Growth and Metastasis of Intraocular Tumors in Aged Mice

Abstract

Purpose: Since deterioration of the immune apparatus is closely associated with cancer, we examined the effect of aging on the growth and metastasis of intraocular melanomas in mice.

Methods: Murine B16LS9 melanoma cells were transplanted into the posterior compartment of the eye (vitreous chamber) and intraocular tumor growth and development of liver metastases were evaluated in young (8-10 weeks of age) and old (>18 months of age) mice. Liver metastases were also induced by intrasplenic injection of melanoma cells. Natural killer (NK) cells from the livers of mice harboring liver metastases were evaluated in vitro for their cytolytic activity.

Results: Tumors grew more rapidly in the eyes of young mice than old mice, yet old mice developed significantly more liver metastases. Increased liver metastasis in old mice was evident even when melanoma cells were injected intrasplenically as a means of bypassing the influence of the ocular immunosuppressive environment. Increased liver metastases in old mice correlated with reduced cytolytic activity of liver NK cells. Lethally irradiated young mice reconstituted with bone marrow from old donors developed significantly more liver metastases than young mice reconstituted with bone marrow from young donors, indicating that bone marrow-derived cells were the root cause of the heightened development of metastases in old mice.

Conclusions: Aging affects the growth and metastasis of intraocular melanomas. Even though intraocular melanomas grow slower in old mice, the development of liver metastases is exacerbated and correlates with a reduction in liver NK cell activity in the old mouse.

Figure 1

Growth of intraocular melanomas in old and young mice. (A) Tumor volume in eyes of old (>18 months) and young (8–10 wks) mice 14 days after intravitreal injection of B16LS9 melanoma cells. (B) Intraocular tumor (arrow) in young mouse at day 14 and (C) in old mouse at day 14. R, retina; L, lens. This experiment was performed three times with similar results. n = 10 mice/group/experiment.

Figure 2

Growth of melanoma cells injected into the AC of T-cell–deficient mice and NK-cell–deficient mice. Growth of B16L69 melanomas in the AC of T-cell–deficient NOD-SCID mice, WT mice depleted of NK cells using anti-asialo GM1 antiserum, and untreated WT mice. There were 10 mice per group. This experiment was performed once.

Figure 3

Effect of macrophage depletion on the growth of intraocular melanomas in old and young mice. Murine B16LS9 melanoma cells were injected intravitreally on day 0. Ocular macrophages were depleted by subconjunctival injections of clodronate-containing on the day of tumor injection and every 3 to 4 days throughout the tumor observation period. Control mice were similarly treated using PBS-containing liposomes. There were 10 mice/group/experiment.

Figure 4

Liver metastases in old and young mice. (A) Number of metastases arising following intravitreal injection of B16LS9 melanoma cells or (B) following intrasplenic injection of B16LS9 melanoma cells. (C) Photograph of livers from old mice and (D) young mice injected intrasplenically with B16LS9 melanoma cells. This experiment was performed twice with similar results. There were 10 mice/group/experiment.

Figure 5

Liver NK cytotoxicity in naïve and tumor-bearing mice. (A) Natural killer cytotoxicity using liver mononuclear cells from naïve mice and Yac-1 target cells and (B) B16LS9 target cells. (C) Natural killer cytotoxicity using NK1.1⁺CD3⁻ NK cells enriched from liver mononuclear cells isolated from mice with liver metastases induced by intrasplenic injection of B16LS9 melanoma cells and tested against Yac-1 target cells and (D) tested against B16LS9 target cells. There were 10 mice/group/experiment.

Figure 6

Number of NK cells in the livers of (A) old naïve mice, (B) young naïve mice, (C) old mice with liver metastases induced by intrasplenic injection of B16LS9 melanoma cells, and (D) young mice injected intrasplenically with B16LS9 melanoma cells. (E) Number of NK cells from naïve mice and old mice and (F) young and old mice with liver metastases. This experiment was performed three times with similar results. There were five mice/group/experiment.

Figure 7

Bone marrow chimeric mice reconstituted with bone marrow (BM) from old donors display heightened liver metastases produced by intrasplenic injection of B16LS9 melanoma cell. (A) Old BM- > young recipients and (B) young BM - > young recipients and untreated young mice. (C) Untreated young mice used as a control group for comparison with young bone marrow chimera mice reconstituted with old bone marrow (old BM-> young). (D) Young mice that were either untreated (control) or were lethally irradiated and reconstituted with young bone marrow (young BM-> young). There were 20 mice in each bone marrow transplantation group and 14 mice in the control group.

Figure 8

Murine CD11b⁺Gr1⁺ MDSC populations in livers of old and young mice following intrasplenic injection of B16LS9 melanoma cells. Murine CD11b⁺ MDSCs were isolated based on their expression of the Gr1epitope. The Gr1high + low group was a combination of both high and low Gr1-expressing CD11b⁺ MDSCs. This experiment was performed three times with similar results. There were five mice/group/experiment.