An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis
Summary
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.
Abstract
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 >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.
Introduction
Ovarian cancer is a deadly disease with the 5th highest mortality rate of all cancers in women1. Most women with ovarian cancer are diagnosed at an advanced stage, with metastatic spread present in 70% of patients at the time of diagnosis. Factors such as early metastases and advanced stage at diagnosis contribute to the high mortality rates seen with this disease. Moreover, these unique disease characteristics have posed a challenge for establishing ovarian cancer mouse models, including reproducing rapid disease migration into the peritoneal cavity2,3,4.
Ovarian cancer pathogenesis, including peritoneal spread, is facilitated by the formation of a supportive tumor microenvironment (TME) that comprises many elements5. One critical component of the ovarian cancer TME is the carcinoma-associated mesenchymal stem cell (CA-MSC). CA-MSCs are stromal progenitor cells that enhance ovarian cancer initiation, growth, chemotherapy resistance, and metastasis6,7. CA-MSCs also drive the formation of the ovarian cancer TME through stimulating tumor-associated fibrosis, inducing angiogenesis, and altering the immune microenvironment6,8,9. Given the powerful functions of CA-MSCs within the ovarian cancer TME, modeling human ovarian cancer within a physiologically relevant stromal microenvironment is critical in studying ovarian cancer progression and metastasis.
Recently, transgenic mouse models have gained attraction in the study of spontaneous ovarian cancer metastases. However, transgenic mice are costly and exhibit a prolonged time course for the development of metastatic disease. While other available mouse models such as the intraperitoneal model and patient derived xenografts (PDX) have relatively short metastatic time intervals, they do not fully recapitulate ovarian cancer metastases due to the lack of a relevant stromal microenvironment10,11,12. In an attempt to overcome this challenge, this study presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived CA-MSCs. In the model described here, combining CA-MSCs with ovarian cancer cells generates tumors with early and diffuse metastases, as demonstrated by the presence of intra-abdominal metastases in 100% of mice within 30 days post-injection.
Protocol
Representative Results
Discussion
Despite significant clinical and research efforts, minimal headway has been made in the treatment and prevention of ovarian cancer1. While a variety of mouse models have been used to study ovarian cancer progression and metastases, these models have faced significant limitations. In particular, previous mouse models have been unable to fully recapitulate the natural history of ovarian cancer progression, including the hallmark feature of early, diffuse intra-abdominal metastases1…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We would like to thank the Gynecologic Oncology Biospecimen Program-Promark for help with tissue collection. LGC is supported by Tina's Wish Rising Star Grant and The Mary Kay Foundation.
Materials
| 0.05% trypsin/0.02% EDTA | Sigma | SLCD2568 | |
| Anti-human CD105 | BD Pharmingen | 560847 | |
| Anti-human CD73 | BD Pharmingen | 555596 | |
| Anti-human CD90 | BD Pharmingen | 561443 | |
| B27 | Gibco | 17504-044 | |
| β-FGF | Gibco | PHG0261 | |
| β-mercaptoethanol | MP Biomedicals | 194834 | |
| Carprofen | Henry Schein | 11695-6934-1 | |
| D-luciferin | PerkinElmer | 122799 | |
| DMED | Gibco | 11995-065 | |
| EGF | Gibco | PHG0311 | |
| Gentamicin | Gibco | 15710072 | |
| Heat-inactivated FBS | Gibco | 16000069 | |
| Insulin | Gibco | 12585014 | |
| Insulin syringe | BD Pharmingen | 324704 | |
| In vivo imaging system IVIS | PerkinElmer | IVIS Lumina X5 | |
| Matrigel | Corning | 354230 | |
| MEBM (mammary epithelial cell basal medium) | ATCC | PCS-600-030 | |
| Mycoplasma test kit | ABm | G238 | |
| NSG mice | The Jackson Laboratory | 5557 | |
| OVCAR3 | ATCC | HTB-161 | |
| Penicillin/streptomycin | Gibco | 15070063 | |
| Polyglycolic Acid suture | ACE | 003-2480 |
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