Name: Author Spotlight: Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction
Uploaded: 2024-01-26T14:15:00
Description: Watch this Scientific Journal Video about Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction at JoVE.com

This study is funded in part by The Janet Burros Memorial Foundation. We acknowledge the patients and the Karmanos Cancer Institute Gynecologic Oncology Department for the collection of omentum samples. We also acknowledge the Biobank and Correlative Sciences Core at Karmanos Cancer Institute for coordination of patient recruitment and preparation of pathology slides. The Biobank and Correlative Sciences Core is supported in part by NIH Center grant P30 CA22453 to the Karmanos Cancer Institute at Wayne State University.

The following research protocol was reviewed and approved by the Wayne State University Institutional Review Board (IRB). Informed consent was obtained from all patients prior to surgery. Figure 1 illustrates the three general steps in this protocol.
1. Preparation of human omentum tissue
<ol>
	<li>Prepare omentum culture media (DMEM/F12 + 10% fetal bovine serum + 1% penicillin-streptomycin) and store at 4 &#176;C. Aliquot 30-40 mL of this media...

Establishing a 3D Ex Vivo Model for Ovarian Cancer-Omentum Interaction

<ol>
	<li>Siegel, R. L., Miller, K. D., Wagle, N. S., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J Clin. 73 (1), 17-48 (2023).</li><li>Berek, J. S., Renz, M., Kehoe, S., Kumar, L., Friedlander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+of+the+ovary,+fallopian+tube,+and+peritoneum:+2021+update.">Cancer of the ovary, fallopian tube, and peritoneum: 2021 update.</a> Int J Gynaecol Obstet. 155 (Suppl 1), 61-85 (2021).</li><li>Ledermann, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+diagnosed+and+relapsed+epithelial+ovarian+carcinoma:+ESMO+clinical+practice+guidelines+for+diagnosis,+treatment+and+follow-up.">Newly diagnosed and relapsed epithelial ovarian carcinoma: ESMO clinical practice guidelines for diagnosis, treatment and follow-up.</a> Ann Oncol. 24 (Suppl 6), vi24-32 (2013).</li><li>Jelovac, D., Armstrong, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+diagnosis+and+treatment+of+ovarian+cancer.">Recent progress in the diagnosis and treatment of ovarian cancer.</a> CA Cancer J Clin. 61 (3), 183-203 (2011).</li><li>Morgan, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer.+Clinical+practice+guidelines+in+oncology.">Ovarian cancer. Clinical practice guidelines in oncology.</a> J Natl Compr Canc Netw. 6 (8), 766-794 (2008).</li><li>Nieman, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipocytes+promote+ovarian+cancer+metastasis+and+provide+energy+for+rapid+tumor+growth.">Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth.</a> Nat Med. 17 (11), 1498-1503 (2011).</li><li>Motohara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evolving+story+of+the+metastatic+voyage+of+ovarian+cancer+cells:+cellular+and+molecular+orchestration+of+the+adipose-rich+metastatic+microenvironment.">An evolving story of the metastatic voyage of ovarian cancer cells: cellular and molecular orchestration of the adipose-rich metastatic microenvironment.</a> Oncogene. 38 (16), 2885-2898 (2019).</li><li>Di Nicola, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Omentum+a+powerful+biological+source+in+regenerative+surgery.">Omentum a powerful biological source in regenerative surgery.</a> Regen Ther. 11, 182-191 (2019).</li><li>Meza-Perez, S., Randall, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+functions+of+the+omentum.">Immunological functions of the omentum.</a> Trends Immunol. 38 (7), 526-536 (2017).</li><li>Liu, M., Silva-Sanchez, A., Randall, T. D., Meza-Perez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+immune+responses+in+the+peritoneal+cavity+and+omentum.">Specialized immune responses in the peritoneal cavity and omentum.</a> J Leukoc Biol. 109 (4), 717-729 (2021).</li><li>Lee, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+facilitate+ovarian+cancer+premetastatic+niche+formation+in+the+omentum.">Neutrophils facilitate ovarian cancer premetastatic niche formation in the omentum.</a> J Exp Med. 216 (1), 176-194 (2019).</li><li>Cardenas, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipocyte+microenvironment+promotes+Bclxl+expression+and+confers+chemoresistance+in+ovarian+cancer+cells.">Adipocyte microenvironment promotes Bclxl expression and confers chemoresistance in ovarian cancer cells.</a> Apoptosis. 22 (4), 558-569 (2017).</li><li>Wu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+adipocytes:+key+players+in+breast+cancer+progression.">Cancer-associated adipocytes: key players in breast cancer progression.</a> J Hematol Oncol. 12 (1), 95 (2019).</li><li>Zhang, Z., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue:+The+dysfunctional+adipocyte+-+a+cancer+cell's+best+friend.">Adipose tissue: The dysfunctional adipocyte - a cancer cell's best friend.</a> Nat Rev Endocrinol. 14 (3), 132-134 (2018).</li><li>Alvero, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRX-E-002-1+Induces+c-Jun-dependent+apoptosis+in+ovarian+cancer+stem+cells+and+prevents+recurrence+in+vivo.">TRX-E-002-1 Induces c-Jun-dependent apoptosis in ovarian cancer stem cells and prevents recurrence in vivo.</a> Mol Cancer Ther. 15 (6), 1279-1290 (2016).</li><li>Alvero, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+approach+for+the+detection+of+intraperitoneal+micrometastasis+using+an+ovarian+cancer+mouse+model.">Novel approach for the detection of intraperitoneal micrometastasis using an ovarian cancer mouse model.</a> Sci Rep. 7, 40989 (2017).</li><li>Craveiro, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+modifications+in+ovarian+cancer+stem+cells+following+Paclitaxel+treatment.">Phenotypic modifications in ovarian cancer stem cells following Paclitaxel treatment.</a> Cancer Med. 2 (6), 751-762 (2013).</li><li>Sumi, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+for+non-invasive+imaging+to+detect+and+monitor+ovarian+cancer+recurrence.">Murine model for non-invasive imaging to detect and monitor ovarian cancer recurrence.</a> J Vis Exp. (93), e51815 (2014).</li><li>Agarwal, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+migration+inhibitory+factor+expression+in+ovarian+cancer.">Macrophage migration inhibitory factor expression in ovarian cancer.</a> Am J Obstet Gynecol. 196 (4), 348.e1-348.e5 (2007).</li><li>Kelly, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR-4+signaling+promotes+tumor+growth+and+paclitaxel+chemoresistance+in+ovarian+cancer.">TLR-4 signaling promotes tumor growth and paclitaxel chemoresistance in ovarian cancer.</a> Cancer Res. 66 (7), 3859-3868 (2006).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CBX7+binds+the+E-box+to+inhibit+TWIST-1+function+and+inhibit+tumorigenicity+and+metastatic+potential.">CBX7 binds the E-box to inhibit TWIST-1 function and inhibit tumorigenicity and metastatic potential.</a> Oncogene. 39 (20), 3965-3979 (2020).</li><li>Tedja, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+epithelial-mesenchymal+hybrid+cancer+cells+with+tumorigenic+potential.">Generation of stable epithelial-mesenchymal hybrid cancer cells with tumorigenic potential.</a> Cancers (Basel). 15 (3), 15030684 (2023).</li><li>Dauleh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+cultured+mesothelial+cells+derived+from+the+murine+adult+omentum.">Characterisation of cultured mesothelial cells derived from the murine adult omentum.</a> PLoS One. 11 (7), e0158997 (2016).</li></ol>

Using this protocol, a preclinical model of peritoneal carcinomatosis for ovarian cancer was developed using a combination of basic in vitro and ex vivo techniques. A progressive tumor growth was observed across 50 days of co-culture after seeding omentum specimens with mCherry+ OCSC1-F2 human ovarian cancer cells. This method was developed and optimized across several experimental trials using different omentum specimens. Successful tumor growth depended on the quality of omentum, viability of cancer c...

Ovarian cancer is the deadliest gynecologic malignancy worldwide1. The lifetime risk of developing this cancer is approximately 1 in 70, with the median age of diagnosis at 63 years old2. Primary ovarian malignancies are classified histologically as either epithelial or non-epithelial. Epithelial ovarian cancers (EOC) represent over 90% of tumors, and the most common subtype is high-grade serous carcinoma (HGSC), which accounts for approximately 70%-80% of EOCs. Currently, there are no effective screening methods to detect disease early. So most patients are diagnosed at an advanced stage (i.e., F&#233;d&#233;ration Internationale de Gyn&#233;cologie et d&#39;Obst&#233;trique [FIGO] stage III or IV) after the cancer has spread throughout the peritoneal cavity2.
Standard frontline treatment is cytoreductive surgery to remove all visible macroscopic disease, followed by adjuvant platinum-based chemotherapy to destroy any residual microscopic disease. While there have been many advances in ovarian cancer treatment over the last two decades, approximately 70% of patients with advanced disease will relapse within 3 years of treatment3. Given the overall poor prognosis of these patients, ongoing and future translational research efforts in EOC aim to identify biomarkers for early detection, prevent metastasis, improve current therapies to evade resistance and develop new personalized cancer treatments.
Generalized metastasis within the peritoneal cavity and its associated chemoresistance are two of the major limitations for the improvement of the treatment of patients with ovarian cancer4,5. The omentum, a fatty apron-like structure that hangs down from the stomach over the intestines, is a main site of ovarian cancer metastasis6,7. In addition to its function as a physical barrier, the omentum has been shown to have regenerative and angiogenic capacities and possess immune activities, which together promote vascularization, accelerate wound healing, and limit infection8. It contains a high concentration of stem cells that can differentiate into various cell types and can help repair damaged tissues. The omentum can become inflamed in response to injury or infection, which triggers the migration of immune cells to the site of injury9. These immune cells release growth factors and other molecules that help to promote the repair and regeneration of damaged tissue. Immune cells, such as macrophages, lymphocytes, and plasma cells, localized in the omentum are structures known as &#34;milky spots&#34;, which are responsible for detecting and attacking pathogens and regulating peritoneal immunity. The omentum has also been shown to play a role in inducing immune tolerance10, which is the ability of the immune system to tolerate self-antigens and not attack healthy tissues. However, the same immune-related activities are also involved in pathological responses, such as the growth of omental tumors, metastasis, and escape of immune surveillance9,11. Previous studies from our lab and others have demonstrated a unique and active role of the adipose microenvironment in the inhibition of anti-tumoral immune responses and in the acquisition of chemoresistance12,13,14. Unfortunately, we have limited information on the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment.
To better understand the interactions between cancer cells and the omentum, a 3D culture system consisting of human ovarian cancer cells and patient-derived omentum explants was developed. The protocol described here represents a novel ex vivo model of peritoneal carcinomatosis. This model mimics the natural progression of ovarian cancer tumorigenesis in this adipose-rich tissue. The proposed model is easy to generate, inexpensive, and potentially applicable to translational investigations in ovarian cancer research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x)</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Insulin Syringe with 26 G detachable needle</td>
			<td>BD</td>
			<td>329652</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological Pipets</td>
			<td>CELLTREAT</td>
			<td>229010B</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Tissue Culture Dish</td>
			<td>Fisherbrand</td>
			<td>FB012924</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tube</td>
			<td>CELLTREAT</td>
			<td>229411</td>
			<td></td>
		</tr>
		<tr>
			<td>24 Well Cell Culture Plate</td>
			<td>Costar</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>CELLTREAT</td>
			<td>229421</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 Tissue Culture Flask</td>
			<td>CELLTREAT</td>
			<td>229341</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Cell Counter</td>
			<td>Corning</td>
			<td>9819000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 5 imager</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1) (1x), +L-Glutamine, +2.438 g/L Sodium Bicarbonate</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Qualified</td>
			<td>Gibco</td>
			<td>1043028</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>No. 10 Stainless Steel Disposable Scalpel</td>
			<td>Integra-Miltex</td>
			<td>4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline, pH 7.4 (1x)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Revolve microscope</td>
			<td>Echo</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo model of ovarian cancer peritoneal metastasis using human omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

Author Spotlight: Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction 

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Current studies use co-culture systems comprised of isolated adipocytes and ovarian cancer cells. These adipocytes are differentiated in vitro from pre adipocytes. These cells are then separated through a transwell insert and therefore lack direct cell to cell contact.This ex vivo culture method offers a model to study the direct interaction of cancer cells with the adipose microenvironment. Our model preserves the structure of the human momentum and therefore maintains the various cell types that are typically in this organ. In addition to adipocytes, these cells include supporting fibroblasts as well as immune cells.This model also maintains these cells in their inherent matrix. This model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche.

Successful establishment of ovarian cancer cells into omentum specimens was evident by about day 14 (Figure 3A-C). At least 24 replicates were prepared and injected per collected specimen to allow for further experimentation. Tumor growth was monitored by taking fluorescent images (Figure 3D,E). Images had to be carefully interpreted as a monolayer of cancer cells also grew at the bottom of each well that was no...

Watch this Scientific Journal Video about Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction at JoVE.com

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...