Name: generation of organ-conditioned media and applications for studying organ-specific influences on breast cancer metastatic behavior
Uploaded: 2016-06-13T15:00:00
Description: Watch this Scientific Journal Video about Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior at JoVE.com

This work was supported by grants from the Canadian Breast Cancer Foundation-Ontario Region, the Canada Foundation for Innovation (No. 13199), and donor support from John and Donna Bristol through the London Health Sciences Foundation (to A.L.A.). Studentship and fellowship support were provided by the Ontario Graduate Scholarship program (Province of Ontario, to G.M.P. and J.E.C.), the Canada Graduate Scholarship-Master's program (to M.M.P), the Canadian Institutes of Health Research (CIHR)-Strategic Training Program (to M.M.P., G.M.P and J.E.C.) and the Pamela Greenaway-Kohlmeier Translational Breast Cancer Research Unit at the London Regional Cancer Program (to M.M.P., G.M.P., J.E.C. and Y.X.). A.L.A. is supported by a CIHR New Investigator Award and an Early Researcher Award from the Ontario Ministry of Research and Innovation.

All animal studies were conducted in accordance with the recommendations of the Canadian Council on Animal Care, under protocols approved by the Western University Animal Use Subcommittee.
1. Organ Isolation (Lung, Brain, Bone, Lymph Node)
<ol>
	<li>Prepare four sterile 50 ml conical tubes (one for each organ to be isolated) containing approximately 30 ml of sterile phosphate-buffered saline (PBS). Pre-weigh each tube of PBS using an electronic balance.</li>
	<li>Euthanize 6-12 week old mous...

<ol>
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Metastasis is a complex process by which a series of cellular events are ultimately responsible for tissue invasion and distant tumor establishment4,30,31. The ex vivo model system presented here can be utilized to study two important aspects of metastatic progression: cancer cell homing or migration to a specific organ (&#34;getting there&#34;) and growth in that organ (&#34;growing there&#34;). Many studies have previously focused on identifying key molecular characteristics associated with the canc...

Breast cancer is the most frequently diagnosed cancer in women and the second leading cause of cancer-related deaths1. Breast cancer&#39;s high mortality rate is mainly due to the failure of conventional therapy to mitigate and eliminate metastatic disease; approximately 90% of cancer-related deaths are due to metastasis2. Understanding the underlying molecular mechanisms of the metastatic cascade is paramount to the development of therapeutics effective in both early and late-stage breast cancer.
Past research has helped elucidate the multistep nature of breast cancer metastasis and it is hypothesized that the outcome of both cancer progression and metastasis is largely dependent upon interactions between cancer cells and the host environment3. Clinical observations indicate that many cancers display organ tropism, i.e., the tendency to preferentially metastasize to specific organs.In the case of breast cancer, a patient&#39;s disease typically spreads or metastasizes to 5 main sites, including the bone, lungs, lymph node, liver, and brain4-6. Many theories have been developed to explain this process, but only a few have withstood the test of time. Ewing&#39;s theory of metastasis, proposed in the 1920s, hypothesized thatthe distribution of metastasis was strictly due to mechanical factors; whereby tumor cells are carried throughout the body by normal defined physiological blood flow patterns and simply arrest in the first capillary bed they encounter7. In contrast, Stephen Paget&#39;s 1889 &#34;seed and soil&#34; hypothesis suggested that additional molecular interactions were responsible for survival and growth of metastases, whereby cancer cells (&#34;seeds&#34;) can only establish themselves and proliferatein organ microenvironments that produce appropriate molecular factors (&#34;soil&#34;)8. Almost a century later, Leonard Weiss undertook a meta-analysis of previously published autopsy data and confirmed Ewing&#39;s prediction that many metastatic tumors detected at the time of autopsy were found in the anticipated proportions that would be expected if metastatic organ tropism was determined by blood flow patterns alone. However, in manyinstances there were fewer or more metastases formed at certain sites then would be expected by Ewing&#39;s proposed mechanical factors9. These accounts and theories suggest that specific organ microenvironments play a critical role in the dissemination patterns and subsequent growth and survival of many cancers, including breast cancer.
Past research efforts have mainly focused on tumor-cell derived factors and their contribution to the organ tropism observed in breast cancer metastasis10-12, however little research has explored factors derived from the organ microenvironment that may provide a favorable niche for the establishment of breast cancer metastases. This is largely attributable to the technical challenges of studying components of the organ microenvironment in vitro. 
The current article describes a comprehensive ex vivo model system for studying the influence of soluble components of the lymph node, bone, lung, and brain on the metastatic behavior of human breast cancer cells. Previous studies have validated this model system by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential13. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and to evaluate the role of organ-derived soluble factors in driving the process of cancer metastasis.

<table border="0" cellpadding="0" cellspacing="0" width="1195">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">50 ml conical tubes</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td style="width:223px;">339652</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">1X Phosphate-buffered saline</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:223px;">10010-023</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Nude mice</td>
			<td style="width:159px;">Harlan Laboratories</td>
			<td style="width:223px;">Hsd:Athymic Nude-Foxn1nu</td>
			<td>Use at 6-12 weeks of age</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:339px;">Polystyrene foam pad</td>
			<td style="width:159px;">N/A</td>
			<td style="width:223px;">N/A</td>
			<td style="width:475px;">The discarded lid (~4 x 8 inches or larger) of a polystyrene foam shipping container can be used for this purpose. Sterilize by wiping with ethanol.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:339px;">Forceps</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:223px;">11050-10</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:339px;">Scissors</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:223px;">14058-11</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:339px;">Gauze pads</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:223px;">22-246069</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:339px;">60 mm2 glass petri dishes</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">CLS7016560</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:339px;">Scalpel blades</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:223px;">S95937A</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM:F12</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">21331-020</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">1 x Mito+ Serum Extender</td>
			<td style="width:159px;">BD Biosciences</td>
			<td style="width:223px;">355006</td>
			<td style="width:475px;">Referred to as &#34;concentrated mitogen supplement&#34; in the manuscript. Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">15140-122</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">Rosewell Park Memorial Institute 1640 (RPMI 1640)</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">11875-093</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Fetal Bovine Serum</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">F1051-500ML</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">Trypsin/EDTA solution</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:223px;">R-001-100</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">6-well tissue culture plates</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td>140675</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">0.22 &#956;m syringe filters</td>
			<td>Sigma-Aldrich</td>
			<td>Z359904</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">T75 tissue culture flasks</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td style="width:223px;">178905</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Transwells</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">CLS3464</td>
			<td>Keep sterile, use for migration assays</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse Sca-1</td>
			<td>R&#38;D Systems</td>
			<td>FAB1226P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD105</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB1320P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD29</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB2405P-025</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD73</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB4488P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD44</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">MAB6127-SP</td>
			<td>use at 0.25 &#181;g/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD45</td>
			<td>eBioscience</td>
			<td style="width:223px;">11-0451-81</td>
			<td>use at 5 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse gp38</td>
			<td>eBioscience</td>
			<td style="width:223px;">12-5381-80</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">M6250&#160;</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Protein arrays</td>
			<td style="width:159px;">RayBiotech Inc.</td>
			<td style="width:223px;">AAM-BLM-1-2</td>
			<td>Use 1 array per media condition (including negative control), in triplicate</td>
		</tr>
	</tbody>
</table>

generation of organ-conditioned media and applications for studying organ-specific influences on breast cancer metastatic behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

Generation of Organ-conditioned Media
An overview diagram/schematic of the process of organ isolation and generation of conditioned media is presented in Figure 1, with representative photographic images of the procedure shown in Figure 2. It should be noted that when this protocol was first under development, liver was included in our analysis because it is a common site of bre...

Watch this Scientific Journal Video about Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior at JoVE.com

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

The overall goal of this procedure is to generate conditioned media using mouse lymph nodes, bones, lungs, and brains to identify and study organ derived soluble factors and their effects on the metastatic behavior of cancer cells. This method can help answer key questions in the metastasis field, such as, how do organ specific factors influence cancer cell growth, migration, and gene expression? This technique provides a straightforward ex vivo platform representative of organ specific in vivo soluble factors that influence the phenotype and behavior of cancer cells.To isolate the lymph nodes, begin by placing a six to twelve week old mouse on a polystyrene foam pad in a sterile tissue culture hood, and secure the limbs in an outstretched position. Next, use forceps and scissors to make a midline incision in the abdominal skin from the genitalia to the mouth. Then, gently pull the skin from the abdominal muscles and pin it to the dissecting pad.Using scissors, carefully remove the lymph nodes from the skin, fat, and vessels, placing the nodes in 30 milliliters of ice cold PBS as they are harvested. When all of the lymph nodes have been collected, use the forceps and scissors to carefully cut through the exposed abdominal wall in an upward motion through the sternum, exposing the thoracic cavity. Cut the diaphragm.Lift the lungs from underneath, and cut the underlying tissue toward the trachea. Then, transfer the lungs into a new container of ice cold PBS. The heart can be removed from the lungs during this step or prior to organ weighing.To harvest the leg bones, place the mouse in the prone position, and cut the skin across the lower back from flank to flank. Next, use a sterile piece of gauze to grasp the torso and the skin over the legs and feet. Using the same piece of gauze, hold the lower leg while carefully breaking the ankle joint of one foot, and peel the skin over the joint proximally towards the knee.Then, using scissors, remove the tibia from the knee joint and place the leg bone in ice cold PBS. After harvesting the second tibia, hold the femur in place with forceps and use scissors to cut away the surrounding muscle tissue. Then, place the leg bone in ice cold PBS.To harvest the brain, use a new piece of sterile gauze to grasp the head, and gently remove the skin with scissors and forceps to expose the skull. Next, use scissors to carefully the occipital bone from the top center of the skull in a straight and downward line to expose the posterior brain. Then, scooping underneath the tissue toward the anterior of the skull, transfer the whole brain into ice cold PBS.To generate the lung conditioned medium, invert the tubes of lung tissue three times to remove any residual blood from the organs. Then, replace the wash with fresh cold PBS and repeat until the saline is blood free. Next, transfer the lungs into one 60 square millimeter glass Petri dish per mouse, and use two sterile scalpel blades to mince the tissues with repeated back and forth slicing.When the tissue fragments are approximately one millimeter cubed in size, re-suspend the pieces in the appropriate volume of DMEM:F12 medium supplemented with antibiotics, and incubate the tissues in one well of a six well plate per mouse at 37 degrees Celsius and five percent CO2. After 24 hours, transfer the entire contents of each well into individual 50 milliliter conical tubes, and dilute the conditioned media with three equivalent volumes of fresh DMEM:F12 medium per tube. Centrifuge the tubes to remove any large pieces of tissue debris.Then, pool the conditioned media through a 0.22 micron syringe strainer into a single 50 milliliter conical tube. To generate bone marrow conditioned medium, transfer the bones under a sterile tissue culture hood, and trim the excess tissue from the bone. Use scissors to remove the epiphyses.Then, use a 27 and a half gauged needle to flush one milliliter of PBS through the medullary cavity of each bone into a tube of fresh PBS. When all of the bone marrow stromal cells have been collected, wash the cells three times in PBS. After the last wash, re-suspend the pellet in 20 millilters of bone stromal cell growth medium supplemented with 10%fetal bovine serum, and seed 10 milliliters of cells per T75 flask.When the cells have reached approximately 70%confluency, wash the cultures two times with three milliliters of PBS. Then, detach the cells with three milliliters of 0.025%trypsin/EDTA solution, taking care that the trypsin covers the entire flask surface. After two to three minutes, arrest the enzymatic reaction with three milliliters of bone stromal cell growth medium, and pool the cells in a 50 milliliter tube.Spin down the cells. Then, re-suspend the pellet in 10 milliliters of fresh medium. Now, pass into the cultures at a one to five ratio into three new T75 flasks until they reach 70%confluency.Then, wash the cells three times with PBS as just demonstrated, and feed the cultures with serum free bone stromal cell collection medium supplemented with mitogen and antibiotics for 72 hours. After the third day of culture, pool the media through a 0.22 micron strainer. The stromal cells should be adherent in culture with an elongated and fibroblastic morphology observed in the lymph node stromal cell cultures, and a smaller and more rounded morphology exhibited by the bone marrow stromal cell cultures.Flow cytometric analysis confirms that the lymph node stromal cells are largely CD45 negative, and gp38 positive. Bone marrow stromal cells should be positive for CD-44 and CD-29, weakly positive for CD-105 and Sca-1, and negative for CD-73. Human breast cancer cells with varying genetic backgrounds exhibit differential migration and growth patterns towards specific organ conditions.For example, in this representative experiment, the bone-seeking 231-BoM variant preferentially migrates to the bone marrow conditioned medium over the brain and lung conditioned media. Similarly, the lung-seeking 231-LM2 variant preferentially migrates to lung conditioned medium over the bone marrow and brain conditioned media. Further, RT qPCR analysis demonstrates that the exposure of parental MDA-MB-231 breast cancer cells to bone or lung conditioned medium induces an up regulation of the genes associated with bone or lung specific metastases of the breast cancer cells in vivo.Once mastered, the tissue harvesting and conditioned medium set ups can be completed in two to three hours if they are performed properly. The sub culturing steps on subsequent days take 15 to 30 minutes per day. While attempting these procedures, it is important to remember to keep everything sterile.Following the conditioned medium generation, other methods like protein arrays, cell growth and migration assays, tumorsphere formation, and RT-PCR can be performed to answer additional questions about the effect of organ specific factors on cancer cell behavior. After its development, this technique paved the way for researchers in the field of breast cancer research to explore organ specific metastatic behavior in an ex vivo model system. After watching this video, you should have a good understanding of how to generate conditioned media using the bones, lungs, and brains of mice.Don't forget that working with sharp dissection tools can be hazardous, and that precautions, such as using precise and careful dissection technique, should always be taken while performing this procedure.

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Research

JoVE Journal

Medicine

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

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 ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

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 ...

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

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 ...