Impact of tumor-associated macrophages in LH(BETA)T(AG) mice on retinal tumor progression: relation to macrophage subtype

Abstract

Purpose: To determine the distribution of tumor-associated macrophages (TAMs) during retinoblastoma tumor development, examine the contribution of bone marrow-derived TAMs in retinoblastoma tumors, and evaluate the supportive role of TAMs in tumor growth in a transgenic retinoblastoma mouse model.

Methods: The time course of macrophage infiltration in transgenic retinoblastoma tumors was assessed by immunohistochemistry at different time points in tumorigenesis. The origin of TAMs in transgenic retinoblastoma tumors was determined by transplanting 10(7) bone marrow cells from green fluorescent protein (GFP)-positive 16-week-old mice into age-matched, irradiated LH(BETA)T(AG) mice via tail vein injections. Macrophage depletion was performed by subconjunctival (SC) delivery of liposomal clodronate.

Results: The density of TAMs increased from 4 to 12 weeks of age in mice with small to medium tumors (P = 0.037) and remained stable in the later stages of disease (i.e., 16 weeks old with large tumors; P = 0.20). In 16-week-old mice, 38% (2.5 +/- 3.2 cells per 400x high-power field) of TAMs were GFP-positive, bone marrow-derived macrophages. Total TAM depletion was associated with a significant decrease in the expression levels of MMP-9 (P = 0.014) and mature vessels (P < 0.001) and a nonsignificant decrease in the density of neovessels (P = 0.94). The density of M2-polarized TAMs did not change significantly after TAM depletion (P = 0.68). After M1-polarized TAM depletion, the tumor burden increased (P = 0.056).

Conclusions: This work extends understanding of the complex role that macrophages play in retinoblastoma. Macrophage modulation in the tumor microenvironment is a critical factor in retinoblastoma tumor progression.

Figure 1.

TAMs were recruited or activated early in the disease course. They were detected in the tumor microenvironment of the LH_BETAT_AG mice as early as 4 weeks of age. At this time point, the density of TAMs was significantly higher in the retinal regions composed of microscopic tumor foci (rosettelike) than in the normal retina (P = 0.013). The density of TAMs showed a steady statistically significant increase in size from 4 to 12 weeks of age, with small to medium tumors, respectively (P = 0.037), and a continued steady increase in size in the later stages of the disease (i.e., from 12 to 16 weeks of age, with large tumors present at 16 weeks of age; P = 0.20): 1.3%, 5.4%, and 3.7% of the tumor area was composed of macrophages in the small, medium, and large tumors, respectively.

Figure 2.

Levels of MMP-9 activity at different time points of tumor development in LH_BETAT_AG retinal tumors. MMP-9 activity levels significantly increased from 4 to 16 weeks of age (P = 0.042). MMP-9 activity levels did not significantly differ in the LH_BETAT_AG retinal tumors from that in the wild-type, negative control tumors at 4 weeks of age (i.e., small-size tumors; P = 0.33); however, these levels significantly increased in the LH_BETAT_AG retinal tumors when compared with the wild-type, negative control tumors at 8 (small to medium tumors; P = 0.035), 12 age (medium tumors; P = 0.016), and 16 (large tumors; P = 0.019) weeks of age.

Figure 3.

Bone marrow–derived macrophages are present in advanced LH_BETAT_AG retinal tumors. The image shows a retinal tumor from a 16-week-old LH_BETAT_AG chimera with numerous GFP-positive cells (green, top left). The tumors were stained with F4/80, to visualize microglia/macrophages (red, bottom left). Right: merged images; bottom: DAPI-stained nuclei. Magnification, ×400.

Figure 4.

A fraction of TAMs in LH_BETAT_AG retinal tumors were bone marrow–derived. Retinal tumors in 16-week-old LH_BETAT_AG mice had 8.50 ± 1.29 macrophages per 400× high-power field (hpf), irradiated mice had 6.06 ± 2.68, and chimeras had 6.45 ± 4.20. The difference in macrophage density between the untreated, nonirradiated control group and the irradiated control group is not significant (P = 0.15). GFP-positive cells in chimeras comprised 38% of the macrophage population (2.5 ± 3.2 cells per 400× hpf). Bars, SD (n = 5; n = 10 for chimeras).

Figure 5.

Macrophage depletion with clodronate liposomes. There was a significant decrease in TAMs in the group treated with clodronate-encapsulated liposomes compared with the group treated with PBS-encapsulated liposomes (P = 0.037). The difference between the PBS-treated and the nontreated control groups was not significant (P = 0.64).

Figure 6.

The tumor burden increased after macrophage depletion. There was a borderline statistically significant increase in tumor burden in the group treated with clodronate-encapsulated liposomes compared with the group treated with PBS-encapsulated liposomes (P = 0.056). There was no significant difference between the PBS and the nontreated control groups (P = 0.52).

Figure 7.

Neovessel and mature vessel density did not change after macrophage depletion. A trend toward a decrease in neovessels and mature vessels was noted after depletion of TAMs. The difference between TAM-depleted tumors and the nontreated control is statistically significant for mature vessels (P < 0.001), but not for neovessels (P = 0.94).

Figure 8.

The expression levels of MMP-9 decreased after macrophage depletion. There was a significant decrease in the density of MMP-9 in the group treated with clodronate-encapsulated liposomes compared with the group treated with PBS-encapsulated liposomes (P = 0.014).

Figure 9.

M2-polarized macrophages were present in the apex. M2-polarized macrophages were present only in the apical regions of the tumor. The density of this type of macrophage did not change after macrophage depletion (P = 0.68).