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 into a sterile 50 mL conical tube or surgical specimen container.</li>
	<li>Obtain surgical specimens from omental biopsy or omentectomy surgery. Immerse the sterile specimen in omentum culture media immediately after removal in the operating room. If the workflow does not allow this, place the specimen in omentum culture media as soon as possible. Transfer the sample by placing it on ice and store at 4 &#176;C until processing. 
	NOTE: The size of the omentum specimen removed will determine the amount of workable tissue.</li>
	<li>Process the omentum specimen within 1-2 h of collection using a laminar flow hood. Ensure that all materials and tools are sterile or sterilized.</li>
	<li>Remove the omentum from the collection container and transfer it into a 100 mm culture dish. Immerse the specimen in 1x phosphate-buffered saline (1x PBS) and gently wash the specimen to remove any blood clots or debris.</li>
	<li>Cut the tissue into pieces (0.5 cm x 0.5 cm or 100-200 mg; Figure 2A) using either small surgical scissors or a 10-15 mm scalpel. Use small surgical forceps to help manipulate the tissue and avoid crushing the omentum. If an energy device was used to surgically remove the omentum specimen, then avoid using the distorted tissue at the sealed edge.</li>
	<li>Place each piece of cut omentum into individual wells of a 24-well culture plate and fill the wells with 500 &#956;L of omentum culture media or to a volume that is enough to cover the omentum without allowing the omentum piece to float above the well floor (Figure 2B).</li>
	<li>Store omentum pieces in fresh culture media at 4 &#176;C until ready for injection.</li>
</ol>
2. Preparation of ovarian cancer cells
<ol>
	<li>Culture human ovarian cancer cells in a 75 cm2 culture flask at 37 &#176;C and 5% CO2 to at least 75% confluency by the day of omentum collection. 
	NOTE: This protocol utilizes mCherry-positive OCSC1-F2 human ovarian cancer cells previously described15,16,17,18. It can be modified to any cancer cell line tagged with a fluorescence signal.</li>
	<li>Prepare cells inside a laminar flow hood immediately after collection of the omentum specimen. Ensure that all materials and tools are sterile or sterilized.</li>
	<li>Add 10 mL of sterile 1x PBS to the culture flask and wash the cells by gently rocking the flask. Remove the 1x PBS and then add 3 mL of 0.05% Trypsin-EDTA.</li>
	<li>Gently rock the culture flask to coat all cells, and then place the flask in an incubator (37 &#176;C and 5% CO2) for no more than 5 min. Tap the culture flask to completely detach the cells, and then neutralize trypsin by adding 3 mL of omentum culture media.</li>
	<li>Mix the cell suspension by pipetting, and then transfer the suspension into a 15 mL conical tube. Centrifuge the suspension for 5 min at 1,200 x g at 24 &#176;C. Remove the supernatant and resuspend the cell pellet in 6 mL of fresh omentum culture media.</li>
	<li>Count the cells using a hemacytometer. Dilute the cell suspension to at least 100,000 cells per 100-200 &#956;L of suspension. This is the volume of injection into each cut piece of omentum.</li>
	<li>Transfer an appropriate number of cells into a new culture flask to continue cell passage.</li>
</ol>
3. Injection of ovarian cancer cells
<ol>
	<li>Use a 1 mL syringe to draw up the cell suspension, then attach a 26-G needle. Do not draw up the cell suspension using the needle, as this can fragment the cells.</li>
	<li>Use small surgical forceps to pick up a piece of cut omentum. Transfer the omentum to a separate working sterile dish to facilitate injection. Gently pierce the omentum with the needle tip and inject a small volume of cell suspension into the tissue (Figure 2C).</li>
	<li>Repeat injection over several areas of omentum to a total volume of at least 100 &#956;L or 100,000 cells. Note that much of the cell suspension may appear to pool around the specimen. If there is concern regarding the quality of the injection, then repeat the injection or inject a larger volume of cell suspension into the specimen.</li>
	<li>Return the injected omentum samples into each well of a 24-well culture plate. Take note that the volume of omentum culture media is to a level that covers the omentum without allowing it to float. Carefully handle the plate and place it inside an incubator (37 &#176;C and 5% CO2).</li>
	<li>OPTIONAL: Use Matrigel (basement membrane matrix) to suspend omentum tissue in a solid matrix to allow easier injection and more concentrated cell suspension delivery.
	<ol>
		<li>Thaw the basement membrane matrix at 4 &#176;C and mix in 1:1 ratio with omentum culture media immediately prior to use. Store the basement membrane matrix mixture on ice or at 4 &#176;C until injection. Fill empty wells with enough basement membrane matrix mixture to immerse a cut piece of omentum.</li>
		<li>Place specimens into the basement membrane matrix mixture and then place the 24 well culture plate in an incubator (37 &#176;C and 5% CO2) for 20 min. Once the mixture has solidified, continue with injection as described above.</li>
	</ol>
	</li>
	<li>Confirm successful injection using fluorescent imaging. Ensure a streak of fluorescent signal is visualized at the injection sites (Figure 2D). Note that the actual number of cells attached to the omentum after injection is unpredictable.</li>
</ol>
4. Co-culture of human omentum and ovarian cancer cells
<ol>
	<li>Change media every 48-72 h by adding 500-2000 &#956;L of fresh omentum culture media. Do not pipet media directly on top of omentum tissue, as this may displace cancer cells. Note that if the media color turns to yellow, then change the media. 
	NOTE: The frequency of media change will depend on the volume of media used and the trajectory of cancer cell growth. Once the cancer cells have seeded the omentum, whether or not the omentum piece is floating is no longer important.</li>
	<li>OPTIONAL: If a basement membrane matrix was used, the matrix would typically be dissolved by the time of the first media change.</li>
	<li>Monitor the growth of ovarian cancer tumors using fluorescent imaging. Frequency of imaging is at the discretion of the investigator. Anticipate evidence of small tumors by about 14 days (Figure 3A-C). Results may vary depending on the quality of the injection.</li>
	<li>Terminate the experiment based on the experiment endpoint. 
	NOTE: Tumor growth and integrity of omentum tissue have been observed past 50 days.</li>
</ol>

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

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	<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. 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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>

The authors have nothing to disclose.

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 cells, and effectiveness of injection. In this report, we only used mCherry+ OCSC1-F2 cells and mCherry+ R182 human ovarian cancer lines previously reported in several publications12,15,16,17,18,19,20,21,22. Using these two cell lines with different doubling times (16 h for OCSC1-F2 and 36 h for R182), a difference in the time to achieve logarithmic growth within the omental tissue was noted. Nevertheless, we were successful in establishing both cell lines in the co-culture system.
Efficient communication between investigators, research coordinators, and surgical teams was critical for the collection and processing of omentum specimens. The investigator identified protocol candidates, the research coordinators obtained consent from patients before surgery, and the investigator collected the specimens from the operating room. Given this workflow, the investigator&#39;s schedule had to be flexible as many patient-sided factors affecting collection were unpredictable (e.g., surgery canceled, time of surgery changed, inadequate or no omentum removed). Direct visualization and selection of tissues from the operating room ensured specimen integrity, surgical sterility, and timely processing. Initially, specimen collection was delegated to research coordinators; however, after a few specimens were collected, we noted that time to processing was delayed (i.e., &#62;4 h) and the quality of omentum was grossly inferior (e.g., tissue was in pieces). In one experimental trial, omental tissue was stored at 4 &#176;C for 48 h prior to injection because cancer cells were not ready in time. Similar tumor growth was still observed, but this sequence was not repeated as temperature changes are known to affect the tumor microenvironment (TME).
Before specimen collection, it was important to have sufficiently confluent cancer cells according to the surgery schedule. Since the number of specimens collected each month varied, active cultures of ovarian cancer cells were not always maintained. The frozen cells were thawed at least 1 week in advance, plated using a standard protocol, and then passed the cells at least once to ensure viability. The cell lines used in this protocol were relatively resilient; however, we still encountered instances when cells had not recovered by the day of surgery and could not be injected. In this study, there was no attempt to inject cancer cells that were less than 50% confluent. Subsequent delays related to cell preparation were mitigated by using good sterile technique and splitting cells in ratios to maintain confluence depending on the anticipated specimen collection schedule. We limited the protocol to only two fluorescently tagged cancer cell lines, but it is important to note that other established cancer cell lines or primary cancer cells without fluorescent proteins could also be utilized.
Lastly, the rate of tumor growth was correlated to the confluence of cancer cells adjacent to and within adipocytes immediately following injection. This was determined by taking fluorescent images of all omentum specimens post-injection. Compared to tumor cell inoculation of solid organs or subcutaneous tissues, injecting cancer cells into omental tissue was more challenging. The loose structure and fatty consistency of omentum prevent the tissue from effectively holding volumes of cell suspension without leaking. When a smaller needle (i.e., 30 G or narrower) was used to inject, there was concern for cell fragmentation as cell proliferation was decreased. We tested various injection techniques, cell numbers, and cell suspension volumes and found consistent results with the protocol described above. At the same time, we acknowledge that such variations to the protocol are likely to still produce similar results given the malignant nature of the cells. The addition of Matrigel made the injection step easier as the extracellular matrix held the cell suspension better; however, this method is more expensive and did not consistently produce a higher tumor burden in the samples. Overall, it was rare for any of the omentum specimens to not grow tumors, but the number of tumors and speed of growth varied.
This model simulated a hallmark of ovarian cancer tumorigenesis by replicating early metastasis to adipose-rich peritoneal tissues. This protocol allows variation with no required skill, is easy to replicate, and generates a system with potential translational value. Compared to existing in vitro and ex vivo models in ovarian cancer, the method described here combines several techniques to produce a model that retains tumor architecture and TME over a long lifespan. Additionally, we observed that when an omentum specimen with multiple tumors was transferred into a new well and cultured with cancer-free omentum (i.e., not injected), the na&#239;ve omentum was seeded with cancer cells within 7 days. This finding suggests that the replicated tumors retain metastatic potential via epithelial-mesenchymal transition. We did not identify any similar ovarian cancer models in the literature that cultured tissue biopsies for extended periods of time. This approach is supported by a previous finding that mesothelial cells isolated from mouse omentum could be cultured for more than 30 passages after collection23.
The model described in this study can be employed to further understand the effect of direct interaction between ovarian cancer cells and the cells within the adipose-rich omentum. Cancer cells can be dissociated from the omentum tissues and obtained for transcriptomic or immunohistochemistry (IHC) analysis. Additionally, cells can be used for drug response studies.
The primary limitations of this model are that the number of tumors, rate of tumor growth, and location of tumors were unpredictable. Lack of standardization and variation across samples could limit the application of this model. We also made several observations that will require further examination during the development of this protocol. For example, the &#34;control&#34; replicates in one experiment were not actually cancer-free as the patient was subsequently diagnosed with omental micrometastasis; however, the non-injected tumors remained viable throughout the experimental period. Future applications of this model will include molecular studies of the TME during early tumorigenesis, manipulation of the TME by injecting immune cells, and drug response investigations. In summary, we believe that the preclinical model described here will add to the repertoire of existing research tools and help elucidate molecular mechanisms of TME in ovarian cancer.

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><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 cm&lt;sup&gt;2&lt;/sup&gt; 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.

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 not attached to the omentum. We preferred taking images of the omentum when it was suspended in media to avoid overlapping fluorescent signals with the cancer cell monolayer.
The absence of a fluorescent signal by day 14 can be considered as failed injections. It was observed that the cancer cells initially spread out across the surface of the omentum during the first 7 days, but over time, they congregated to form tumors (Figure 3A-D). A surrogate measure for tumor burden between samples was the rate at which omentum culture media changed color from red to yellow (Figure 3F). We utilized fluorescent imaging, histologic review, and gross inspection to confirm tumor growth and omental tissue viability past 50 days of co-culture (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66031/66031fig01.jpg" /> 
Figure 1: Illustration of general steps in the protocol. (A) Step 1: Preparation of omentum; (B)&#160;Step 2: injection of cancer cells; (C) Step 3: establishment of cancer cell-omentum microenvironment.&#160;(Figure prepared in BioRender.com).&#160;<a href="https://www.jove.com/files/ftp_upload/66031/66031fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66031/66031fig02.jpg" /> 
Figure 2: Preparation and injection of cancer cells into the omentum. (A) Omentum is cut into 1x1 cm pieces. (B)&#160;Each piece is placed in a well of a 24-well plate and covered with 500 &#956;L of media. (C) Injection of cancer cells into omentum piece. (D) A streak of fluorescent cancer cells is observed after injection. <a href="https://www.jove.com/files/ftp_upload/66031/66031fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66031/66031fig03.jpg" /> 
Figure 3: Representative images after 14 and 25 days of co-culture. (A-C) Representative images after 14 days of co-culture: (A) phase, (B) mCherry channel, (C) merged. (D-E) Representative images after 25 days of co-culture. (F) Change in media color from pink to yellow is an indication of successful cancer cell injection and establishment of co-culture. <a href="https://www.jove.com/files/ftp_upload/66031/66031fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66031/66031fig04.jpg" /> 
Figure 4: Gross morphology and Hematoxylin and Eosin (H&#38;E) staining. (A,B) Representative gross morphology of co-cultures at day 50; arrows point to sites of growth of mCherry+ ovarian cancer cells. (C,D) Representative H&#38;E staining of co-cultures at day 50. <a href="https://www.jove.com/files/ftp_upload/66031/66031fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Cancer Research

2.5K Views.  Karmanos Cancer Institute. Huidige studies maken gebruik van co-cultuursystemen die bestaan uit geïsoleerde adipocyten en eierstokkankercellen. Deze adipocyten worden in vitro onderscheiden van pre-adipocyten. Deze cellen worden vervolgens gescheiden door middel van een transwell-insert en missen daarom direct cel-tot-celcontact.Deze ex vivo kweekmethode biedt een model om de directe interactie van kankercellen met de vetmicro-omgeving te bestuderen. Ons model behoudt de structuur van het menselijk momentum en ...

Video: Auteur in de schijnwerpers: geavanceerd ex vivo-model voor het onderzoeken van de interactie tussen kanker en vethoudende micro-omgeving 

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

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 ...

Medicine

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Orthotopic Inoculation of Ovarian Cancer Cells into Murine Ovary: A Procedure to Establish an Ovarian Cancer Mouse Model to Study Peritoneal Dissemination of Cells

Source: Koya, Y. et al. <a href="https://www.jove.com/t/54353">Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells.</a> J. Vis. Exp. (2016)
This video describes the protocol for creating a murine experimental model to study tumor development and peritoneal metastasis. This ovarian cancer model is generated by the orthotopic inoculation of human epithelial ovarian cancer cells into the ovaries of a female mouse.

Source: Koya, Y. et al. Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian ...

JoVE Encyclopedia of Experiments

Gynecologic Cancer

Animal model studies

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.

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

Assays & techniques

Modeling Ovarian Cancer Metastasis in Peritoneal Cavity Lining: A Method To Establish an In Vitro 3D Model of the Peritoneal Lining

To release the normal omentum fibroblasts from culture, rinse the culture flask with 10 milliliters of PBS followed by 3 milliliters of trypsin for no more than 5 minutes. When the cells have detached, neutralize the trypsin with at least three times the volume of full growth medium and transfer the cells to a 50-milliliter conical tube. Spin down the cells and resuspend the pellet in 5 milliliters of full growth medium. Then, count the cells and dilute them to a 2 to 4 times 10 to the third fibroblast per 100 microliters of full growth medium concentration.
Next, add 0.5 micrograms per 100 microliters of rat-tail collagen I to the cells and plate 100 microliters of cells per well into a black, clear bottom 96-well plate. Then, transfer the plate to a cell culture incubator for at least 4 hours or until the cells adhere to the plate surface. While the normal omentum fibroblasts are settling, detach the HPMC from their culture flasks and dilute these cells to 1 to 2 times 10 to the fourth HPMC per 50 microliters of full growth medium.
Then, add 50 microliters of HPMC per well to the attached normal omentum fibroblasts and return the plate to the cell culture incubator for at least 18 hours before experimental use. The next day, seed the 3D organotypic co-cultures with GFP expressing ovarian cancer cells from an 80% to 90% confluent culture at the appropriate dilution for the desired downstream assay.

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

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