is highly coloured in alkaline medium. The coloured product was read at 540 nm [6].

• Serum Glutamate Pyruvate Transaminase (SGPT)

SGPT was estimated by Reitmann and Frankle method. In this method the amount of pyruvate released, is determined colorimetrically; by the formation of hydrazones with DNPH reagent. The coloured final product was read at 540 nm [6].

• Alkaline Phosphatase (ALP)

ALP was estimated by King & King Method. In this method, the serum is incubated with the buffer substrate under optimum conditions to release phenol, which reacts with 4- animoantipyrine in alkaline medium to give a red coloured compound which is measured at 520nm [6].

• Total Protein:

Total protein was performed by biuret method. Proteins in the serum bind with the cupric ions present in the biuret reagent to form a blue-violet coloured complex, measured at visible wavelength 546 nm.

• Calcium:

The method utilizes the metallochromogen (Arsenazo III) which combines with calcium ions present in the serum to give a highly coloured chromophore. The absorbance measured at 630 nm.

• TRAP

In this method, the serum samples were incubated with citrate buffer or distilled water at 37°C for 1 hour, to inactivate the TRAP released from erythrocyte. The reaction mixture (PNPP p- nitro phenyl phosphate, sodium citrate, sodium tartarate and sodium chloride) was added to the serum samples. Reaction was stopped by adding NaOH [7].

1.5. Antioxidant Enzymes

Seven antioxidant enzymes were estimated.

♣ Catalase:

In the presence of catalase, hydrogen peroxide (H₂O₂) is converted into oxygen and water. The present method is based on measurement of decrease in absorbance of hydrogen peroxide which is measured at 240 nm. The decrease in absorption is directly proportional to the activity of enzyme [8].

♣ Super oxide Dismutase (SOD):

This assay method for SOD is based on the ability of enzyme to inhibit autoxidation of Pyrogallol (1,2,3 benzene triol). Pyrogallol is known to autooxidises rapidly. SOD dismutate the superoxide anion radical. As a result the solution becomes yellow –brown which is read at 420 nm [9].

♣ Reduced Glutathione (GSH):

Glutathione is a smallest thiol molecule, which plays an important role in antioxidant defence mechanisms. In this method, Dithiobis-2-nitrobenzoic acid (DTNB) was used as a colouring reagent. 2100µl of potassium phosphate EDTA buffer, 300 µl sample (tissue sample), equal volume of DTNB and Glutathione Reductase was mixed in cuvette and incubated for 30 seconds. This allows the conversion of GSSG (oxidized form of glutathione) to GSH (reduced form of glutathione). 60 µl of NADPH was added and the reaction mixture was read at 412 nm [10,11].

♣ Oxidized Glutathione (GSSG):

This method is known as GSH recycling assay. The method uses the property of glutathione reductase, to reduce the GSSG to GSH in presence of NADPH. The tissue samples were treated with 2 vinyl pyridine, and incubated for 1 hour before assay. 2 vinyl pyridine was used as derivatizing agent to mask the –SH group of GSH. The reaction starts with conjugation of GSH with DTNB to form the mixed disulphide GS-TNB and chromophore 5-thio-2-nitrobenzoic acid (TNB) which is followed by back-reduction of GS-TNB to GSH by GR and NADPH (prevailing reaction) or by direct reaction of GS-TNB with any GSH still present in the assay mix with formation of GSSG. Then TNB is measured by spectrophotometer at a 412-nm wavelength [12].

♣ Glutathione S Transferase (GST):

GST is an enzyme involved in detoxification of wide range of compounds and in reducing oxidative stress. The assay reaction involves the conjugation of GSH with 1-chloro 2,4 dinitro benzene (CDNB), which is observed by an increase in absorbance at 340 nm. 980 µl PBS (phosphate buffer saline), 10 µl CDNB and 10 µl glutathione was added in the cuvette and increase in absorbance was observed [13].

♣ Glutathione peroxidise (GPX):

GPX activity was measured by a method described by Flohe and Gunzler. The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7), 3.6 mM GSH, 3.6 mM sodium azide, 1 IU/mL glutathione reductase, 0.2 mM NADPH, and 1.2 mM H₂O₂. In this assay, the GSSG is generated by GPX, which was then reduced by GR (glutathione reductase), and NADPH oxidation was monitored at 340 nm [14].

♣ Lipid Peroxidation:

Lipid peroxidation was expressed in terms of MDA/g tissue, as MDA is major degradation product of lipid hydroperoxides. In this assay thiobarbituric acid (TBA) reactive (TBAR) products is measured (varshney and kale) [15]. Two molecules of TBA react with a molecule of MDA to form pink complex which is measured at 530 nm.

1.6. Collagen Staining

Picro Sirius red stain was used for histological visualization of collagen. During cancer metastasis to bone, collagen content decreases. In this study, the bone samples were sent for histology and paraffin embedded bone sections slides were procured [16]. The sections were deparaffinize and then stained with picro Sirius red.

Deparaffinization [17]:

a) 3 changes of xylene for 5 minutes each.

b) 2 changes of 100 % ethanol for 5 minutes each.

c) 2 changes of 95% ethanol for 5 minutes each.

d) 70% ethanol for 5 minutes.

e) 2 changes of deionised distilled water for 1 minute each.

Staining [18]:

a) After deparaffinization, apply adequate picro Sirius red solution to completely cover the tissue section and incubate for 60 minutes.

b) Rinse the slide in 2 changes of acetic acid solution.

c) Rinse slide in absolute alcohol.

d) Dehydrate in 2 changes of absolute alcohol.

e) Clear slide and mount in synthetic stain.

2. Results

• SGOT and SGPT:

SGOT levels increased by 57% as the number of cells administered increased. The highest increase was observed in GroupII (10⁶ cells were administered). There was no significant difference observed in SGPT after insertion of breast cancer cells.

Figure 1. Represents Variation in SGOT (A) and SGPT (B) levels in different groups after two months of insertion of MDA-MB 231 cells

• ALP and TRAP:

After two months of insertion of breast cancer cells, ALP level increased by 14 %. The highest level was observed in Group III (10⁷ cells was administered). 12% Increase in tartarate resistance acid phosphatase was observed after two months of administration of breast cancer cells (MDA-MB 231).

Figure 2. Represents Variation in ALP (A) and TRAP (B) levels in different groups after two months of insertion of MDA-MB 231 cells

• Calcium and Protein:

Calcium levels increased by 60% after two months of administration of breast cancer cells. After insertion of cells in femur bone, the bone releases calcium in blood and becomes fragile. Protein concentration increased by 11.6% after insertion of breast cancer cells.

Figure 3. Represents (A) Variation in calcium (A) and protein (B) levels in different groups after two months of insertion of MDA-MB 231 cells

3.1. Antioxidant Enzymes

• Catalase and SOD:

Catalase levels increased by 55.4% after two months of insertion of breast cancer cells. Highest levels were observed in Group II (10⁶ cells was administered). SOD levels increased by 60% after two months of administration of breast cancer cells.

Figure 4. Represents Variation in catalase (A) and SOD (B) levels in different groups after two months of insertion of MDA-MB 231 cells

• GST and GSH:

GST levels increased by 60 % after two months of insertion of breast cancer cells. Highest increase was observed in Group III (10⁷ cells were administered). 23% increase was observed in GSH after two months of administration of breast cancer cells. Highest increase was observed in Group III (10⁷ cells were administered).

Figure 5. Represents Variation in GST (A) and GSH (B) levels in different groups after two months of insertion of MDA-MB 231 cells

• GSSG and GPX:

There was no significant difference observed in GSSG and GPX after insertion of breast cancer cells.

Figure 6. Represents Variation in GSSG (A) and GPX (B) levels in different groups after two months of insertion of MDA-MB 231 cells. GPX

• MDA:

MDA levels increased by 26 % after two months of insertion of breast cancer cells. Highest increase was found in Group III (10⁷ cells were administered).

Figure 7. Represents (A) Variation in MDA levels in different groups after two months of insertion of MDA-MB 231 cells

3.2. Liver Enzymes

• SGOT and SGPT:

SGOT levels increased in liver homogenate by 24% after two months of administration of breast cancer cells. But the increase is not very significant. There was no significant difference observed in liver homogenate SGPT after insertion of breast cancer cells.

Figure 8. Represents Variation in SGOT (A) and SGPT (B) levels in liver homogenate in different groups after two months of insertion of MDA-MB 231 cells

• ALP and Protein:

There was no significant difference observed in liver homogenate SGPT and protein after insertion of breast cancer cells.

Figure 9. Represents Variation in ALP and protein levels in liver homogenate in different groups after two months of insertion of MDA-MB 231 cells

Liver Histology:

Figure 10. Liver histology of different groups (10 X resolution)

Bone Histology:

Figure 11. Bone histopathology of different groups (10 X resolution)

Collagen Staining:

Figure 12. Bone collagen staining of experimental rats of different groups (10 X resolution)

3. Discussion

In this study, breast cancer cell line MDA-MB 231 was inserted in the femur bone of rat. The serum investigations reveal that, SGOT, ALP, TRAP (Tartarate resistant acid phosphatase) and calcium levels increased from the normal range. Reactive oxygen species (ROS) generated from mitochondria electron transport chain results in oxidative stress [19]. To measure antioxidant stress, the antioxidant enzymes catalase, SOD (superoxide dismutase), GPX (glutathione peroxide), GSH (reduced glutathione), GSSG (glutathione reduced), GST (glutathione–s-transferase) and MDA (malonyl aldehyde) were measured [19]. Catalase, SOD, GST, GSH and MDA showed increased levels. The significant increase in the levels of these antioxidant enzymes indicates their role as key enzymes in the protection of liver from free radicals [19]. An increased level of oxidative stress is further proved by increased level of MDA [15]. The increase in MDA levels inspite of increased activity of antioxidant enzymes could be due to high concentration of ROS, in excess of the capacity of antioxidant enzymes to neutralize the free radicals [19]. Increased levels of MDA also indicate increased lipid peroxidation, which causes impairment of membrane functions and inactivation of membrane bound receptor and enzymes [15]. Increased lipid peroxidation is also reported in case of solid tumors [20]. These results are indicative of increase in ROS or oxidative stress and studies on breast cancer metastasis have reported that ROS are involved in initiation, promotion and progression of carcinogenesis [20]. Major clinical complication of metastatic breast cancer is osteolytic bone destruction, when breast cancer metastasizes to bone. This further leads to increased fracture risk [21]. Breast cancer patients often experience low bone mineral density and accelerated bone loss as unavoidable side effects of cancer therapies [21]. In osteolytic bone destruction, calcium is released from the bone and hence increased levels in serum are observed. In our study we observed increase in the antioxidant liver enzyme levels i.e. SGOT, ALP, TRAP and calcium levels also were increased in serum. This corroborates that the animal model for breast cancer metastasis to bone was generated successfully.

Liver histopathology reports show that, as the number of administered cells increases, inflammation increases in liver. Necrosis was also observed in Group II and Group III experimental animals in which 10⁶ and 10⁷ cells were inserted. This indicates liver damage in Group II and Group III experimental animals.

Bone histopathology results reveal that the bone trabecular region decreases and appears fragmented (necrotic bone) in group II and Group III. The bone marrow content also decreases in Group II and Group III experimental animals. All of the studied parameters indicate changes in experimental animals when bone histology was performed after administration of breast cancer cells.

4. Conclusion

Various experimental animal models for mammary tumor have been developed, but they rarely generate bone metastasis [1]. Most commonly used model of cancer metastasis employ intravenous administration of cells, a route that bypass early steps of invasion metastasis cascade [1]. This model gives insight into the metastatic colonization, in which initially formed micrometastasis succeed in colonizing the marrow and generating osteolytic metastasis [1]. In the present study, animal model for breast cancer metastasis was generated by administration of MDA-MB 231 breast cancer cell line. This was evident by increase in antioxidant enzymes in liver homogenate, increase in calcium, SGOT, TRAP in serum levels. Liver and bone histopathology reveals inflammation and necrosis, which indicates as the number of administered cells increases, damage to liver and bone increases. Since free radicals contribute towards the development of cancer, it can be assumed that antioxidants help reduce the risk of cancer [20]. Further studies on formulating a conditioned Nutrient formulation for prevention and treatment of breast cancer metastasis to bone is envisaged.

Acknowledgements

University Research Project: Development of Murine model for breast cancer metastasis to bone. NO Dev/AH/2135 dated 16 November 2015.

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