In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

Summary

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

Abstract

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

Introduction

Unlike most solid tumors, metastases from high-grade serous ovarian cancer (HGSC) are limited to the peritoneal cavity ¹. Thus, effective peritoneal therapies could potentially control or eradicate HGSC. Currently, a standard therapeutic approach is surgical cytoreduction combined with chemotherapy ^1-3. Unfortunately, the vast majority of patients experience and succumb to complications of disease recurrence. These dismal statistics show the need for improved understanding of metastatic colonization, the process by which cancer cells localize to, utilize, and proliferate within host tissues to form metastases ².

Protocol

All mice were housed, maintained and euthanized according to Institutional Animal Care and Use Committee (IACUC) guidelines and under the supervision of the University of Chicago Animal Resource Center.

1. Preparing Animals for Experimental Studies

1. Allow animals to acclimate to new housing and environment, to recover from potential physiologic effects of transport and handling.
   Note: The following technique is applicable to all commercially available strains of mice. The number of animals to be used for an experiment is dependent on the study design and should be done with consultation from a statistician.

Results

Identification and Histologic Examination of Peritoneal Fat Depots

Gross anatomic dissection allows identification of four of the five primary sources of peritoneal fat (Figure 1A). Moving clockwise from the top center are: the omentum (OM; outlined) located over the stomach and spleen, the gonadal fat (GF) surrounding the left ovary (ov), the uterine fat (UF) attached to the uterine horns (uh) and the mesentery (MY) attached to the small intestine (si). The o.......

Discussion

Development of therapies to target disseminated cells requires a mechanistic understanding of metastatic colonization, the critical first step in the development of peritoneal disease. To address these issues we report approaches that can be used to discern how the omentum’s unique tissue composition and architecture promote ovarian cancer metastatic colonization. Distinguishing features of our approach are: 1) the focus on early events in the colonization process; 2) comparison of milky spot-containing an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dissection Tools	Fine Science Tools	NA
Geimsa	Fluka/Sigma Aldrich	48900
5% Formalin	Sigma	HT501320
DMEM	Corning	10-013-CV
Trypsin	Gibco	25200-056
PBS	Corning	21-040-CV	Without calcium and Magnesium
26 gauge needle	BD	329652
BSA	Sigma	A7906
Collagenase	Worthington	LS004196
Stomacher	Seward Labsystems	Stomacher 80 Biomaster
Microstomacher bag	Stomacher Lab Systems	BA6040/Micro
ACK Lysis Buffer	Gibco	A10492-01
Millicell culture plate insert	Millipore	PICM01250
Cell-Tak	Corning	354240