The authors would like to thank the National Natural Science Foundation of China (grant no. 81873111, 81673924, 81774039, 81503517), the Beijing Natural Science Foundation (grant no. 7172095, 7162084, 7162083), and the Xu Xia Foundation of the Beijing Hospital of Traditional Chinese Medicine (grant no, xx201701).&#160;We thank Prof. Chang-Zhen Liu, from Experimental Research Center, China&#160;Academy of Chinese Medical Sciences, for bioluminescent images.

Animal experiments were conducted in accordance with the Provision and General Recommendation of Chinese Experimental Animal Administration Legislation and were approved by the Institution of Animal Care and Use Committee of Capital Medical University (Ref no. AEEI-2014-052). Female Balb/c mice aged 6 to 8 weeks were used.
1. Preparation of the Cells and Animals
<ol>
	<li>One day before the operation, shave the hair around the fourth nipples to expose the operating area.
	<ol>
		<li>Use one hand to hold the mouse tightly at the back to make sure the mouse will not move freely, turn up the mouse to expose its belly to the operator, and then use another hand to shave the fur around the fourth nipples with an electronic shaver.</li>
		<li>Put a thin layer of hair removal cream around the nipples for 30 - 60 s, clean away the cream with distilled water, and then dry the mouse with soft paper.</li>
	</ol>
	</li>
	<li>On the day of the operation, collect murine breast cancer cells (4T1-luc2), count the cell number, and adjust the cell density to 2 x 105 cells/mL in phosphate-buffered saline (PBS) in sterile microcentrifuge tubes and keep them on ice. For each mouse, 1 x 104 cells/50 &#181;L in a 1-mL syringe is prepared.</li>
</ol>
2. Orthotopic Injection
<ol>
	<li>Anesthesia of mice 
	<ol>
		<li>Induce anesthesia. Put the mice in a closed transparent container and turn on 2% isoflurane gas to anesthetize the mice for about 5 min, till they all lie down and fall asleep.</li>
		<li>Maintain anesthesia. Transfer the mice from the container to individual anesthesia masks, put some vet ointment on their eyes to avoid dryness, keep the mice supinely with their nipples exposed to the operator, and let them continue to inhale isoflurane gas for a few minutes until the injection has been performed.</li>
		<li>Confirm the success of anesthesia by the lack of a toe pinch withdrawal reflex.</li>
		<li>Apply ophthalmic ointment to the eyes of the mice to prevent eye dryness. 
		Note: According to the container size and the number of individual anesthesia masks available, a group of mice can be anesthetized together and injected one by one under individual anesthesia.</li>
	</ol>
	</li>
	<li>Performance of the injection
	<ol>
		<li>Use one hand to tent up the skin around the fourth nipple with tweezers, to set up an uplifted &#8220;tunnel&#8221; for the syringe to follow. Use the other hand to hold the syringe with the needle turned upwards to enter the skin subcutaneously at 5 - 10 mm from nipple; then, follow the &#8220;tunnel&#8221; until the needle tip comes close to the nipple, carefully move the needle tip up into the mammary fat pad till the needle eye comes under the nipple tip, discard the tweezers, and inject the cells slowly.</li>
		<li>Turn around the needle a little and retract the syringe slowly; use a cotton swab to press the needle wound for 10 - 20 s to make sure there is no fluid seepage.</li>
		<li>Observe the fourth nipple to confirm a successful injection: one white transparent flat sphere with the nipple as the center can be observed (Figure 1).</li>
	</ol>
	</li>
	<li>Keep the mice anesthetized for another minute to avoid letting the mice recover and move too quickly, which will cause the injection wound to open and the tumor cells to leak out.</li>
</ol>
3. Analysis of the Tumor Growth and Metastasis
Note: Primary tumors are identified as transparent or blister-like bumps after injections, and as spherical or ellipsoid solid lumps with the nipple a few days later. Tumor growth is evaluated by tumor volume and tumor bioluminescence.
<ol>
	<li>To establish the tumor volume, hold the mouse with one hand. Expose its belly to the operator and use the other hand to caliper the tumor length (L) and width (W). Calculate the tumor volume (V) as follows. 
	V = (L x W2)/2</li>
	<li>To capture the tumor bioluminescence, inject 150 mg/kg D-luciferin at the back of the mouse&#8217;s neck, 10 min before imaging. Anesthetize the mice with 2% isoflurane gas for 5 min and transfer them to individual anesthesia masks to let them inhale isoflurane gas for 5 min longer. Following the manufacturer&#8217;s instructions, capture bioluminescence images of the primary tumor and metastasis, with auto exposure time, medium binning, and f/stop at 1.</li>
</ol>
4. Harvesting of the Primary Tumor and Metastasis Organs for Analysis
Note: When achieving the purpose of this experiment, or reaching the humane endpoint, the primary tumor can be harvested for further study. In this study, tumors are removed at 4 to 5 weeks after the injection, when the tumor volume reaches about 800 mm3. The lungs are removed at week 8.
<ol>
	<li>Harvesting of the primary tumor
	<ol>
		<li>Perform the surgery in a sterile hood to maintain a sterile atmosphere. Anesthetize the animals by isoflurane. Confirm the success of anesthesia by the lack of toe pinch withdrawal reflexes. Apply ophthalmic ointment to the eyes of the mice to prevent them from drying out.</li>
		<li>Dissociate the tumor from the skin with scissors, dropwise painkiller (1 mg/mL tramadol, 1 drop of about 50 &#181;L per mouse) to&#160;relieve postoperative pain.</li>
		<li>Stitch the skin with a surgical suture, cut off excess suture, drape off the incision.</li>
		<li>Cut the primary tumor in half with a scalpel, put 1/2 of the tumor into 4% paraformaldehyde for further hematoxylin and eosin (H&#38;E) staining and immunohistochemistry, and immediately freeze the other 1/2 with liquid nitrogen for western blotting or an RNA isolation study later.</li>
		<li>After suturing, warm the mice with a lamp till they recover, keep the mice in single cages until fully recovered. Continuously&#160;administer 10 mg/kg tramadol with an oral gavage for 3 d after the surgery.</li>
	</ol>
	</li>
	<li>Harvesting of metastasis organs
	<ol>
		<li>Euthanize the mice with an overdose of pentobarbital, open their chests with scissors, gently take out the lungs, and wash them with PBS. Lung metastasis foci can be identified as white transparent bumps which are morphologically different and distinguishable from normal pink lung tissue.</li>
		<li>Put the lungs into 4% paraformaldehyde to verify the metastasis by H&#38;E staining.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The cells used in this study are 4T1-luc2, murine triple-negative breast cancer cells with luciferase labeling, which are a useful tool for investing antitumor and antimetastatic effects of drugs due to their highly invasive nature2,19. Luciferases, stable to the next cell generation, are used to indicate the living tumor cells, both at the mammary gland and at other distant organs20. In some cases, hypoxia and nutrient deficiency within the tumor result in a decrease in the bioluminescent signal, and the volume of the primary tumor is not inconsistent with the bioluminescent signal at a later stage of tumor progression. The technique of the cellular orthotopic injection (COI) of cell suspensions can also be performed with other breast cancer cell lines, such as human cells MDA-MB-231 (unpublished data). In this study, with the mammary gland as its environment, it is a proper animal model to study tumor microenvironment, crossing talking between tumor cells and stromal cells. During the tumor progress, in some mice, the nipple can ulcerate, scab, grow red granulation tissue, flatten, or even disappear, similar to what happens in human nipples during breast cancer. Mice may experience discomfort with ulcerations; humane care should be taken. More nipple change has been found in mice using this COI technique than with the micro-invasive surgery technique. The surgical orthotopic implantation (SOI) of histologically intact human tumor tissue, compared with the cellular orthotopic injection (COI) of cell suspensions, shows higher malignant cancers in the bladder, lung, stomach, kidney, and colon21 that mimic what is seen in patients22.
With a high incidence of metastasis, especially lung metastasis, the murine breast cancer model established in this study is a good syngeneic cancer model to explore the spontaneous migration and invasion, compared with an artificial metastasis model, such as a tail vein injection. The tumor cell activities and characters also play important roles in metastasis. Other cell lines, such as human breast cancer cell MCF-7, even implanted by an orthotopic injection at the mammary fat pad of immune-deficient Balb/c mice, have a lower metastatic ability. With the modification of cells or mice, such as the over-expression or down-expression of a certain gene, this technology can be applied in more research fields.
The crucial operation of this injection is to carefully and slowly move up the needle tip, from subcutaneous into the mammary fat pad. When the syringe tip comes close to the nipple, the operator will feel insistence from the tissue. Since the mouse gland is very small, only a little more pressure is needed to let the syringe tip pass through the insistence. Too much pressure will result in the tip passing through the gland and breaking the skin, which results in a failure of the injection. Logarithmic-phase cells, a single-cell suspension, the standard operation protocol (SOP), and skilled operators are all required to reduce the variation among mice. Tumor cell behavior is important for the formation and the repeatable of tumor models. Harvest logarithmic-phase cells when they grow to about 80% - 90% confluence; too low or too high cell densities will interfere with the tumor model and result in variations between experiments. The single-cell suspension is also important since cell clumps result in miscalculations of the cell number. Trypsin, an EDTA solution, or other appropriate methods are chosen to detach the cells according to the experiment aims and the targets the researcher is interested in. Dependent on the syringe scale and cell density, the injection volume could vary from 25 to 100 &#181;L. The amount of 1 x 104 to 1 x 106 cells is commonly used to irritate tumor growth according to different cell types. Trypan blue staining is used to distinguish viable cells from dead ones. More than 95% cell viability is regarded as a good situation to induce a rapid tumor growth and metastasis in vivo. These are also suitable for any other tumor models when preparing tumor cells. To correctly caliper the primary tumor size and get an accurate bioluminescent signal, the fur around the nipple should be removed again once it grows back. When the primary tumor grows to a large mass and its bioluminescent signal is too strong for researchers to observe other organ metastases (e.g., lung metastasis), cover it with black electrical tape before bioluminescence images are taken, or remove it by surgery. A painkiller can be used for post-surgical pain.
Successful inoculation can result in lung metastasis: with an occurrence rate of more than 90%, metastatic foci at non-injected breasts, bone, or lymph nodes are quite common, while celiac metastasis is rare. The secondary tumors are a translucent mass and can be distinguished from the surrounding normal tissues. An imprecise application of the injection can lead to experimental errors in tumor size and shape and metastasis locations. Any leakage from the flat sphere after the injection will reduce the tumor size, any leakage from the mammary gland into the &#34;tunnel&#34; will result in a long cylindrical tumor which will obstruct the determination of the tumor volume when we use a digital caliper to measure it. Subcutaneous injections around the mammary fat pad seldom lead to lung metastasis and are not suitable for distant metastasis-associated study. To make the inoculation stable and reduce the risk of leakage, Matrigel can be used when suspending tumor cells. However, Matrigel is not a single solvent, it is regarded as a kind of extracellular matrix (ECM), and some types of gel even contain factors which may interfere with the experiments. In the experiment aimed at tumor microenvironment, ECM, crossing talking among tumor cells or with stromal cells, it is better to avoid using Matrigel. For injected cells with the luciferase tag D-luciferin, the substrate of luciferase is injected to bind the living tumor cells in mice. In this study, the dynamics of bioluminescence in vivo suggested that at 10 minutes after D-luciferin injection, the bioluminescent signal reach the peak and can be stable for about 10 - 15 minutes (varying between different mice), whether with an intraperitoneal injection or a subcutaneous injection. Considering the tested drug is injected intraperitoneally, to avoid the interaction of the tested drug with D-luciferin and to reduce the peritoneal irritation, a subcutaneous injection is suggested.
In conclusion, the orthotopic injection of breast cancer cells outlined in this paper is a very useful and powerful tool in studying the pathology and progression of breast cancer. It is easy to handle and can be performed quickly. The primary tumor originates at the mammary gland with a high occurrence of metastasis, which maximumly mimics the pathophysiological process of human breast carcinoma.

Breast cancer is the leading cause of female mortality worldwide. With its progressively increasing incidence, breast cancer has become a serious challenge to public health1. Murine cancer models are good bridges between preclinical and clinical studies, and a good mimic murine disease model will increase the accuracy of research on disease and medicine.
Primary tumor growth starts the progress of malignant disease, while metastasis and complications are the main causes of death and poor life qualities in most cancer patients. Several murine models are used to mimic the pathology of human breast cancer2,3,4. Xenograft models are widely used for cancer study to understand the pathological characters and to screen drugs for safety and efficacy5,6,7. Genetically engineered mice (GEM) are generated to mimic human breast cancer by targeting certain oncogenes or tumor suppressor genes8. GEM have a relatively simple and uniformed background to understand the role of genes in cancerous progress; however, the artificial environment and background are limited to investigate the metastasis pathology and related therapies9. Human cancer cells, although with human pathological features, can only be implanted in immune-deficient mice, and the insufficiency of the tumor-host immune interaction may lead to biased results10.
Where the solid tumor initiates has a direct influence on the biological and pathological characters of the disease11,12,13. Since cancerous progress is the complicated outcome of interactions among tumor cells, stromal cells, immunology cells, inflammatory cells, growth factors, and proteases, primary tumors implanted in situ will provide better insight and mimic the cancerous process more accurately than tumors induced by chemical agents or a subcutaneous injection of tumor cells. Chemical agents used to induce tumors may be harmful to researchers and environment and are even forbidden in some countries. Because of the absence of a mammary fat pad environment, the pathological progress of a subcutaneous injection may differ with that in real breast cancer patients, whose cancer originates and irritates from the mammary fat pad. The disadvantages of subcutaneous injections encourage the use of orthotopic models to study tumor growth. In earlier research, highly metastatic MDA-MB-231 tumors, developed after seven orthotopic transplantations, indicated the importance of the injection location14. Recently, the orthotopic implantation of breast cancer cells into the mammary fat pad with surgery was reported15,16. With the mammary pad environment, the tumor growth and the migration into distant organs cover the entire process of breast cancer at pathologically relevant sites, which makes this model a miniature of the progress of human diseases. However, after the surgery, the skin automatically attempts to heal itself, which may bring the potential risk of interfering with the normal breast cancer origination and bias the results.
We have compared some breast cancer models and established a minimally invasive orthotopic model to investigate the potential effect of drugs on breast cancer progression17,18. In this study, a video protocol on how to orthotopically inject breast cancer cells into the mammary fat pad in a simple, less invasive way is presented. This orthotopic injection method without surgery is advantageous in many ways. First, the operation is simple and rapid, about 1 minute per mouse. Second, with primary tumor foci starting at the right pathological sites, it covers the whole tumorigenic process of breast cancer progression from the tumor growth to other organs metastasis, which provides a good experimental animal model for studying the interaction of tumor cells and tumor microenvironment. Besides, it can be a valuable model to estimate the treatment effects at all stages of breast cancer. The goal of this method is to provide an animal model to maximumly mimic human breast cancer progression.

<table><tbody><tr><td>anesthesia machine</td><td>Midmark Corporation, Dayton, OH, USA</td><td>Matrx VMS</td><td>anesthesia</td></tr><tr><td>In-Vivo Imaging System</td><td>PerkinElmer</td><td>IVIS Spectrum</td><td>used for bioluminescence detecion</td></tr><tr><td>&amp;nbsp;isoflurane</td><td>Hebei yipin chemical reagents&amp;nbsp; company</td><td>O21400</td><td>anesthesia</td></tr><tr><td>1 ml syringe</td><td>Becton,Dickinson and&amp;nbsp; Company</td><td>A257</td><td>cell injection</td></tr><tr><td>digital caliper</td><td>Shang Hai Shen Han Measuring Tools Co., Ltd.</td><td>S-H</td><td>volume measurement</td></tr><tr><td>tramadol</td><td>Mundipharma company</td><td>-</td><td>pain killer</td></tr><tr><td>D-luciferin</td><td>Gold Biotechnology Inc.</td><td>LUCK-1G</td><td>used for bioluminescence detecion</td></tr><tr><td>The primary antibody against cluster of differentiation (CD) 31</td><td>Abcam</td><td>&amp;nbsp;ab28364</td><td>used for MVD detection</td></tr><tr><td>hematoxylin and eosin staining kit</td><td>&amp;nbsp;Beijing Zhong Shan Jinqiao Biotechnology Co., Ltd.</td><td>ZLI-9615</td><td>histology&amp;nbsp;</td></tr><tr><td>hair removal cream</td><td>Veet</td><td>-</td><td>hair removal cream</td></tr><tr><td>Carbomer Eye Gel</td><td>Dr.GerhardMannChem-Pharm.FabrikGmbH</td><td>-</td><td>ophthalmic ointment</td></tr><tr><td>sewing needle</td><td>Shanghai Pudong Jinhuan Medical Products Co., Ltd.&amp;nbsp;</td><td>17U0302J</td><td>suture</td></tr></tbody></table>

orthotopic injection of breast cancer cells into the mice mammary fat pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

After a successful injection, one white transparent flat sphere with the nipple as the out-surface round center can be observed (Figure 1). Primary tumor growth can be measured by tumor volume and living tumor cell bioluminescence (Figure 2). Both the tumor volume and the total flux increased during the experiment before resection. At an early stage, metastasis cannot be found because no secondary tumor has happened or the strong bioluminescent signals of the primary tumor obstruct the detection of small metastasis foci with weak signals. After resection, the bioluminescent signal from the metastatic foci of distant organs can be detected and analyzed. The morphology of both the primary breast tumor and the lung sections were studied with H&#38;E staining, while angiogenesis was investigated by staining the tumor with vessel marker CD31 for microvessel density (MVD) (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58604/58604fig1v2.jpg" /> 
Figure 1: Pictures before and after the orthotopic injection at the mammary fat pad of female Balb/c mice. The operation was performed, and the pictures were taken while the mice were under isoflurane anesthesia. A, before injection,&#160;B, after injection.&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58604/58604fig2.jpg" /> 
Figure 2: Volume and total flux of the primary tumor were measured from 7 days after the implantation. The cell suspension implantation generated an increased primary tumor both in volume, which was visualized and determined using calipers, and in total flux, which was measured by the Spectrum system. The figure shows bioluminescent images of primary tumors and metastasis observed at various time points after day 7 of implantation. p/s = photons/second. This figure has been modified from Zhang et al.17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58604/58604fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58604/58604fig3.jpg" /> 
Figure 3: H&#38;E staining and immunohistochemical staining analysis of primary tumors and lung metastasis. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and stained. (A and B) Sections were stained with hematoxylin and eosin (H&#38;E). (C and D) Sections were stained with antiCD31 antibody. Brownish staining indicated CD31+ vessels. Panels A and C present primary tumor tissue, panels B and D present lung metastasis. CD = cluster of differentiation. The magnification used = 200X. The scale bar = 50 &#181;m. This figure has been modified from Zhang et al.17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58604/58604fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad at JoVE.com

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

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

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Nanyang Technological University

Research

JoVE Journal

Cancer Research

50.5K Views.  Capital Medical University. Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

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

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

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.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

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.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Medicine

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

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

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

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.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

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.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 &#181;L suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&amp;E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

Orthotopic Injection into the Mammary Fat Pad:  Establishing Breast Cancer in Mice

Source: Zhang, G. L., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/58604/orthotopic-injection-breast-cancer-cells-into-mice-mammary-fat?access=v2wuhgcg">Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad.</a>&#160;J. Vis. Exp. (2019).
In this article we discuss how to establish a breast cancer model in female mice by injecting breast cancer cells into the mammary fat pad. The example protocol demonstrates the orthotopic injection.

Wstrzyknięcie ortotopowe do poduszeczki tłuszczowej sutka: ustalenie raka piersi u myszy

Source: Zhang, G. L., et al. Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad.&#160;J. Vis. Exp. (2019).
In this article we ...

JoVE Encyclopedia of Experiments

Breast Cancer

Procedures and techniques

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

Source: Katsuta, E., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57849/generating-murine-orthotopic-metastatic-breast-cancer-model">Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy.</a>&#160;J. Vis. Exp.&#160;(2018).
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 of Breast Cancer Cells into the Mice Mammary Fat Pad

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Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

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

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

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Wstrzyknięcie ortotopowe: wszczepianie komórek rakowych specyficznych tkankowo dorosłej myszy