Name: Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy (Video) | JoVE Journal
Uploaded: 2018-11-29T13:00:00.000+00:00
Duration: 5 min 5 s
Description: Learn to generate a murine orthotopic metastatic breast cancer model using bioluminescence imaging for tumor burden quantification. This method enables researchers to study human-like cancer progression and treatment responses.

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5 minutes

This work was supported by NIH grant R01CA160688 and Susan G. Komen Foundation Investigator Initiated Research grant (IIR12222224) to K.T. Mice bioluminescence images were acquired by shared resource Translational Imaging Shared Resource at Roswell Park Comprehensive Cancer Center, which was supported by the Cancer Center Support Grant (P30CA01656) and Shared Instrumentation grant (S10OD016450).

Approval from the Roswell Park Comprehensive Cancer Center Institutional Animal Care and Use Committee was obtained for all experiments.
NOTE: Nine&#160;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
<ol>
	<li>Thaw a frozen gelatinous protein mixture (e.g., Matrigel) on ice in a tissue culture hood.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Prepare the procedure table, which can be immediately used for the operation.
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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 &#176;C in 5% CO2.</li>
	<li>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 &#176;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.</li>
	<li>Aspirate the supernatant and resuspend the cells in 2 mL of PBS; then, count the cells using the hemocytometer.</li>
	<li>Suspend 2 x 106 4T1-luc2 cells in 40 &#956;L of cold PBS (pH 7.4, 4 &#176;C) in the tissue culture hood.</li>
	<li>Mix the 40-&#956;L cell suspension with 360 &#956;L 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 &#956;L (1:9 PBS:gelatinous protein mixture). For the orthotopic model (no mastectomy), 1 x 104/20 &#956;L of the final concentration was used, to avoid reaching euthanasia criteria (tumor size &#62; 2 cm) within two weeks.</li>
</ol>
3. Cancer Cell Inoculation
<ol>
	<li>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&#8211;3 min).</li>
	<li>Grasp the mouse and inject 0.05 mg/kg buprenorphine into its shoulder subcutaneously.</li>
	<li>Confirm adequate anesthesia by the lack of reaction to a toe pinch. Insert the mouse&#8217;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.</li>
	<li>Restrain the mouse&#8217;s limbs using lab tape and sterilize its skin using chlorhexidine, iodine, and 75% ethanol, using cotton swabs.</li>
	<li>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.</li>
	<li>Carefully inject 20 &#956;L 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.</li>
	<li>Close the skin incisions by stitching, using sterile 5-0 non-absorbable sutures.</li>
	<li>After surgery, return the animals to a clean cage and monitor them until they have recovered and are moving freely (after ~1&#8211;2 min).&#160;If an animal does not appear to be in good health within 24 h&#160;of surgery, administer buprenorphine (0.2 mg/kg).</li>
	<li>Remove the sutures under anesthesia (see step 3.1) 7 d after the surgery.</li>
</ol>
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.
<ol>
	<li>Anesthetize a mouse with 2-4% inhaled isoflurane and inject buprenorphine (see steps 3.1 and 3.2).</li>
	<li>Restrain the mouse and sterilize its skin (see step 3.4).</li>
	<li>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.</li>
	<li>Close the skin defects by stitching, using sterile 5-0 non-absorbable sutures in the shape of a &#8220;Y&#8221;.</li>
	<li>The same as in step 3.8, return the mouse to a clean cage and monitor until they have recovered.</li>
	<li>Remove the sutures under anesthesia (see step 3.1) 7 days after surgery.</li>
</ol>
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.
<ol>
	<li>Dissolve D-luciferin in Dulbecco&#8217;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 &#176;C. 
	NOTE: For a 20 g mouse, 200 &#181;L of diluted D-luciferin is required.</li>
	<li>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.</li>
	<li>Anesthetize the mice with 2-4% isoflurane in a dedicated induction chamber prior to imaging (see step 3.1).</li>
	<li>Weigh the mice.</li>
	<li>Inject 150 mg/kg D-luciferin intraperitoneally at the point of the mid-abdomen, using a 28.5 G needle.</li>
	<li>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.</li>
	<li>Capture an image every 5 min to detect the peak bioluminescence for 50 min (or up to the confirmed peak bioluminescence).
	<ol>
		<li>Select Luminescence as Auto, Binning as Medium, and Field of View as D.</li>
		<li>Click Acquire to capture the image.</li>
	</ol>
	</li>
	<li>Return the mice to their cage(s) and monitor them until they have recovered (see step 3.8).</li>
</ol>
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 (&#62; 2 cm) approximately 21 days after inoculation.
<ol>
	<li>Quantify lung metastatic lesions 21 days after the cancer cell inoculation by ex vivo imaging.</li>
	<li>Anesthetize the mice with 2-4% isoflurane in a dedicated induction chamber (see step 3.1).</li>
	<li>Weigh the mice.</li>
	<li>Inject 150 mg/kg D-luciferin intraperitoneally (see step 5.6).</li>
	<li>Euthanize the mice by cervical dislocation, 15 min after the injections.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Remove the heart from the lung using microdissection scissors.</li>
	<li>Put the lungs in a 10 cm Petri dish.</li>
	<li>Capture the bioluminescence image (see steps 5.7.1 and 5.7.2) 5 min after euthanasia (20 min after the luciferin injection).</li>
</ol>

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</ol>

The authors have nothing to disclose.

For the last decade, we have been establishing multiple murine cancer models, including breast cancer models3,7,13,16,20,21. 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...

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 development1,2. 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 profiles6,7. 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 reported3,7.
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 mastectomy10,11. 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 bloc10,11. 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 &#34;experimental metastasis&#34;. 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.

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

generating a murine orthotopic metastatic breast cancer model and performing murine radical mastectomy

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

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.

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...

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.

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

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JoVE Journal

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The video demonstrates the breast cancer orthotopic inoculation technique in a mouse model. This technique produces a high tumorigenesis rate with less variability in tumor size and shape compared to subcutaneous or non-surgical injection.

Orthotopic Injection: Implanting Tissue Specific Cancer Cells into an Adult Mouse

This video describes a technique of radical mastectomy, which is used for the surgical removal of the entire mammary gland from a mouse to study cancer progression.

Radical Mastectomy: Surgical Removal of the Entire Mammary Gland from a Mouse to Study Cancer Progression

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

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An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.<br...

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

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Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Learn to generate a murine orthotopic metastatic breast cancer model using bioluminescence imaging for tumor burden quantification. This method enables researchers to study human-like cancer progression and treatment responses.

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

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