Name: generating a murine orthotopic metastatic breast cancer model and performing murine radical mastectomy
Uploaded: 2018-11-29T13:00:00
Description: Watch this Scientific Journal Video about Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy at JoVE.com

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).
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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

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...

Watch this Scientific Journal Video about Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy at JoVE.com

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

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 ...

This method can help answer key questions in the breast cancer field about how to mimic human breast cancer progression and treatment in a mouse model. The main advantage of this mouse breast cancer model is that it has a high tumorigenesis rate after orthotopic implantation and a low local recurrence rate after mastectomy. Begin by confirming a lack of response to toe pinch in an anesthetized female mouse and taping the animal's limbs to a surgical board.Using sterile cotton swabs sterilize the surgical area with chlorhexidine, iodine and 75%ethanol. And make a five-millimeter skin incision in the middle of the anterior chest wall. Next lift the right side of the skin adjacent to the incision and use sterile microdissection scissors to detach the skin from the chest wall.Invert the skin to expose the right number two mammary fat pad and use a one-milliliter insulin syringe equipped with a 28.5-gauge needle to carefully inject 20 microliters of cancer cell suspension through the wound into the fat pad. Hold the needle within the fat pad for five seconds to allow the gelatinous cancer cell protein mixture to solidify before removing the needle, and closing the incision with sterile 5-0 nonabsorbable sutures. Then place the animal in a clean cage with monitoring until full recumbency.To perform the mastectomy eight days after cancer inoculation prepare the mouse for surgery as just demonstrated, and make a five-millimeter skin incision two millimeters to the left of the surgical scar from the cancer cell inoculation. Extend the incision toward the root of the forelimb to remove the tumor, and the skin, including the surgical scar, and the lesion in contact with the tumor, and the axillary lymph node basin in which typically no visible lymph nodes are present at the time of the mastectomy. Then close the skin defects with sterile 5-0 nonabsorbable sutures in the shape of a Y.And return the animal to its cage with monitoring until full incumbency.For lung metastasis quantification 21 days after inoculation inject 150 milligrams per kilogram of D-Luciferin IP 15 minutes before euthanasia. And use curved Mayo scissors to open the skin and peritoneum at the mid-abdomen. Press down the liver until the diaphragm can be visualized, and cut the diaphragm and the bilateral ribs from caudad to cephalad.Flip the anterior thorax wall to expose the lungs and lift the lungs to identify the thoracic esophagus, which looks like a cord connecting the lungs to the spine. Cut the esophagus with microdissection scissors and use forceps to pull the bilateral lungs and heart toward the caudad. Then cut the trachea and major vessels to the lung apex at the cephalad and place the lungs into a 10-centimeter Petri dish for bioluminescent imaging.Use the scissors to remove the heart from the lung tissue. Beginning at about eight days after mammary fat pad cancer cell inoculation the bioluminescence in the lungs is deeper and smaller than in the primary lesion reflecting the primary tumor burden in live mice. Measurement of the tumor size with calipers allows estimation of the tumor volume in situ.This mastectomy model also allows serial metastatic tumor burden monitoring without having to euthanize the animals. The tumor burden of the lung metastases can also be quantified by ex vivo imaging, but the limitation is that the mice must be euthanized. Mouse survival after surgical treatment can also be monitored as a translatable clinical endpoint.After its development this technique paved the way for researchers in the field of breast cancer to explore not only new drug development, but also breast cancer research in general.

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Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...