Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

Summary

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

Abstract

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

Introduction

Animal models play a key role in cancer research. When a hypothesis is proven in vitro, it should be tested in vivo to evaluate its clinical relevance. Cancer progression and metastasis are often better captured by animal models as compared to in vitro models, and it is essential to test a new drug in an animal model as a preclinical study for drug development^1,2. However, the technical details of animal experiments are often not well described in published articles, making it challenging to reproduce the model successfully. Indeed, the authors who established these orthotopic inoculation and mastectomy models went through long and rigorous processes of trial and error. The success rate of tumorigenesis after cancer cell inoculation is one of the key factors to determine the success and efficiency of an animal study³. The cell line and the number of cells to inoculate, the inoculation site, and the strain of the mice are all important factors. It is well known that there are huge variations in the results of animal experiments due to individual differences, compared to in vitro techniques. Therefore, using a well-established model with a standard technique is important to obtain stable results, to improve the efficiency of animal experiments, and to avoid misleading results.

This paper provides well-established techniques⁴ to generate breast cancer orthotopic and mastectomy mouse models. The aims of these methods are 1) to mimic human breast cancer progression and treatment courses, and 2) to conduct in vivo experiments with greater efficiency and higher success rates compared to other breast cancer inoculation or mastectomy techniques. In orthotopic cancer cell inoculation, to mimic human breast cancer progression, we choose the #2 mammary fat pad as an inoculation site, which is located in the chest. In most of the studies, breast cancer cells are inoculated subcutaneously⁵. This technique does not require surgery and, thus, it is simple and straightforward. However, the subcutaneous microenvironment is quite different from the mammary gland microenvironment, which results in different cancer progression and even molecular profiles^6,7. Some studies use the #4 mammary gland, which is located in the abdomen, as an inoculation site⁶. However, since #4 mammary glands are located in the abdomen, the most common metastatic pattern is peritoneal carcinomatosis⁷, which occurs with less than 10% of metastatic breast cancer⁸. Breast cancer generated by the technique presented here, in the #2 mammary gland, metastasizes to the lung, which is one of the most common breast cancer metastatic sites⁹.

With this technique, the goal is also to achieve a higher tumorigenesis rate with minimal tumor size variability compared to other breast cancer inoculation techniques. To do so, cancer cells suspended in a gelatinous protein mixture are inoculated under direct vision through a median anterior chest wall incision. This technique produces a high tumorigenesis rate with less variability in tumor size and shape compared to subcutaneous or non-surgical injection, as previously reported^3,7.

We also introduce a mouse radical mastectomy technique in which the orthotopic breast tumor is resected with the surrounding tissues and axillary lymph nodes. In the clinical setting, the standard of care for breast cancer patients without distant metastasis disease is mastectomy^10,11. Before a mastectomy, axillary lymph node metastasis is surveyed by imaging and sentinel lymph node biopsy. If there is no evidence of axillary lymph node metastasis, the patient is then treated with a total or partial mastectomy, in which the axillary lymph node resection is omitted. Total mastectomy is a technique to resect breast cancer with the whole breast tissue en bloc, whereas partial mastectomy is to resect breast cancer with a margin of surrounding normal breast tissue only, thus conserving the remaining normal breast tissue in the patient. However, patients who preserve remaining normal breast tissue after a partial mastectomy require postoperative radiotherapy to avoid local recurrence¹⁰. Patients who have axillary lymph node metastasis undertake radical mastectomy which removes the breast cancer with all normal breast tissue and axillary lymph nodes and invaded tissues en bloc^10,11. In the mouse model, surveillance for axillary lymph node metastasis and/or post-operative radiation is not reasonable or feasible. Thus, we utilize the radical mastectomy technique to avoid local or axillary lymph node metastasis.

Cancer cell inoculation via the tail vein is the most common lung metastasis mouse model¹², the so-called "experimental metastasis". This model is easy to generate and does not require surgery; however, it does not mimic human breast cancer progression which may result in different metastatic disease behavior. In order to mimic the human breast cancer treatment course where metastasis often occurs after mastectomy, the primary tumor is removed after orthotopic cancer cell inoculation. This technique produces less local recurrence compared to simple tumor resection, as previously reported¹³, and is useful for novel therapeutics, preclinical studies, and for metastatic breast cancer research studies. The techniques described here are applicable for most breast cancer orthotopic model experiments. However, it is important to consider that the gelatinous protein mixture can affect the microenvironment and surgery can affect the stress/immune response¹⁴. Therefore, investigators studying the microenvironment and/or the stress/immune response should be aware of potential confounding factors.

Protocol

Approval from the Roswell Park Comprehensive Cancer Center Institutional Animal Care and Use Committee was obtained for all experiments.

NOTE: Nine to twelve weeks-old female BALB/c mice are obtained. 4T1-luc2 cells, a mouse mammary adenocarcinoma cell line derived from BALB/c mice that has been engineered to express luciferase, are used. These cells are cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS).

1. Preparation of Instruments

1. Thaw a frozen gelatinous protein mixture (e.g., Matrigel) on ice in a tissue culture hood.
2. Clean and autoclave two sets of surgical instruments (microdissection scissors, Adson forceps, and a needle holder) prior to surgery. Prepare sterilized 5-0 silk sutures and dry sterilant (when serial surgeries are planned).
3. Clip the middle chest hair of the mice using a clipper and mark the mice for identification by punching the ear prior to the time of surgery.
4. Prepare the procedure table, which can be immediately used for the operation.
   1. Spread an absorbent pad and fix the corners with tape, fix anesthesia nose cones with tape, put sterilant and disinfectant (chlorhexidine, iodine, and 75% ethanol) beside the operating space, and place the mice in operation order.

2. Preparation of Cells (for 10 Mice)

NOTE: The cells should be inoculated within 1 h after being detached from the dish to avoid decreased cell viability. Specifically, the cell suspension should be mixed into the gelatinous protein mixture within 15 min after detaching the cells from the dish to maintain their viability.

1. Culture 4T1-luc2 cells, a mouse mammary adenocarcinoma cell line expressing luciferase, in RPMI 1640 media with 10% FBS in a humidified incubator at 37 °C in 5% CO₂.
2. Wash the adherent 4T1-luc2 cells in a 10-cm dish with phosphate-buffered saline (PBS) using a 10 mL serological pipette. Add 1 mL of 0.25% trypsin using a P1000 pipette and, then, incubate the sample at 37 °C for 5 min. Then, add 4 mL of growth media (RPMI-1640 with 10% FBS) using a 5 mL serological pipette and transfer the cell suspension to a 15-mL conical tube in a tissue culture hood. Centrifuge the cell suspension at 180 x g for 5 min.
3. Aspirate the supernatant and resuspend the cells in 2 mL of PBS; then, count the cells using the hemocytometer.
4. Suspend 2 x 10⁶ 4T1-luc2 cells in 40 μL of cold PBS (pH 7.4, 4 °C) in the tissue culture hood.
5. Mix the 40-μL cell suspension with 360 μL of the gelatinous protein mixture in a 1.5 mL microcentrifuge tube on ice in the tissue culture hood.
   NOTE: The final concentration is 1 x 10⁵/20 μL (1:9 PBS:gelatinous protein mixture). For the orthotopic model (no mastectomy), 1 x 10⁴/20 μL of the final concentration was used, to avoid reaching euthanasia criteria (tumor size > 2 cm) within two weeks.

3. Cancer Cell Inoculation

1. Put the mice in the anesthesia induction chamber with 2-4% isoflurane and 0.2 L/min oxygen flow until the mice breathe calmly (2–3 min).
2. Grasp the mouse and inject 0.05 mg/kg buprenorphine into its shoulder subcutaneously.
3. Confirm adequate anesthesia by the lack of reaction to a toe pinch. Insert the mouse’s nose into the hole of the mouse mask, which allows inhalational anesthesia with 2-4% isoflurane and 2 L/min oxygen flow attached to a charcoal canister unit.
4. Restrain the mouse’s limbs using lab tape and sterilize its skin using chlorhexidine, iodine, and 75% ethanol, using cotton swabs.
5. Make a 5 mm skin incision in the middle of the anterior chest wall utilizing sterile microdissection scissors, lift the right-side skin next to the incision and detach the skin from the chest wall using the scissors, and then, invert the skin to expose the right #2 mammary fat pad.
6. Carefully inject 20 μL of cancer cell suspension using a 1 mL insulin syringe with a 28.5 G needle into the fat pad under direct vision through the wound.
   NOTE: The needle goes through the wound, not the skin. Keep holding the needle in the fat pad for 5 s prior to pulling it out, which allows time for the gelatinous protein mixture to solidify.
7. Close the skin incisions by stitching, using sterile 5-0 non-absorbable sutures.
8. After surgery, return the animals to a clean cage and monitor them until they have recovered and are moving freely (after ~1–2 min). If an animal does not appear to be in good health within 24 h of surgery, administer buprenorphine (0.2 mg/kg).
9. Remove the sutures under anesthesia (see step 3.1) 7 d after the surgery.

4. Mastectomy

NOTE: The timing of the mastectomy is very important. If it is done too early, lung metastasis does not occur. If it is done too late, the primary tumor has invaded major blood vessels, which make a complete oncologic resection challenging. Thus, multiple time points were tested for mastectomy to determine which time point produced the appropriate balance in waiting for metastasis before resection became too challenging. After doing so in over 50 mouse experiments, it was demonstrated that mastectomy at 8 days after cancer cell inoculation (or when the tumor size reaches 5 mm) was the ideal time point to achieve that balance¹³.

1. Anesthetize a mouse with 2-4% inhaled isoflurane and inject buprenorphine (see steps 3.1 and 3.2).
2. Restrain the mouse and sterilize its skin (see step 3.4).
3. Make a 5 mm skin incision 2 mm to the left from the surgical scar that was made at the initial cancer cell inoculation, using the microdissection scissors. Extend the incision toward the root of the forelimb to remove the tumor, the skin including the surgical scar, and the lesion in contact with the tumor, as well as the axillary lymph node basin in which most of the time no visible lymph node exists at the time of the mastectomy¹³. Make sure not to damage the axillary vein.
4. Close the skin defects by stitching, using sterile 5-0 non-absorbable sutures in the shape of a “Y”.
5. The same as in step 3.8, return the mouse to a clean cage and monitor until they have recovered.
6. Remove the sutures under anesthesia (see step 3.1) 7 days after surgery.

5. Bioluminescent Quantification of the Primary Tumor (Orthotopic Inoculation Without Mastectomy) or Lung Metastasis (Mastectomy Model)

NOTE: For primary tumor burden quantification, the bioluminescence is measured 2x a week from the day after the orthotopic inoculation. For lung metastasis quantification, the bioluminescence is measured 2x a week from the day after the mastectomy.

1. Dissolve D-luciferin in Dulbecco’s phosphate-buffered saline (DPBS) to a final concentration of 15 mg/mL in a tissue culture hood. Aliquot it into light-shielded 1.5 mL microcentrifuge tubes. Store the diluted solution at -80 °C.
   NOTE: For a 20 g mouse, 200 µL of diluted D-luciferin is required.
2. Open the imaging software and click Initialize.
   NOTE: It takes about 15 min to cool the charge-coupled device (CCD). When the CCD reaches the set temperature, the color of the Temperature bar changes from red to green.
3. Anesthetize the mice with 2-4% isoflurane in a dedicated induction chamber prior to imaging (see step 3.1).
4. Weigh the mice.
5. Inject 150 mg/kg D-luciferin intraperitoneally at the point of the mid-abdomen, using a 28.5 G needle.
6. Fit each mouse with a nose cone inside the imaging system in the supine position (place a maximum of five mice at the same time). Maintain anesthesia at 1% - 3% isoflurane (in 100% oxygen) through the nose cones during imaging.
7. Capture an image every 5 min to detect the peak bioluminescence for 50 min (or up to the confirmed peak bioluminescence).
   1. Select Luminescence as Auto, Binning as Medium, and Field of View as D.
   2. Click Acquire to capture the image.
8. Return the mice to their cage(s) and monitor them until they have recovered (see step 3.8).

6. Lung Metastasis Tumor Burden Quantification by Ex Vivo Imaging

NOTE: Lung metastasis quantification is applicable for orthotopic inoculation both with and without mastectomy models. In the mastectomy model, ex vivo imaging or survival observation is chosen, depending on the purpose. In the orthotopic inoculation (without mastectomy) model, most cases produce primary tumor size euthanasia criteria (> 2 cm) approximately 21 days after inoculation.

1. Quantify lung metastatic lesions 21 days after the cancer cell inoculation by ex vivo imaging.
2. Anesthetize the mice with 2-4% isoflurane in a dedicated induction chamber (see step 3.1).
3. Weigh the mice.
4. Inject 150 mg/kg D-luciferin intraperitoneally (see step 5.6).
5. Euthanize the mice by cervical dislocation, 15 min after the injections.
6. Open the abdomen by cutting the skin and peritoneum at the mid-abdomen, using curved Mayo scissors. Extend the incision to both the right and the left. Pull out the liver with forceps until the diaphragm is visualized; then, cut the diaphragm.
7. Using the curved Mayo scissors, cut the bilateral ribs from caudad (12th ribs) to cephalad (1st ribs) to expose the lungs by flipping the anterior thorax wall.
8. Identify the thoracic esophagus, which looks like a cord connecting the lungs to the spine, by lifting the lungs using forceps and, then, cut the esophagus using the microdissection scissors.
9. Lift the bilateral lungs and heart using forceps (applying traction pulling down, in the direction of cephalad to caudad) and, then, cut the trachea and major vessels to the lung apex at the cephalad, using microdissection scissors.
   NOTE: This allows for the isolation of the lung and heart from the body.
10. Remove the heart from the lung using microdissection scissors.
11. Put the lungs in a 10 cm Petri dish.
12. Capture the bioluminescence image (see steps 5.7.1 and 5.7.2) 5 min after euthanasia (20 min after the luciferin injection).

Results

The purpose of the orthotopic model is to mimic human cancer progression (i.e., the growth of the primary tumor followed by lymph node metastasis and then distant lung metastasis)¹⁵. After cancer cell inoculation, the bioluminescence is quantified regularly (two to three times/week) (Figure 1A). The bioluminescence in the lungs is deeper and smaller than the primary lesion. The bioluminescence mainly reflects the primary tumor...

Discussion

For the last decade, we have been establishing multiple murine cancer models, including breast cancer models^{3,7,13,16,20,21}. Previously, we demonstrated that breast cancer cell orthotopic inoculation into the mammary gland tissue under direct vision produced a larger tumor with less size variability compared to injecting cell...

Materials

Name	Company	Catalog Number	Comments
Micro Dissection Scissors	Roboz	RS-5983	For cancer cell inoculation and masstectomy
Adson Forceps	Roboz	RS-5233	For cancer cell inoculation and masstectomy
Needle Holder	Roboz	RS-7830	For cancer cell inoculation and masstectomy
Mayo	Roboz	RS-6873	For ex vivo
5-0 silk sutures	Look	774B	For cancer cell inoculation and masstectomy
Dry sterilant (Germinator 500)	Braintree Scientific	GER 5287-120V	For cancer cell inoculation and masstectomy
Clipper	Wahl	9908-717	For cancer cell inoculation and masstectomy
Matrigel	Corning	354234	For cancer cell inoculation
D-Luciferin, potassium salt	GOLD-Bio	LUCK-1K	For bioluminescence quantification
Roswell Park Memorial Insitute 1640	Gibco	11875093	For cell culture
Fetal Bovine Serub	Gibco	10437028	For cell culture
Trypsin-EDTA (0.25%)	Gibco	25200056	For cell culture