Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Summary

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.

Abstract

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.

Introduction

Epithelial ovarian cancer (EOC) is the most common cause of death from gynecologic malignancy, with an estimated 21,290 new diagnoses in the U.S. in 2015 and an estimated 14,180 deaths¹. The vast majority (> 75%) of women are diagnosed with late-stage disease (stage III or IV) characterized by diffuse intra-peritoneal metastasis and poor prognosis. Disease recurrence in the peritoneal cavity following first-line chemotherapy is also common and represents a major cause of mortality^2,3. EOC metastasizes by an unique mechanism involving both direct extension from the primary tumor to neighboring peritoneal organs as well as by dissociation or sh....

Protocol

All in vivo studies were approved by the University of Notre Dame Animal Care and Use Committee and used female C57/BL6J mice.

1. Murine Ovarian Cancer Cell Culture

1. Make the ID8 murine ovarian cancer cell culture medium as follows: 1 L of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 4% Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin, 5 µg/ml Insulin, 5 µg/ml Transferrin and 5 ng/ml Sodium Selenite.
2. Transduce ID8 murine ovarian cancer cells¹³ to express Red Fluorescent Protein (RFP) using commercially procured lentiviral particles expressing RFP and a selection marker ....

Results

The metastatic mechanism of ovarian cancer is characterized by highly diffuse intra-peritoneal metastasis comprised of numerous lesions of varying size, including multiple small (< 2mm) lesions. Thus, use of RFP-labelled tumor cells (Figure 1) and optical imaging provides an alternative method to manual counting and measurement of lesion size. The development of tumor burden over time can be determined by weekly weighing of mice and measurement of abdominal girth to ga.......

Discussion

In contrast to studies using human ovarian cancer cells that must be conducted in immunocompromised mice, the protocol described above utilizes immunocompetent C57/Bl6 mice and syngeneic murine ovarian cancer cells. While this enables assessment of the potential role of immune infiltrates in tumor progression and metastasis, the presence of dark hair on the abdominal surface renders imaging less sensitive. Use of a depilatory to remove hair prior to imaging enhances image acquisition, but is time-consuming, particularly .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium	Corning	10-014-CM
Fetal bovine serum	Gibco	10437-028
penicillin/streptomycin
Insulin-transferrin-sodium selenite media supplement	Sigma	I-1884
Bruker Xtreme small animal imaging system	Bruker Corp.
Bruker Multispectral software	Bruker Corp
lentiviral particles with Red fluorescent protein	GenTarget, Inc.	LVP023
trypsin for cell culture	Corning	25-053-CI
PBS	Corning	21-040-CM
depilatory cream (such as Nair Hair Remover Lotion)	purchases from drugstore	n/a
ImageJ software	http://imagej.nih.gov/ij/		free download
dissecting tools (forceps)	Roboz Surgical Instrument	RS 5130
dissecting tools (Scissors)	Roboz Surgical Instrument	RS 5910