Abstract AP24: ALTERNATIVELY ACTIVATED MACROPHAGE SECRETOME MODULATES METASTATIC POTENTIAL OF OVARIAN CANCER CELLS
Authors: Kaitlin Fogg, Will Olson, Jamison Miller, Pamela Kreeger
Journal: Clinical Cancer Research (2019)

Abstract High-grade serous ovarian cancer (HGSOC) metastasizes when tumor spheroids detach from the primary tumor and re-attach throughout the peritoneal cavity. Once spheroids have attached in a new site, the top layer of mesothelial cells is lost and the cancer cells expand across the underlying extracellular matrix (ECM). The factors that influence this process are unclear, but may provide therapeutic targets to slow disease spread. As HGSOC progresses an increase in alternatively activated macrophages (AAMs) in the surrounding ascites fluid has been observed and AAMs enhance tumor invasion and growth in a wide range of cancers. Thus we hypothesized that soluble factors from the AAMs in the peritoneal microenvironment promote the disaggregation of HGSOC spheroids across the underlying ECM. In order to model interactions between primary human AAMs and HGSOC spheroids (OVCAR3, OVCA433, OV90), an in vitro micro-culture device was used that allows for the examination of paracrine signaling in a controlled environment. After two days of co-culture on an 8 mg/mL collagen I hydrogel the area covered by the HGSOC cells was measured and normalized to the initial area of the spheroid. We determined that co-culture with AAMs significantly increased HGSOC spheroid spreading across the collagen matrix for all the HGSOC cell lines examined. To identify the AAM-derived soluble factors responsible for this increased spreading, media was collected from the device and screened for 35 cytokines using a Bioplex assay. The correlation between spheroid spreading and AAM-derived soluble factors was examined using PLSR; a one component PLSR model captured the co-variation between soluble factors and spheroid spreading (R2Y = 0.9) and was able to predict spheroid spreading given the cytokine profile (Q2Y = 0.66). Analysis of the PLSR model identified five ligands (Flt-3L, HB-EGF, IL-6, IL-8, and leptin) that strongly correlated with increased spheroid spreading. These findings were experimentally validated by adding recombinant versions of these proteins in the absence of AAMs as well as inhibiting the receptors for these factors in the presence of AAMs. From these experiments we discovered that each of the HGSOC cell lines was responding to a different AAM-secreted factor; Flt-3L induced spreading in the OVCAR3 cell line, leptin induced spreading in the OVCA433 cell line, and HB-EGF induced spreading in the OV90 cell line. The downstream pathway of these ligands was then interrogated by adding growth factors that induced spreading in combination with cucurbitacin-I, a selective JAK2/STAT3 inhibitor, ab142180, a selective MMP9 inhibitor, or verteporfin, a YAP inhibitor. The effect of these stimulants was inhibited by both cucurbitacin-I and ab142180 yet not by verteporfin, indicating that the AAM-derived factors utilize a common signaling pathway to mediate their effect on spheroid spreading: JAK2/STAT3 activation followed by MMP-9 mediated spreading. These data suggest that inhibiting singular soluble factors will not inhibit AAM-induced effects across a broad group of patients and that downstream pathways should instead be examined as potential therapeutic targets to slow metastasis in HGSOC. Citation Format: Kaitlin Fogg, Will Olson, Jamison Miller, and Pamela Kreeger. ALTERNATIVELY ACTIVATED MACROPHAGE SECRETOME MODULATES METASTATIC POTENTIAL OF OVARIAN CANCER CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP24.