M2 macrophages induce ovarian cancer cell proliferation via a heparin binding epidermal growth factor/matrix metalloproteinase 9 intercellular feedback loop

Abstract

In ovarian cancer, a high ratio of anti-inflammatory M2 to pro-inflammatory M1 macrophages correlates with poor patient prognosis. The mechanisms driving poor tumor outcome as a result of the presence of M2 macrophages in the tumor microenvironment remain unclear and are challenging to study with current techniques. Therefore, in this study we utilized a micro-culture device previously developed by our lab to model concentrated paracrine signaling in order to address our hypothesis that interactions between M2 macrophages and ovarian cancer cells induce tumor cell proliferation. Using the micro-culture device, we determined that co-culture with M2-differentiated primary macrophages or THP-1 increased OVCA433 proliferation by 10-12%. This effect was eliminated with epidermal growth factor receptor (EGFR) or heparin-bound epidermal growth factor (HB-EGF) neutralizing antibodies and HBEGF expression in peripheral blood mononuclear cells from ovarian cancer patients was 9-fold higher than healthy individuals, suggesting a role for HB-EGF in tumor progression. However, addition of HB-EGF at levels secreted by macrophages or macrophage-conditioned media did not induce proliferation to the same extent, indicating a role for other factors in this process. Matrix metalloproteinase-9, MMP-9, which cleaves membrane-bound HB-EGF, was elevated in co-culture and its inhibition decreased proliferation. Utilizing inhibitors and siRNA against MMP9 in each population, we determined that macrophage-secreted MMP-9 released HB-EGF from macrophages, which increased MMP9 in OVCA433, resulting in a positive feedback loop to drive HB-EGF release and increase proliferation in co-culture. Identification of multi-cellular interactions such as this may provide insight into how to most effectively control ovarian cancer progression.

Keywords: bi-directional communication; co-culture; heparin binding epidermal growth factor; matrix metalloproteinase 9; paracrine signaling.

Figure 1. Overview of micro-culture device

(A) Schematic of PDMS ring construction. (B) Schematic of OVCA433 and M2 macrophages in co-culture device.

Figure 2. Paracrine signaling between M2 macrophages and OVCA433 increases tumor proliferation via EGFR

(A) Example of Click iT EdU fluorescent microscopy images from monoculture and co-culture with primary macrophages (CC: Primary Mϕ), scale bar = 100 μm. (B) Impact of M2 MDM co-culture (CC: Primary Mϕ) on OVCA433 proliferation. Shown are results from three unique donors, different symbols indicate each donor, *p < 0.05 compared to monoculture. (C) HBEGF expression in PBMCs from a separate cohort of 23 ovarian cancer patients relative to 21 healthy donors, *p < 0.05 compared to healthy donors. (D) Impact of mAb225 (10 μg/mL) on OVCA433 proliferation in monoculture and co-culture with three unique donors (CC: Primary Mϕ), different letters indicate that two conditions are significantly different, p < 0.05. (E) Impact of M2 THP-1 co-culture (CC: THP-1) on OVCA433 proliferation. Shown are results from three biological replicates, *p < 0.05 compared to monoculture. (F) Impact of mAb225 (10 μg/mL) on OVCA433 proliferation in monoculture and co-culture (CC: THP-1) from three biological replicates, different letters indicate that two conditions are significantly different, p < 0.05.

Figure 3. M2 macrophage-secreted HB-EGF drives tumor cell proliferation in co-culture

(A) Levels of EGFR ligands secreted by M2 THP-1, N/D indicates not detected. (B) Comparison of proliferation in OVCA433 monoculture co-culture with THP-1 (CC: THP-1), and co-culture with HB-EGF neutralizing antibody (10 μg/mL; CC + HB-EGF Ab) after 48 hours, different letters indicate that two conditions are significantly different, p < 0.05. (C) Impact of treatment with HB-EGF (400 pg/mL; + HB-EGF) on OVCA433 proliferation in monoculture compared to M2 THP-1 co-culture (CC: THP-1) after 48 hours, different letters indicate that two conditions are significantly different, p < 0.05.

Figure 4. M2 macrophage-secreted matrix metalloproteinases contribute to tumor cell proliferation

(A) Impact of batimastat (10 μM) on OVCA433 proliferation in co-culture with M2 THP-1 (CC: THP-1) after 48 hours, different letters indicate that two conditions are significantly different, p < 0.05. (B) Concentration of MMP-2, MMP-7, and MMP-9 in M2 THP-1 monoculture (THP-1), OVCA433 monoculture (433), and OVCA433 co-culture with M2 THP-1 (CC), *p < 0.05 compared to M2 THP-1 monoculture, N/D indicates not detected.

Figure 5. MMP-9 impacts tumor cell proliferation via control of HB-EGF secretion

(A) Impact of MMP-9 inhibitor (5 nM) on OVCA433 proliferation in co-culture with M2 THP-1 (CC: THP-1) after 48 hours, different letters indicate that two conditions are significantly different, p < 0.05. (B) Impact of MMP9 siRNA knockdown in M2 THP-1 (siMMP9) on HB-EGF release, *p < 0.05 compared to siRNA control transfected M2 THP-1 (siC). (C) Impact of 800 pg/mL of active MMP-9 on OVCA433 proliferation (+ MMP-9) compared to monoculture and THP-1 co-culture (CC: THP-1) after 48 hours, different letters indicate that two conditions are significantly different, p < 0.05.

Figure 6. MMP-9 in tumor cells is up-regulated via EGFR signaling

(A) Impact of THP-1 conditioned media on OVCA433 proliferation (+CM) compared to monoculture and THP-1 co-culture (CC: THP-1) after 48 hours, different letters indicate that two conditions are significantly different, p < 0.05. (B) MMP9 expression in M2 THP-1 and OVCA433 mono/co-cultures, *p < 0.05 compared to monoculture for each cell type. (C) Impact of 10 μg/mL mAb225 on MMP9 expression in OVCA433 in monoculture or co-culture with THP-1, different letters indicate that two conditions are significantly different, p < 0.05.

Figure 7. MMP-9 creates a feedback loop with HB-EGF to increase its bioavailability and drive tumor cell proliferation

(A) Impact of MMP9 (siMMP9) versus control (siC) siRNA knockdown in OVCA433 on OVCA433 proliferation in co-culture with M2 THP-1 (CC: THP-1). (B) Impact of MMP9 (siMMP9) versus control (siC) siRNA knockdown in M2 THP-1 on OVCA433 proliferation in co-culture (CC: THP-1). (C) Impact of MMP9 (siMMP9) versus control (siC) siRNA knockdown in OVCA433 on OVCA433 proliferation in co-culture with M2 MDMs (CC: Primary Mϕ). Different letters indicate that two conditions are significantly different p < 0.05. (D) Schematic of proposed HB-EGF feedback loop between tumor cells and M2 macrophages.