Alternatively activated macrophage-derived secretome stimulates ovarian cancer spheroid spreading through a JAK2/STAT3 pathway

Abstract

High-grade serous ovarian cancer (HGSOC) metastasizes when tumor spheroids detach from the primary tumor and re-attach throughout the peritoneal cavity. Once the cancer cells have implanted in these new sites, the development of metastatic lesions is dependent on the disaggregation of cancer cells from the spheroids and subsequent expansion across the collagenous extracellular matrix (ECM). As HGSOC progresses an increase in alternatively activated macrophages (AAMs) in the surrounding ascites fluid has been observed and AAMs have been shown to enhance tumor invasion and growth in a wide range of cancers. We hypothesized that soluble factors from AAMs in the peritoneal microenvironment promote the disaggregation of ovarian cancer spheroids across the underlying ECM. We determined that co-culture with AAMs significantly increased HGSOC spheroid spreading across a collagen matrix. Multivariate modeling identified AAM-derived factors that correlated with enhanced spread of HGSOC spheroids and experimental validation showed that each individual cell line responded to a distinct AAM-derived factor (FLT3L, leptin, or HB-EGF). Despite this ligand-level heterogeneity, we determined that the AAM-derived factors utilized a common signaling pathway to induce spheroid spreading: JAK2/STAT3 activation followed by MMP-9 mediated spreading. Furthermore, immunostaining demonstrated that FLT3, LEPR, EGFR, and pSTAT3 were upregulated in metastases in HGSOC patients, with substantial patient-to-patient heterogeneity. These results suggest that inhibiting individual soluble factors will not inhibit AAM-induced effects across a broad group of patients; instead, the downstream JAK2/STAT3/MMP-9 pathway should be examined as potential therapeutic targets to slow metastasis in ovarian cancer.

Keywords: MMP-9; Macrophages; Ovarian cancer; STAT3; Tumor microenvironment.

Figure 1

AAMs promote HGSOC spheroid spreading. (A) Representative images of HGSOC spheroids at day 0 and day 2 +/− AAMs. Scale bar = 500 µm. (B) Quantification of HGSOC spheroid spreading after two days +/− AAMs. Circles represent average values of 6 micro-culture devices per AAM donor, lines represent the average of 5 primary human monocyte donors ± SD; *p < 0.05 vs. - AAMs by paired two-tailed t-test for each cell line. The dashed line indicates fold change in spheroid area equal to one.

Figure 2

A one component PLSR model of HGSOC spheroid spreading identified soluble factors of interest. (A) Soluble factors (Z-score normalized) in the presence of AAMs. Data are an average of n=6 micro-culture devices per AAM donor. Each column within a cell line represents one AAM donor. (B) PLSR-predicted spheroid spreading compared to experimentally-observed spheroid spreading. (C) Scores plot for principal component 1 separates the HGSOC cell lines based on responsiveness to AAMs. (D) VIP>1 are filled in, and VIP>1 that were investigated further are highlighted in blue. (E) Loadings plot for principal component one showing the ligands that vary similar to spheroid spreading. Ligands with a VIP>1 are filled in and ligands that were investigated further are highlighted in blue.

Figure 3

Each HGSOC cell line responded to different AAM-derived factors. (A) HGSOC spheroid spreading after two days +/− vehicle control (Veh.) or PLSR-identified factors: 5 ng/mL IL-2, 2 ng/mL FLT3L, 20 ng/mL IL-8, 100 ng/mL leptin, or 20 ng/mL HB-EGF; n = 6 micro-culture devices, *p < 0.05 vs. Veh. by one-way ANOVA and t-test with Bonferroni correction for each cell line. (B) HGSOC spheroid spreading after two days +/− AAMs +/− inhibitor (Inh.): OVCAR3 + 40 nM CHMFL-FLT3–122, OVCA433 + 20 µg/mL mAb398, OV90 + 10 µg/mL mAb225; n = 6 micro-culture devices, *p < 0.05 vs. - AAMs and ^p < 0.05 vs. + AAMs by two-way ANOVA and t-test with Bonferroni correction for each cell line. (C) qRT-PCR analysis of FLT3, LEPR, and EGFR in response to co-culture with AAMs. Data are expressed as average ± SD, n = 3 pooled samples of 24 spheroids each, *p < 0.05 vs. -AAMs for each gene within each HGSOC cell line by two-way ANOVA and t-test with Bonferroni correction.

Figure 4

All three HGSOC cell lines utilized JAK2/STAT3 to mediate AAM-induced spheroid spreading. A - C: HGSOC spheroids were treated with vehicle control (Veh.) or stimulus (Stim.) and phosphorylated STAT3 (pSTAT3) was examined via confocal microscopy. (A) Representative images of pSTAT3 expression in HGSOC spheroids 4 hours post-treatment. Scale bar = 50 µm. (B) Expression of pSTAT3 in HGSOC spheroids at the edge (the area from the boundary of the spheroid to four nuclei in from the boundary of the spheroid) and the center (the remaining area of the spheroid). Data are expressed as average ± SD, n = 3 spheroids per HGSOC cell line, 8 images of a 40 micron z-stack were analyzed for each spheroid, *p < 0.05 vs. Veh. Center and Veh. Edge, ^p < 0.05 vs. Stim. Center. Significance within each cell line determined by two-way ANOVA and t-test with Bonferroni correction. (C) Proportion of pSTAT3 present in the nucleus vs. the cytoplasm. Data are expressed as average ± SD, n = 3 spheroids per HGSOC cell line, 8 images of a 40 micron z-stack were analyzed for each spheroid, *p < 0.05 vs. Veh. by t-test for each cell line. (D) HGSOC spheroid spreading after two days with vehicle control (Veh.), cucurbitacin I (CCI), stimulus: OVCAR3 + 2 ng/mL FLT3L, OVCA433 + 100 ng/mL leptin, OV90 + 20 ng/mL HB-EGF, or stimulus plus CCI. Data are expressed as average ± SD, n = 6 micro-culture devices; *p < 0.05 vs. - AAMs and ^p < 0.05 vs. + AAMs for each cell line by two-way ANOVA and t-test with Bonferroni correction.

Figure 5

AAM-derived factors induce MMP9-mediated spreading via JAK2/STAT3. (A) Scatter plot of gene expression in HGSOC spheroids after two days +/− AAMs. Changes greater than 2-fold were considered significant. MMP9 is emphasized for each HGSOC cell line as a larger circle. (B) HGSOC spheroid spreading after two days +/− vehicle control (Veh.), AB142180, and stimulus (OVCAR3 + 2 ng/mL FLT3L, OVCA433 + 100 ng/mL leptin, OV90 + 20 ng/mL HB-EGF, Stim). Data are expressed as average ± SD, n = 6 micro-culture devices per cell line; *p < 0.05 vs. Veh. and ^p < 0.05 vs. Stim. by for each cell line by two-way ANOVA and t-test with Bonferroni correction. (C) MMP-9 concentration in conditioned media from HGSOC spheroid spreading after two days +/− vehicle control (Veh.), cucurbitacin I (CCI), and stimulus (Stim). Data are expressed as average ± SD, n = 6 micro-culture devices per cell line; *p < 0.05 vs. Veh. and ^p < 0.05 vs. Stim. for each cell line by two-way ANOVA and t-test with Bonferroni correction. (D) Schematic of proposed mechanism where AAM-derived FLT3L, leptin, and HB-EGF activate the JAK2/STAT3 pathway, leading to MMP-9 secretion, which results in increased HGSOC spheroid spreading.

Figure 6

FLT3, LEPR, EGFR, and pSTAT3 were upregulated in HGSOC patients. (A) Immunofluorescence detection of FLT3, LEPR, EGFR, and pSTAT3 (magenta) in omental and peritoneal wall tissue from patients with either benign or HGSOC tumors. Tissue is counterstained with cytokeratin 7 (CK7, green), a cell surface marker of HGSOC and DAPI (blue). Scale bar = 100 µm. (B) Median fluorescent intensity (MFI) of FLT3, LEPR, EGFR, and pSTAT3. Data are internally normalized to the average MFI of the non-HGSOC tissue for each antibody. Data are expressed as average ± SD, n = 12 non-HGSOC, 16 HGSOC omentum, 15 HGSOC peritoneal wall; *p < 0.05 vs. non-HGSOC by two-way ANOVA and t-test with Bonferroni correction. (C) Proportion of pSTAT3 present in the nucleus vs. the cytoplasm. Data are expressed as average ± SD, n = 12 non-HGSOC, 16 HGSOC omentum, 15 HGSOC peritoneal wall; *p < 0.05 vs. non-HGSOC by two-way ANOVA and t-test with Bonferroni correction. (D) MFI of FLT3, LEPR, and EGFR for each patient (Z-score normalized). Each column represents one site from a HGSOC patient, “O” denotes omental tissue, “P” denotes peritoneal wall tissue, and different numbers represent unique patient.