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.
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Method Article
An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum
In This Article
Summary
Abstract
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.
Introduction
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., Fédération Inte....
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Protocol
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
- Prepare omentum culture media (DMEM/F12 + 10% fetal bovine serum + 1% penicillin-streptomycin) and store at 4 °C. Aliquot 30-40 mL of this media into a sterile 50 mL conical tube or surgical specimen container.
- Obtain surgical specimens from omental biopsy or omentectomy surgery. Imm....
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Results
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.......
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Discussion
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.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 0.05% Trypsin-EDTA (1x) | Gibco | 25300054 | |
| 1 mL Insulin Syringe with 26 G detachable needle | BD | 329652 | |
| 10 mL Serological Pipets | CELLTREAT | 229010B | |
| 100 mm Tissue Culture Dish | Fisherbrand | FB012924 | |
| 15 mL Centrifuge Tube | CELLTREAT | 229411 | |
| 24 Well Cell Culture Plate | Costar | 3524 | |
| 50 mL Centrifuge Tube | CELLTREAT | 229421 | |
| 75 cm2 Tissue Culture Flask | CELLTREAT | 229341 | |
| Corning Cell Counter | Corning | 9819000 | |
| Cytation 5 imager | Biotek | ||
| DMEM/F12 (1:1) (1x), +L-Glutamine, +2.438 g/L Sodium Bicarbonate | Gibco | 11320033 | |
| Fetal Bovine Serum, Qualified | Gibco | 1043028 | |
| Matrigel | Corning | 356230 | Basement membrane matrix |
| No. 10 Stainless Steel Disposable Scalpel | Integra-Miltex | 4410 | |
| Penicillin Streptomycin | Gibco | 15140122 | |
| Phosphate Buffered Saline, pH 7.4 (1x) | Gibco | 10010023 | |
| Revolve microscope | Echo |
References
- Siegel, R. L., Miller, K. D., Wagle, N. S., Jemal, A. Cancer statistics. CA Cancer J Clin. 73 (1), 17-48 (2023).
- Berek, J. S., Renz, M., Kehoe, S., Kumar, L., Friedlander, M. Cancer of the ovary, fallopian tube, and peritoneum: 2021 update. Int J Gynaecol Obstet. 155 (Suppl 1), 61-85 (2....
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