Transiently entrapped circulating tumor cells interact with neutrophils to facilitate lung metastasis development

Abstract

It is unknown why only a minority of circulating tumor cells trapped in lung capillaries form metastases and involvement of immune cells remains uncertain. A novel model has been developed in this study showing that neutrophils regulate lung metastasis development through physical interaction and anchoring of circulating tumor cells to endothelium. Human melanoma cells were i.v. injected into nude mice leading to the entrapment of many cancer cells; however, 24 hours later, very few remained in the lungs. In contrast, injection of human neutrophils an hour after tumor cell injection increased cancer cell retention by approximately 3-fold. Entrapped melanoma cells produced and secreted high levels of a cytokine called interleukin-8 (IL-8), attracting neutrophils and increasing tethering beta(2) integrin expression by 75% to 100%. Intercellular adhesion molecule-1 on melanoma cells and beta(2) integrin on neutrophils interacted, promoting anchoring to vascular endothelium. Decreasing IL-8 secretion from melanoma cells lowered extracellular levels by 20% to 50%, decreased beta(2) integrin on neutrophils by approximately 50%, and reduced neutrophil-mediated extravasation by 25% to 60%, resulting in approximately 50% fewer melanoma cells being tethered to endothelium and retained in lungs. Thus, transendothelial migration and lung metastasis development decreased by approximately 50%, showing that targeting IL-8 in melanoma cells has the potential to decrease metastasis development by disrupting interaction with neutrophils.

Figure 1. Neutrophils increase number of melanoma cells retained in lungs

(A) Intravenously injected melanoma cells become transiently entrapped in lungs 30 minutes after injection (left panel). Twenty-four hours later very few remained (middle panel). Intravenous injection of neutrophils 1-hour later, increased melanoma cell retention in the lungs (right panel). Arrowheads show GFP-tagged melanoma cells (48x). (B) Melanoma cells retained in lungs was scored in the presence or absence of injected neutrophils showing a ~3-fold increase following neutrophil injection. (C) Melanoma cells migrated more frequently across an endothelial-like cell layer in a flow chamber when co-cultured with neutrophils. All data are mean ± SEM, representing at least 2 independent experiments.

Figure 2. Increased IL-8 was present in metastatic variants compared to less aggressive parental melanoma cell lines

Endogenous (A) and secreted IL-8 (B) tended to be higher in melanoma cell lines with greater metastatic potential (labeled bold; § and ‡) compared to matched less metastatic parental cells. (C) Human recombinant IL-8 (12.5-125 ng/ml) led to a corresponding rise in endogenous IL-8 expression in neutrophils. (D) Co-cultured melanoma and neutrophils led to increased IL-8 secretion. Anti-CXCR1/2 antibodies neutralized secreted IL-8 compared to negative control mouse anti-IgG. All data are mean ± SEM, representing at least 2 independent experiments.

Figure 3. IL-8 does not regulate cellular growth or tumorigenesis

SiRNA-mediated knockdown of IL-8 protein expression (A) and secretion (B) from melanoma cells. Decreasing IL-8 levels did not affect growth of cultured metastatic melanoma cells (C) or the cells’ tumorigenic potential (D). All data are mean ± SEM, representing at least 2 independent experiments.

Figure 4. Decreasing IL-8 protein levels in metastatic melanoma cells reduced neutrophil-mediated extravasation across an endothelial like cell layer under flow conditions by disrupting ICAM-1/Mac-1 binding

siRNA-mediated targeting of IL-8 in melanoma cells decreased extracellular protein present in neutrophil co-cultures (A) and reduced Mac-1 expression on neutrophils (B). Values were normalized to background Mac-1 expression on neutrophils alone. (C) Decreasing secreted IL-8 using siRNA or neutralizing the chemokine using antibodies directed against CXCR-1/2 or IL-8, decreased melanoma cell migration across an endothelial-like layer under flow conditions. All data are mean ± SEM, representing at least 2 independent experiments.

Figure 5. Decreasing IL-8 secretion from melanoma cells reduced interaction with exogenously added human neutrophils in lungs

(A) Neutrophils facilitate melanoma cell migration through an adhesion-mediated mechanism. Decreased IL-8 secretion from melanoma cells, reduced human neutrophil co-localization with melanoma cells in the lungs of nude mice (B and C). (B) Co-localized neutrophils (red; arrow heads) and melanoma cells (green; arrows) were more abundant in controls compared to melanoma cells having reduced IL-8 secretion (100x). All data are mean ± SEM, representing at least 2 independent experiments.

Figure 6. Decreasing IL-8 expression in melanoma cells reduced lung metastases formation

SiRNA-mediated targeting of IL-8 in melanoma cells, decreased development of GFP-tagged 1205 Lu (A), or UACC 903M (B) lung metastasis development in the presence of endogenous mouse neutrophils. (C and D) Injection of exogenous human neutrophils doubled rate at which melanoma lung metastases developed. Decreasing IL-8 secretion from melanoma cells reduced lung metastasis formation to control levels. All data are mean ± SEM, representing at least 2 independent experiments.