Increased circulation of galectin-3 in cancer induces secretion of metastasis-promoting cytokines from blood vascular endothelium

Abstract

Purpose: Cytokines such as interleukin (IL)-6 and granulocyte colony-stimulating factor (G-CSF) are important metastasis promoters. This study has investigated the functional significance of the increased circulation of galectin-3, a common feature in patients with cancer and in particular those with metastasis, on cytokine secretion from the blood vascular endothelium in cancer.

Experimental design: The effects of galectin-3 on secretion of cytokines from human microvascular lung endothelial cells were assessed in vitro by cytokine array and in vivo in mice. The consequences of galectin-3-induced cytokine secretion on endothelial cell behaviors were determined, and the relationship between the levels of circulating galectin-3 and cytokines in patients with colorectal cancer with and without metastasis was investigated.

Results: Galectin-3 at pathologic concentrations found in patients with cancer induces secretion of IL-6, G-CSF, sICAM-1, and granulocyte macrophage colony-stimulating factor from blood vascular endothelial cells in vitro and in mice. These cytokines autocrinely/paracrinely interact with the vascular endothelium to increase the expressions of endothelial cell surface adhesion molecules integrinα(v)β(1), E-selectin, ICAM-1, and VCAM-1, resulting in increased cancer cell-endothelial adhesion and increased endothelial cell migration and tubule formation. In patients with metastatic colon cancer, higher serum galectin-3 levels correlated significantly with increased serum G-CSF, IL-6, and sICAM1 concentrations.

Conclusion: The increased circulation of galectin-3 in patients with cancer induces secretion of several metastasis-promoting cytokines from the blood vascular endothelium that enhances endothelial cell activities in metastasis. Targeting the actions of circulating galectin-3 in patients with cancer therefore represents a promising therapeutic strategy to reduce metastasis and improve survival.

Fig 1. Galectin-3 induces endothelial secretion of soluble molecules that increase cancer cell-endothelial adhesion

Lengthy (24 hr, A) but not short (1hr, B) presence of 1μg/ml galectin-3 increases ACA19⁻ and HCT116 cell adhesion to HMVECs. Galectin-3 induces secretion of soluble molecules from endothelial (C), but not cancer (D), cells that cause cancer cell-endothelial adhesion. The 24 hr-culture media (CM) from HMVEC (C), ACA19- or HCT116 (D) cells treated with or without 1μg/ml galectin-3 under suspension were used as culture medium to assess adhesion of fresh ACA19- or HCT116 to fresh HMVEC monolayer. Data are expressed as percentage compared to BSA-treated controls (mean±SD) from 3 independent experiments, each in triplicate. *P<0.05.

Fig 2. Galectin-3 induces secretion of IL-6, G-CSF, GM-CSF and sICAM-1 from endothelial but not cancer cells

Cytokine profile in the CM of 24 hr-1μg/ml galectin-3, or BSA-treated HMVECs (A) or ACA19- cells (B). Galectin-3 (1.5μg/ml) induces dose- (C) and time- (D) dependent secretion of IL-6, G-CSF, GM-CSF and sICAM-1 from HMVECs. Data are expressed as mean ± SD of triplicate. *P<0.05, **P<0.01, ***p<0.001.

Fig 3. Galectin-3-mediated cytokine secretion increases cancer cell-endothelial adhesion

A and B: Galectin-3(1μg/ml)-mediated cytokine secretion (A) and cancer cell adhesion to HUVECs (B) is inhibited by lactose. C. Galectin-3-mediated cancer cell-endothelial adhesion is inhibited by anti-cytokine neutralizing antibodies. HMVECs were treated with 1μg/ml galectin-3 or BSA for 24 hr, the culture media was harvested and used for assessing ACA19- and HCT116 cell adhesion to fresh HMVECs with or without a combination of neutralizing antibodies against G-CSF (25ng/ml), GM-CSF (300pg/ml), IL-6 (2ng/ml) and sICAM-1 (5ng/ml). D: A combination of recombinant G-CSF (2.5ng/ml), GM-CSF (30pg/ml), IL-6 (200pg/ml) and sICAM-1 (500pg/ml) increases ACA19⁻ and HCT116 cell adhesion to HMVECs. The data are expressed as percentage compared to BSA-treated controls from 3 independent experiments, each in triplicate. P*<0.05, **p<0.01, ***p<0.001.

Fig 4. Galectin-3-induced cytokine secretion enhances expressions of endothelial cell surface adhesion molecules which are responsible for galectin-3-mediated cancer cell-endothelial adhesion

A: The presence of 1.5μg/ml galectin-3 (green) for 24 hr induces expressions of cell surface integrinα_vβ₁, E-selectin, VCAM-1 and ICAM-1 but not CD44 nor integrin α_vβ₃ compared with control 1.5μg/ml BSA-treated cells (red). B and C: Galectin-3-mediated increase of endothelial cell adhesion molecules is the consequence of galectin-3-induced cytokine secretion. HMVECs were treated without (red) or with 1.5μg/ml galectin-3 in the absence (green) or presence of a combination of neutralizing antibodies against IL-6, G-CSF, GM-CSF and ICAM-1 (purple), a combination of recombinant IL-6, G-CSF, GM-CSF or ICAM-1 (black) (B), or in the presence of each individual recombinant GM-CSF (dark red), ICAM-1 (green), G-CSF (light blue) or IL-6 (orange) (C) for 24hr before the expression of integrin α_vβ₁ on HMVECs were analyzed. D: The presence of neutralizing antibodies against integrinα_vβ₁(10μg/ml), E-selectin(10μg/ml), VCAM-1(10μg/ml), and ICAM-1 (10μg /ml) inhibits galectin-3 (1.5μg/ml)-mediated ACA19- cell adhesion to HMVECs. IgG control shown in blue.

Fig 5. Galectin-3 induces secretion of cytokines in vivo and their secretion promotes angiogenesis

Galectin-3-induced cytokine secretion promotes endothelial cell migration (A) and tubule formation (B-D). HMVECs were treated with 1.5μg/ml BSA or galectin-3 with or without lactose for 24 hr. The culture media was collected and used for subsequent assessment of fresh HMVECs migration through matrix proteins or HUVEC tubule formation, with or without the presence of a combination of antibodies against G-CSF, GM-CSF, IL-6 and sICAM-1, or a combination of recombinant G-CSF, GM-CSF, IL-6 and sICAM-1. Tubule length (B) and branch points (C) were quantified. Data are expressed as percentage compared with BSA-treated controls from 3 independent experiments, each in triplicate. Representative images are shown in D. E: Intravenous injection of galectin-3 increases serum concentrations of sICAM-1, G-CSF, GM-CSF and IL-6 cytokine in mice. P*<0.05, **p<0.01, ***p<0.001.