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 5^th 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 cavity^2,3,

Protocol

Patient samples were obtained in accordance with the protocols approved by the University of Pittsburgh's IRB (PRO17080326). Animal experimental methods were conducted under the protocol approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.

1. Isolation and validation of patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs)

NOTE: CA-MSCs are derived from surgically resected human ovarian cancer tissue (this study uses high grade serous carcinoma), including the fallopian tube, ovary and/or omental metastatic deposits. CA-MSC medium is prepared from MEBM (ma....

Results

The described approach closely mimics the supportive microenvironment of ovarian cancer (in particular, high grade serous carcinoma) by co-injection of patient-derived mesenchymal stem cells (CA-MSCs) and ovarian cancer cells into the ovarian bursa. First, CA-MSCs were isolated from primary, surgically resected human high grade serous ovarian cancer involving the omentum (all CA-MSCs used in this experiment were derived from the same patient). After 2 weeks of plating the tissue samples, CA-MSCs were tested to meet the c.......

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 metastases^1.......

Materials

Name	Company	Catalog Number	Comments
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