eoe sub articles

EoE Sub Articles

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Milky Spot Colonization Ex Vivo
<ol>
	<li>Establishment of basic ex vivo omental organ culture conditions
	<ol>
		<li>Place each omentum into a single culture plate insert and place within one well of a 12-well tissue culture plate containing 500 &#181;l of DME/F12 media containing 20% FCS. Maintain organ cultures at 37 &#176;C in a 5% CO2 environment for 24 to 48 hr and/or additional time points. Use three independent omenta (triplicate samples) for each condition (e.g., media type/time point).</li>
		<li>To confirm the integrity of tissues at the experimental endpoints, fix and process samples as described in Step 1.1.3. Counting healthy versus necrotic adipocytes on H&#38;E sections can assess tissue integrity. A minimum of ~120 cells from five biological samples is required to formulate a percentage of live tissue present.</li>
		<li>To measure tissue function, place omenta (n=3) in separate wells of a 24-well plate containing 500 &#181;l of DME/F12 media with 20% FCS. Allow omenta to condition the media at 37 &#176;C in a 5% CO2 environment for 24 hr and then remove 250 &#181;l for use in an IL-6 ELISA assay.</li>
	</ol>
	</li>
	<li>Ex vivo co-culture of the mouse omentum with SKOV3ip.1-GFP cells
	<ol>
		<li>Grow and prepare fluorescently tagged cells by pipetting 1x106 &#160;thawed cells in 10 ml of media in a T-75 flask. Allow the cells to grow at 37 &#176;C in a 5% CO2 environment until they reach 70% confluence. Trypsinize and resuspend at a concentration of 2 x 106&#160;cells per ml.</li>
		<li>Apply ~6 &#181;l of the tissue adhesive to the membrane of the culture insert and allow it to air dry. Wash the membrane twice with sterile water to remove any excessive adhesive. Air dry the membranes under a laminar hood.</li>
		<li>Carefully excise the omenta and attach it to the adhesive-coated membrane using sterile forceps. Allow the tissue to adhere to the membrane for 1 min prior to adding media. Add 500 &#181;l of the cell suspension on top of each omentum in each culture insert. Fill the area around the transwell chamber with 2.5 ml of DME/F-12 .</li>
		<li>Incubate omenta with cell suspension for 6 hr at 37 &#176;C in a 5% CO2 environment. Carefully remove and wash the omenta with ~10 ml of PBS. Visualize fluorescent cancer cell foci using an appropriate fluorescent imaging system.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>Dissection Tools</td><td>Fine Science Tools</td><td>NA</td><td></td></tr><tr><td>Giemsa</td><td>Fluka/Sigma Aldrich</td><td>48900</td><td></td></tr><tr><td>5% Formalin</td><td>Sigma</td><td>HT501320</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td>Without calcium and Magnesium</td></tr><tr><td>Collagenase</td><td>Worthington</td><td>LS004196</td><td></td></tr><tr><td>Millicell culture plate insert</td><td>Millipore</td><td>PICM01250</td><td></td></tr><tr><td>Cell-Tak</td><td>Corning</td><td>354240</td><td></td></tr></tbody></table>

modeling ovarian cancer cell colonization of omentum: an ex vivo method to establish milky spot colonization of fluorescently labeled ovarian cancer cells in murine omentum explants

Source: Krishnan, V. et al. <a href="https://www.jove.com/t/52721">In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.</a> J. Vis. Exp. (2015)
In this video, we establish an ex vivo omental organ culture overlaid with GFP-expressing ovarian cancer cells to study ovarian cancer colonization within the omental explant. This protocol analyzes immune cell structures within the omentum known as milky spots that promote metastases.

Modeling Ovarian Cancer Cell Colonization of Omentum: An Ex Vivo Method to Establish Milky Spot Colonization of Fluorescently Labeled Ovarian Cancer Cells in Murine Omentum Explants

Source: Krishnan, V. et al. In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose. ...

The omentum, an adipose-rich tissue within the peritoneal cavity, consists of fat depots that provide a suitable microenvironment for metastatic cancer cells to colonize and form secondary tumors. To study this colonization ex vivo, begin by positioning a permeable membranous cell culture insert inside a culture plate.
Apply a tissue adhesive over the inner region of the insert membrane. Place a mouse omental explant on the adhesive to immobilize the tissue. Gently seed a suspension of fluorescently-labeled ovarian cancer cells on top of the omentum.
Next, fill the sides of the well with a suitable medium to supplement cell growth and incubate for the desired time. The omental tissue contains specialized structures called milky spots, which are clusters of immune cells packed around networks of blood vessels. These immune cells secrete certain chemical factors that attract cancer cells and promote their implantation. The colonized cancer cells proliferate, simulating metastatic conditions.
Finally, discard the spent media. Observe the culture using a fluorescent microscope to assess fluorescent cancer cell colonies within the omental tissue.

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Research

JoVE Encyclopedia of Experiments

Cancer Research

Gynecologic Cancer

Assays & techniques

1.0K Views. Source: Krishnan, V. et al. In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose. J. Vis. Exp. (2015) In this video, we establish an ex vivo omental organ culture overlaid with GFP-expressing ovarian cancer cells to study ovarian cancer colonization within the omental explant. This protocol analyzes immune cell structures within the omentum known as milky spots that promote metastases. 

Video: Modeling Ovarian Cancer Cell Colonization of Omentum: An Ex Vivo Method to Establish Milky Spot Colonization of Fluorescently Labeled Ovarian Cancer Cells in Murine Omentum Explants - Concept

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

JoVE Journal

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

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 ...

Modeling Ovarian Cancer Cell Colonization of Omentum: An Ex Vivo Method to Establish Milky Spot Colonization of Fluorescently Labeled Ovarian Cancer Cells in Murine Omentum Explants

Transcript

Modeling Ovarian Cancer Cell Colonization of Omentum: An Ex Vivo Method to Establish Milky Spot Colonization of Fluorescently Labeled Ovarian Cancer Cells in Murine Omentum Explants

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

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