The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.
Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.
The identification of the FT as a candidate site of origin for SOC provides the opportunity for basic and translational research aimed at deciphering the mechanisms linking known risk factors and the actual serous carcinogenic process. Critical for this is the development of tractable model systems that will enable us to begin testing the hypothesis that the FTSEC is a cell-of-origin for pelvic serous carcinomas. The ex vivo culture model described herein is a novel system that permits the isolation and co-culture of primary FT secretory and ciliated cells in a manner that preserves the morphology and biology of native FT epithelium. Using this system, we recently characterized the secretome of this epithelium and how the epithelium responds to mechanical and genotoxic injury 10. Ovulation, a major risk factor associated with ovarian tumorigenesis, is characterized by a combination of tissue injury, inflammatory mediators, growth factors, and hormones 11. This system could be used to study the effect of an ovulatory milieu on the response and viability of secretory and ciliated cells. In addition, the impact of other cell types (i.e. inflammatory cells) could be examined to allow a better understanding of the events that drive cell-type differentiation and the factors that contribute to the neoplastic transformation of these cells. Similar models have been described in other tissues where polarized epithelium is present. For instance, in polarized primary cultures of airway epithelium, the effect of heregulin on cell growth and response to cellular injury was assessed 12. These types of model systems provide a new and useful way to study the initiation and pathogenesis of tumors which arise from epithelial tissues, and this is of critical importance in tissues such as the FT where no cell lines currently exist, and where there is a great need for the identification of key pathways and development of novel treatment strategies.
The authors have nothing to disclose.
We thank the faculty, fellows, residents, and physician assistants of the Brigham and Women’s Hospital, Department of Pathology for making tissue available for these studies. This work was supported by research grants from the NIH/National Cancer Institute (P50 CA105009, K08 CA108748, U01 CA152990), Ovarian Cancer Research Fund, The May Kay Foundation, Novartis Pharmaceuticals, Robert and Deborah First Fund, Randi and Joel Cutler Ovarian Cancer Research Fund, Marsha Rivkin Foundation – Scientific Scholar Award, AACR – George and Patricia Sehl Fellowship for Cancer Genetics Research, and the American Physician Fellowship for Medicine in Israel – Claire and Emmanuel G. Rosenblatt Foundation Grant.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Collagen from human placenta (Bornstein and Traub Type IV) | Sigma | C7521 | ||
DMEM:F12 media | Cellgro | 15-090-CV | ||
Ultroser G | Crescent Chemical Company | 67042 | Add 20 ml of DMEM:F12 to stock bottle and use 10mls per 500 ml of media (2% final concentration) | |
Pen Strep | Invitrogen | 15140-122 | ||
BD Primaria culture plates | BD Bioscience | 353802 | ||
24 well Transwell Permeable supports | Corning (Costar) | 3470 | Clear, 6.5 mm insert, 0.4 μm pore size, treated polyester | |
MEM (Minimal Essential Media) | Cellgro | 10-010-CV | ||
Pronase | Roche | 11459643001 | ||
DNAse | Sigma | DN25 | ||
Sall2 antibody | Dr T. Benjamin, Harvard | Gift | 1:20 dilution | |
Pax8 antibody | Proteintech | 10336-1-AP | 1: 1000 dilution |