Abstract B33: Omental milky spots serve as a niche for ovarian cancer metastatic colonization
Authors: Krishnan Venkatesh , Clark Robert , Schoof Michael , Rodriguez Irving , Chekmareva Marina , Rinker-Schaeffer Carrie 
Published: August 2014

Abstract
Introduction: Metastatic ovarian cancer remains an urgent clinical problem. The homing and invasion of cancer cells into the omentum, the preferred site of ovarian cancer metastasis, is a rate-limiting step in disease progression. We have recently shown that immune cell-containing structures known as milky spots are required for colonization of the omental adipose. Experiments were designed using well-established ovarian cancer models and experimental metastasis assays to answer the following questions: Does cancer cell localization depend upon the immune composition of the milky spots? Do cancer cells utilize the native milky spot microenvironment, or does it undergo remodeling during colonization? Answers to these questions will be the foundation for mechanism-based studies aimed at identifying ovarian cancer-omental interactions that can be targeted therapeutically.
Methods: Mouse strains with varying immune-competency were used to: 1) test the effect of immune composition on volume of milky spots within the omentum; 2) determine the requirement of specific immune cell types on ovarian cancer cell lodging and invasion into milky spots; and assess the effect of ovarian cancer cell lodging on milky spot composition. Strains included: C57BL/6 (immunocompetent), Athymic Nude (T cell deficient), Beige Nude (NK and T cell deficient), Rag1-/- (no mature T or B cells, no CD3+ or T cell receptor a-b positive cells), and Igh6-/- (lack mature B cells). Experimental metastasis assays used intraperitoneal injection of 1 x 106 ID8 (mouse) or SKOV3ip.1 (human) ovarian cancer cells into C57BL/6 mice, or immunodeficient mice, respectively. Milky spot volume in naïve omenta was calculated from digital whole-mounts constructed from Giemsa-stained tissue sections. Milky spot composition was evaluated using immunohistochemistry (IHC) for lymphocytes (CD45), T cells (CD3), B cells (B220), macrophages (F4/80), and endothelial cells (CD31).
Results: ID8 and SKOV3ip.1 ovarian cancer cells localized to and proliferated in the milky spots. Studies using a panel of immune-deficient mice showed that the mouse genetic background does not alter omental milky spot number and size, nor does it affect ovarian cancer colonization. Analysis of digital whole mounts found no difference in the volume of milky spots in omenta from C57BL/6, Athymic Nude, Beige Nude, Rag1-/-, Igh6-/- mice. IHC of naïve omenta show the presence of B cells, T cells, and macrophages consistent with the genotype of the mouse. Abundant endothelial cells were present in milky spots corresponding to the dense vasculature that is a conserved feature of milky spots. Finally, evaluation of milky spot composition during metastatic colonization showed an influx of macrophages 24 hours and 3 days post injection (dpi). Staining for activated macrophages in metastases increased from week 1 to week 5; staining persisted until the experimental endpoint.
Conclusions: Targeting the host tissue microenviroment is crucial to contain or reduce cancer spread, prolonging patient life. Use of genetic models ruled out a requirement of B, T, and NK cells in ovarian cancer cell homing to milky spots suggesting a role for macrophages in this process. A preliminary study indicates a temporal increase in activated macrophages upon cancer colonization. We speculate that cancer cells co-opt the normal developmental role of macrophages to promote their own growth.
Work was supported by grants from the DOD (W81XWH-09-1-0127), NCI/NIH (2RO1CA089569), and Elsa U. Pardee Foundation; A Rivkin Center for Ovarian Cancer Research Pilot Study Award, and funds from the Section of Urology (The University of Chicago)
Citation Format: Venkatesh Krishnan, Robert Clark, Michael Schoof, Irving Rodriguez, Marina Chekmareva, Carrie Rinker-Schaeffer. Omental milky spots serve as a niche for ovarian cancer metastatic colonization. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B33.