Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis

Abstract

P-selectin facilitates human carcinoma metastasis in immunodeficient mice by mediating early interactions of platelets with bloodborne tumor cells via their cell surface mucins, and this process can be blocked by heparin [Borsig, L., Wong, R., Feramisco, J., Nadeau, D. R., Varki, N. M. & Varki, A. (2001) Proc. Natl. Acad. Sci. USA 98, 3352-3357]. Here we show similar findings with a murine adenocarcinoma in syngeneic immunocompetent mice but involving a different P-selectin ligand, possibly a sulfated glycolipid. Thus, metastatic spread can be facilitated by tumor cell selectin ligands other than mucins. Surprisingly, L-selectin expressed on endogenous leukocytes also facilitates metastasis in both the syngeneic and xenogeneic (T and B lymphocyte deficient) systems. PL-selectin double deficient mice show that the two selectins work synergistically. Although heparin can block both P- and L-selectin in vitro, the in vivo effect of a single heparin dose given before tumor cells seems to be completely accounted for by blockade of P-selectin function. Thus, L-selectin on neutrophils, monocytes, and/or NK cells has a role in facilitating metastasis, acting beyond the early time points wherein P-selectin mediates interactions of platelet with tumor cells.

Figure 1

L-selectin deficiency attenuates metastasis of human adenocarcinoma cells in immunodeficient mice. Mice were injected i.v. with 3–4 × 10⁵ LS180 cells and studied 6 weeks later. Human-specific Alu-PCR was conducted on genomic DNA isolated from dissected lungs and densitometrically quantified as described in Materials and Methods. The number of animals studied were 9–10 in each group. Statistical significance was determined by the Student's t test.

Figure 2

Characterization of selectin ligands on MC-38 mouse adenocarcinoma cells by flow cytometry. MC-38 cells were probed with recombinant mouse selectins and studied by flow cytometry as described in Materials and Methods. The gray areas represent control profiles with secondary Ab only. (A--C) Bold solid line represents selectin-stained cells (selectin as indicated); dashed line represents selectin staining in presence of 5 mM EDTA, and thin line represents selectin staining in presence of 30 mM EDTA. (D) L- and P-selectin staining of cells after O-sialoglycoprotease treatment as indicated. (E and F) Bold solid line represents selectin-stained cells (selectin as indicated); dashed line represents selectin-stained cells after heparinase plus chondroitinase treatment, and thin solid line represents selectin-stained cells after sialidase treatment.

Figure 3

Characterization of selectin ligands in the lipid extracts of MC-38 mouse adenocarcinoma cells. Total lipid extracts of MC-38 cells and control bovine sulfatide were coated onto ELISA plates and probed with selectins as described in Materials and Methods. (A) Selectins were incubated in absence (black bars) or presence (white bars) of 5 mM EDTA. Data shown are mean ± SD of triplicates (B) Treatment of lipid-coated ELISA with arylsulfatase before addition of P- and L-selectin as described in Materials and Methods. Wells coated with bovine sulfatide were used as a positive control. Solid bars represent selectin only; white bars represent selectin binding after arylsulfatase treatment at 37°C, and gray bars represent selectin binding after arylsulfatase treatment at 4°C. Data from one of three representative experiments is presented.

Figure 4

Effect of selectin deficiencies on the metastatic progression of MC-38 mouse adenocarcinoma cells. Mice were injected i.v. with 2–3 × 10⁵ MC-38-GFP cells and examined 22 days later. Lungs were dissected, photographed, and homogenized, and the homogenate was diluted for a fluorescence read-out. (A) Examples of dissected lungs from each mouse genotype. (B) Quantitation of metastasis by GFP fluorescence. All mice were in a syngeneic C57BL6 background. The number of animals studied were six to eight per group. Statistical significance was determined by the Bonferonni multiple compare test.

Figure 5

Effect of heparin on metastatic progression of MC-38 cells in selectin-deficient mice. Mice were i.v. injected with 100 units of heparin or saline 10 min before injection of 2–3 × 10⁵ GFP-expressing MC-38 cells, and studied 22 days later; the lungs were dissected, homogenized, and diluted for quantitation of metastasis by GFP fluorescence. (A) wt = C57BL6 wt mice; P^−/− mice in C57BL6 background, and PL^−/− mice in C57BL6 background. The number of animals studied were five to eight per group. Statistical significance was determined by the Bonferonni multiple compare test. (B) wt = C57BL wt mice and L^−/− mice in C57BL background. The number of animals studied were 8–10 per group. Statistical significance was determined by Student's t test.