February 2011
Volume 52, Issue 2
Free
Anatomy and Pathology/Oncology  |   February 2011
Detection of M2-Macrophages in Uveal Melanoma and Relation with Survival
Author Affiliations & Notes
  • Inge H. G. Bronkhorst
    From the Departments of Ophthalmology,
  • Long V. Ly
    From the Departments of Ophthalmology,
  • Ekaterina S. Jordanova
    Pathology, and
  • Johannes Vrolijk
    Molecular Cell Biology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
  • Mieke Versluis
    From the Departments of Ophthalmology,
  • Gregorius P. M. Luyten
    From the Departments of Ophthalmology,
  • Martine J. Jager
    From the Departments of Ophthalmology,
  • Corresponding author: Martine J. Jager, Department of Ophthalmology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands; m.j.jager@lumc.nl
Investigative Ophthalmology & Visual Science February 2011, Vol.52, 643-650. doi:10.1167/iovs.10-5979
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      Inge H. G. Bronkhorst, Long V. Ly, Ekaterina S. Jordanova, Johannes Vrolijk, Mieke Versluis, Gregorius P. M. Luyten, Martine J. Jager; Detection of M2-Macrophages in Uveal Melanoma and Relation with Survival. Invest. Ophthalmol. Vis. Sci. 2011;52(2):643-650. doi: 10.1167/iovs.10-5979.

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      © 2015 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: The presence of a high number of infiltrating macrophages in uveal melanoma is associated with a bad prognosis. However, there are several known types of macrophages, of which the M2 is considered to be proangiogenic and tumor-promoting. This study was conducted to determine whether the tumor-infiltrating macrophages in uveal melanoma are of this M2 subtype.

Methods.: Macrophages were identified in sections from 43 uveal melanomas by immunofluorescence histochemistry, using monoclonal antibodies directed against CD68 and CD163. The immunopositive cell density was measured visually and with a confocal microscope and calculated per square millimeter. Results were compared with clinical and tumor characteristics.

Results.: Infiltrating macrophages in uveal melanoma were predominantly CD68+CD163+, thus of the M2 phenotype. The density of CD68+, CD163+, and CD68+CD163+ cells was significantly increased in uveal melanomas with monosomy 3 compared with cases with disomy of chromosome 3 and was associated with ciliary body involvement. High CD68+CD163+ staining was associated with an increased microvascular density. Survival was significantly better among patients with low CD68+ and CD68+CD163+ staining.

Conclusions.: The main type of macrophage present in uveal melanoma was the M2 type. Tumors with monosomy of chromosome 3 contained a higher number of M2-macrophages than tumors with disomy of chromosome 3. Infiltration of M2-type macrophages gives a worse prognosis for survival. As M2-type macrophages are proangiogenic, a high density of these cells may contribute to the previously noticed positive association between the density of CD68+ macrophages and blood vessels.

Uveal melanoma is the most frequently occurring primary tumor in the eye, with an estimated incidence of seven cases per million per year in the Western world. 1,2 After treatment of the primary tumor, approximately 40% of patients will develop liver metastases, even up to 15 years after diagnosis, which suggests the presence of occult micrometastases at the time of primary treatment of the uveal melanoma. 3 Several prognostic factors have been described for this type of cancer, such as large tumor size, involvement of the ciliary body, increased age, extrascleral extension, monosomy of chromosome 3, and the presence of a high number of macrophages. 4 7 A high density of infiltrating macrophages and lymphocytes, increased HLA class I and II expression, and high microvascular density are components of the inflammatory phenotype that is associated with monosomy of chromosome 3. 6,8  
Mature tissue macrophages are capable of phagocytosis, degradation of self and foreign materials, establishment of cell–cell interactions, and the production of inflammatory mediators. 9 Depending on the type of tumor, macrophages are often associated with increased malignancy because of their capacity to enhance tumor growth by promoting tumor cell proliferation, metastasis, and angiogenesis. 10,11 Different types of macrophages have been identified: The classically activated macrophage can stimulate immune responses and has antibacterial and antiangiogenic functions. The alternatively activated macrophage, on the contrary, displays anti-inflammatory and proangiogenic functions. These two types of macrophages have been named M1- and M2-type macrophages, respectively. 12,13 Several studies have shown that tumor-associated macrophages (TAMs) exhibit predominantly the anti-inflammatory M2 phenotype and that these M2-macrophages highly express CD163. 14 16  
We hypothesize that the tumor-supportive functions of M2-macrophages can explain the relation between poor survival and macrophage density in uveal melanoma. A better understanding of the molecular and cellular immunology of uveal melanoma is important, because it can lead to the identification of novel targets for treatment of metastases. Shifting the macrophage balance from the immunosuppressive phenotype into a tumoricidal phenotype represents an interesting therapeutic approach. We therefore studied the presence and prognostic importance of M2-macrophages in human uveal melanoma. 
Methods
Study Population
Tissue specimens were obtained from 43 patients with uveal melanoma who had undergone enucleation between 1999 and 2004 at the Leiden University Medical Center, the Netherlands. Patient data and survival were updated to December 2009 from the patients' charts and from the database of the Integral Cancer Center West, which records death data. Deaths were also obtained from the central institution for epidemiologic data (Statistics Netherlands, The Hague, The Netherlands). The mean follow-up at the time of analysis was 59 months (range, 14–121 months). During this period, 20 patients died, 16 due to metastatic disease. There were no patients with metastasis who were still alive at the end of follow-up, and no patients were lost to follow-up. All patients were informed about the potential use of their eyes for research and had signed an informed consent form. Collecting material from uveal melanoma patients for research had been agreed on by the Medical Ethics Committee of the LUMC. The research protocol adhered to the current revision of the tenets of the Declaration of Helsinki (World Medical Association Declaration of Helsinki 1964; ethical principles for medical research involving human subjects). 
Analysis of chromosome 3 status (FISH on isolated nuclei) and examination of other histologic parameters of this patient tissue was performed and described previously by Maat et al. 8 Microvascular density (MVD) was assessed with immunostaining for the CD34 epitope as described previously by Mäkitie et al. 17  
Immunohistology
Enucleated eyes were fixed in 4% neutral-buffered formalin for 48 hours. After the eyes were embedded in paraffin, 4-μm serial sections were cut and mounted on a slide. Phenotypic characterization of macrophages was performed by using immunofluorescence (IF) double staining to enable evaluation in pigmented tumors. The antigen-retrieval method used was boiling in citrate buffer (DakoCytomation, Glostrup, Denmark) for 10 minutes. After two additional washing steps with distilled water and two with phosphate-buffered saline (PBS), mouse anti-human macrophage CD68 mAb (clone 514H12; ab49777; Abcam, Cambridge, UK) was used as a marker for all macrophages, and mouse anti-human CD163 mAb (clone 10D6, Novocastra NCL-CD163, Newcastle-upon-Tyne, UK) as a marker for M2-type macrophages. The CD163 antibody has been shown to label not only mature monocytes and macrophages, but also neoplastic cells in tumors of monocyte/macrophage lineage, such as acute myelomonocytic leukemia, and is also a helpful marker in diagnosing soft tissue tumors, such as atypical fibroxanthoma. However, no immunoreactivity is shown with nonhematopoietic neoplasms. 14,15 Monoclonal antibodies were diluted in PBS/bovine serum albumin (BSA) 1%; the optimal antibody concentration was 1:50 for the CD68 mAb and 1:100 for the CD163 mAb, as determined on tonsils, which were used as the positive control. The next day, after three 5-minute washes with PBS, the sections were incubated for 60 minutes at room temperature with the secondary antibodies AlexaFluor IgG2a (488) goat-anti-mouse for CD68 and IgG1(546) goat-anti-mouse for CD163 (Invitrogen-Molecular Probes, Eugene, OR), diluted in PBS/BSA1% 1:200. The slides were washed three times with PBS in the dark for 5 minutes each time. The slides were mounted in Mowiol (Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands). In negative control experiments, the primary antibody was replaced by PBS/BSA 1%. 
Detection of Immunostaining
Pictures of the slides were obtained with a confocal laser scanning microscope (LSM510; Carl Zeiss Meditec, Jena, Germany) in a multitrack setting in which the slide is scanned multiple times, with a fixed laser–filter pair. AlexaFluor 488 was excited at 488 nm and detected with a 505- to 530-nm band-pass filter. AlexaFluor 546 was excited at 543 nm and detected with a 560- to 615-nm band-pass filter. The computerized scans presented each fluorochrome signal with an artificial color: green for AlexaFluor 488 (CD68) and red for AlexaFluor 546 (CD163). Before each scan, the microscope was focused on the maximum amount of fluorescent signal. A positive control, containing the two fluorescent labels, was scanned after the microscope was set to detect the widest range of fluorescence intensities possible. These settings were then used to scan the negative controls. Scan settings were adjusted to receive the best view of the macrophages. All pictures were 512 × 512 pixels, 8-bit depth, and stack size 368.5 × 368.5 μm. A ×25 objective was used (PH2 Plan-NEOFluar 25×/0.80 Imm Korr; Carl Zeiss Meditec, GmbH). Images were viewed and saved as merged images or as a set of two separate panels in LSM files. 
Assessment of Staining
Analysis was performed blind with respect to the clinical outcome. Ten representative high-power fields (×250 magnification) per slide were manually selected. Images of the tumor were exported as JPG files by an image-converting software program (LSM Data Server, ver. 3.2.0.70; Carl Zeiss Meditec, GmbH). Since macrophages are irregular in shape, with sometimes numerous, long dendrites, it is hard to analyze the shape by means of image analyses. For digital analysis of the sections, we used an in-house image-analysis software program, Stacks (Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands). The images with the green and red signals of the corresponding staining CD68 and CD163 were first thresholded and in the resulting binary images, the presence of the subtype of macrophages was calculated by the number of pixels expressing green, red, or both colors (yellow) (Fig. 1). Each scan represented one square optical field (area, 0.137 mm2), with a maximum count of 262,144 pixels. For verification of single- and double-positive cells, we counted 15 slides manually and compared the results with the digitalized counting. Previously, our group determined the density of infiltrating macrophages by immunohistochemical staining with CD68 only. 8 The staining was visualized by using fast red (Scytec, Logan, UT). The macrophage infiltration was scored semiquantitatively according to the classification of Mäkitie et al. 6 To verify the grade of macrophage infiltration, we compared the scores obtained visually with immunohistochemistry (IHC) with those of the IF image analysis. 
Figure 1.
 
Immunofluorescence staining of macrophages in uveal melanoma using two antibodies directed against CD68 or CD163. (A) CD68 (green); (B) CD163 (red). (C) Double-positive cells in merged images of both stains (yellow). Images were thresholded to perform digital analysis of the overlay area (D). Magnification: ×250.
Figure 1.
 
Immunofluorescence staining of macrophages in uveal melanoma using two antibodies directed against CD68 or CD163. (A) CD68 (green); (B) CD163 (red). (C) Double-positive cells in merged images of both stains (yellow). Images were thresholded to perform digital analysis of the overlay area (D). Magnification: ×250.
Statistical Analysis
All statistical analyses were performed with a statistical software program (SPSS for Windows, ver. 16.0; SPSS Inc., Chicago, IL). For the comparisons, a P < 0.05 was considered statistically significant. For categorical analysis, the first tertile value was used as a cutoff point to dichotomize the series. The χ2 test was used to test for relationships between categorical variables, and for the numerical data Student's t-test was used. ANOVA was used for analyzing a numerical variable distribution among multiple groups. Kaplan-Meier survival curves were made for survival analysis by using the log rank statistic. A multivariate Cox proportional hazards model was used to determine independent prognostic factors. 
Results
The 7th edition of AJCC-UICC (American Joint Committee on Cancer-International Union on Cancer) criteria 18,19 for T staging, including the anatomic extent of the tumor based on involvement of the ciliary body and extrascleral tissues, was used for classification in anatomic stages and prognostic groups: 3 (7%) were stage I, 9 (21%) were stage IIA, 13 (30%) were stage IIB, 15 (35%) were stage IIIA, and 3 (7%) were stage IIIB. The average age of the 20 female and 23 male patients was 60 years (range, 27–88 years; Table 1). 
Table 1.
 
Baseline Characteristics of Patients and Histological Data of Primarily Enucleated Eyes
Table 1.
 
Baseline Characteristics of Patients and Histological Data of Primarily Enucleated Eyes
Categorical Variables Baseline Data (N = 43) Associations (P)
n % CD68+ CD163+ CD68+ CD163+
Sex
    Male 23 53 0.32 0.74 0.74
    Female 20 47
Prognostic groups
    Stage I 3 7 0.011 0.09 0.18
    Stage IIA 9 21
    Stage IIB 13 30
    Stage IIIA 15 35
    Stage IIIB 3 7
Cell type
    Spindle 10 23 0.07 0.07 0.07
    Mixed+epithelioid 33 77
Ciliary body involvement
    Not present 25 58 0.001 0.001 0.001
    Present 18 42
Chromosome 3 status
    Disomy 17 40 <0.001 0.003 <0.001
    Monosomy 26 60
CD68 quantity (IHC)
    Low 14 33 <0.001 0.002 <0.001
    Moderate 16 37
    Many 13 30
Numerical Variables Baseline Data Associations (P)
Mean SD CD68+ CD163+ CD68+ CD163+
Mean age at enucleation 59.9 15.4 0.52 0.25 0.08
Mean diameter (mm) 13 3 0.004 0.023 0.023
Mean prominence (mm) 8 2 0.72 0.86 0.45
Mean MVD (CD34/mm2) 93.7 42.4 0.14 0.026 0.004
Mean CD68+ (pixels × 103 × mm−2) 12.5 7.6
Mean CD163+ 20.3 11.0
Mean CD68+ CD163+ 9.7 5.6
Distribution of CD68+ and CD163+ Cells in Uveal Melanoma
Double-IF staining, using anti-CD68 and anti-CD163 monoclonal antibodies, was performed on slides from 43 primary uveal melanomas. Macrophages were mainly distributed diffusely throughout the section, as has been shown by immunohistochemical analysis in uveal melanoma before. 6,20 In all uveal melanomas, we observed larger areas of CD163+ than of CD68+ staining, as CD163 immunoreactivity is characterized not only by granular cytoplasmic or cytoplasmic staining patterns, but also by strong membrane-staining patterns and is expressed by monocytes. Usually, coexpression of CD68 and CD163 markers was seen, with smaller areas of CD68+ staining only (Fig. 2). The presence of positive cells was measured as the total amount of staining per section. 
Figure 2.
 
Distribution of CD68 and CD163 IF staining in 43 uveal melanomas. The amount of pixels is the total of 10 scans calculated per square millimeter. Green, CD68+; red, CD163+; yellow, CD68+CD163+ double staining.
Figure 2.
 
Distribution of CD68 and CD163 IF staining in 43 uveal melanomas. The amount of pixels is the total of 10 scans calculated per square millimeter. Green, CD68+; red, CD163+; yellow, CD68+CD163+ double staining.
The number of single- and double-positive cells counted manually in 15 slides corresponded to the digitalized counting. The number of CD68+ cells by IHC was low in 14 (33%) tumors, moderate in 16 (37%), and high in 13 (30%). To make sure that the staining indeed identified macrophages, we compared scores obtained visually and by image analysis and found a good association (Fig. 3). As these corresponded significantly, CD68/CD163 double-stained cells were assumed to be M2-macrophages, thereby excluding monocytes. 
Figure 3.
 
A comparison of IHC CD68-macrophage staining according to the semiquantitative classification of Mäkitie 17 and CD68 IF staining, expressed in number of pixels, as determined by image-calculation software.
Figure 3.
 
A comparison of IHC CD68-macrophage staining according to the semiquantitative classification of Mäkitie 17 and CD68 IF staining, expressed in number of pixels, as determined by image-calculation software.
Relation with Chromosome 3 Status, MVD, and Other Histologic Parameters
The variable levels of staining as shown in Figure 2 indicate that there was quite a large variability between tumors. As immunohistochemical staining for CD68+ macrophages had previously shown an association between high density of macrophages and the presence of monosomy 3, 8 we reanalyzed the presence of this association and determined potential associations with other tumor characteristics. Using FISH analysis on isolated nuclei to identify cells with monosomy 3, we found that 17 (40%) of the uveal melanomas showed disomy for chromosome 3 and 26 (60%) showed monosomy. Tumors with monosomy 3 showed significantly more IF-CD68+ staining (P = 0.0023), CD163+ staining (P = 0.026), and CD68+CD163+ double-staining (P = 0.0049) than those in the group without monosomy 3 (Student's t-test, Fig. 4). When the positively-stained areas were divided into the first tertile for low staining and the second and third tertiles combined for high staining, as these two groups did not significantly differ, there were significant positive associations related to ciliary body involvement and largest basal diameter (Table 1). We compared MVD in patients with and without metastasis. The mean MVD was significantly higher in the group with metastasis (120/mm2) than in the group without (78/mm2; Fig. 5A). There was no significant difference in CD68+ IF staining in relation with MVD. However, the MVD was significantly higher in the high CD68+CD163+ double-stained group than in the low CD68+CD163+ double-staining group (P = 0.004; Fig. 5B). CD68+ staining was also associated with the AJCC-UICC prognostic groups; however, the minimum expected cell count in this sub-table was <1, and the χ2 results may be invalid. 
Figure 4.
 
Chromosome 3 status by FISH on isolated nuclei of uveal melanoma sections plotted against (A) CD68+ staining (P = 0.0023), (B) CD163+ staining (P = 0.025), and (C) CD163+CD68+ double staining (P = 0.0048) with IF.
Figure 4.
 
Chromosome 3 status by FISH on isolated nuclei of uveal melanoma sections plotted against (A) CD68+ staining (P = 0.0023), (B) CD163+ staining (P = 0.025), and (C) CD163+CD68+ double staining (P = 0.0048) with IF.
Figure 5.
 
Comparison of MVD. (A) MVD was significantly increased in cases with metastasis compared with those with no metastasis. (B) MVD was significantly increased in uveal melanoma with high CD68+CD163+ staining compared with uveal melanoma with a low number of CD68+CD163+ staining.
Figure 5.
 
Comparison of MVD. (A) MVD was significantly increased in cases with metastasis compared with those with no metastasis. (B) MVD was significantly increased in uveal melanoma with high CD68+CD163+ staining compared with uveal melanoma with a low number of CD68+CD163+ staining.
Univariate Survival Analysis
According to Kaplan-Meier analysis and log rank testing, low CD68+ and CD68+CD163+ staining were found to associate significantly with a better survival (P = 0.027 and P = 0.008, Figs. 6A, 6C), whereas CD163+ staining (P = 0.08; Fig. 6B) did not (Table 2). The presence of monosomy 3 was associated with decreased survival (P < 0.001), as were involvement of the ciliary body (P = 0.002) and the presence of epithelioid cells (P = 0.006). Kaplan-Meier analysis showed that AJCC-UICC staging in prognostic groups predicted a worse survival (P = 0.015); however, this was not seen in univariate Cox regression analysis. There were no events in the stage I and IIA groups. Univariate Cox regression showed significant associations between worse survival and largest basal diameter, ciliary body involvement, high MVD, and presence of monosomy 3. P values and hazard ratios (HRs) are shown in Table 2
Figure 6.
 
Kaplan-Meier curves showing melanoma-specific survival. Prognosis of mortality due to metastasis was significantly better among patients with low CD68+ staining (P = 0.027) (A) and low CD68+CD163+ staining (P = 0.008) (C), while low CD163+ staining (P = 0.081) was not (B).
Figure 6.
 
Kaplan-Meier curves showing melanoma-specific survival. Prognosis of mortality due to metastasis was significantly better among patients with low CD68+ staining (P = 0.027) (A) and low CD68+CD163+ staining (P = 0.008) (C), while low CD163+ staining (P = 0.081) was not (B).
Table 2.
 
Analysis of the Relationship between Different Patient and Tumor Characteristics and Survival, with Death Due to Metastasis as the End Point
Table 2.
 
Analysis of the Relationship between Different Patient and Tumor Characteristics and Survival, with Death Due to Metastasis as the End Point
Cox Univariate Kaplan-Meier
P HR 95% CI
Sex (male*/female) 0.60 1.30 0.49–3.49 0.598
Age at enucleation (for each year) 0.08 1.03 1.00–1.07
Prognostic groups (AJCC-UICC criteria)
    Stage I 0.015
    Stage IIA
    Stage IIB* 1
    Stage IIIA 0.15 2.35 0.73–7.53
    Stage IIIB 0.24 2.77 0.50–15.38
Largest basal diameter (for each mm) <0.01 1.39 1.11–1.70
Prominence (for each mm) 0.40 0.91 0.75–1.15
Ciliary body involvement (not present*/present) <0.01 4.80 1.64–14.00 0.002
Mixed/epithelioid cell type (not present*/present) 0.11 37.31 0.44–3184.09 0.006
Microvascular density (for each CD34/mm2) <0.01 1.02 1.01–1.03
Chromosome 3 status (disomy*/monosomy) <0.01 16.77 2.17–129.43 <0.001
CD68+ macrophages IHC
    Few* 1 0.152
    Moderate 0.15 2.40 0.73–7.89
    Many 0.86 0.87 0.19–3.92
CD68+ (IF) (low*/high staining) 0.03 5.06 1.14–22.50 0.027
CD163+ (IF) (low*/high staining) 0.10 2.95 0.83–10.52 0.081
CD68+CD163+ (IF) (low*/high staining) 0.03 5.29 1.19–23.58 0.008
Multivariate Survival Analysis
Multivariate Cox regression analysis was performed to determine which parameters were independent significant prognostic factors for survival. We analyzed a series of parameters that had all shown a significant correlation in univariate survival analysis and that included CD68+ and CD68+CD163+ staining (Table 2). This analysis showed four parameters to be significant predictors of death due to metastatic disease, which did not include macrophage staining: largest basal diameter (P = 0.015; HR = 1.52), ciliary body involvement (P = 0.011; HR = 6.87), high MVD (P = 0.008; HR = 1.03) and, the most important prognostic factor, monosomy of chromosome 3 (P = 0.012; HR = 50.80; Table 3). 
Table 3.
 
Multivariate Analysis Performed for Several Significant Prognostic Factors, According to the Cox Proportional Hazard Survival Analysis
Table 3.
 
Multivariate Analysis Performed for Several Significant Prognostic Factors, According to the Cox Proportional Hazard Survival Analysis
B P HR 95% CI
Largest basal diameter (for each mm) 0.42 0.015 1.52 1.08–2.14
Ciliary body involvement 1.92 0.011 6.87 1.55–30.52
High microvascular density (for each CD34/mm2) 0.02 0.008 1.03 1.01–1.05
Monosomy 3 3.93 0.012 50.80 2.36–1086.38
Low CD68+ staining (IF) NS
Low CD68+CD163+ (IF) NS
Discussion
Hematopoietic cells are recruited to most tumors, and the TAMs constitute a large portion of a tumor mass. 21 TAMs in various cancer tissues assist tumor growth by producing various growth factors and proangiogenic cytokines. The presence of these cells in aggressive melanomas may be an indication of a host response against the tumor or it may be indirect evidence of aggressiveness of the tumor, with a high cell turnover rate, leading to a greater demand for phagocytosing cells. Uveal melanoma can grow for a prolonged time before being recognized by the innate immune system and can escape from T-cell-mediated immune surveillance, despite sufficient HLA class I expression. 22 The presence of a high number of CD68+ macrophages as described in this article was already known to carry a bad prognosis, but the reason was unknown. 6 In this study, we determined the presence of cells that expressed CD68 as well as CD163, which are assumed to be exclusively M2-macrophages. We observed that CD68+CD163+ double staining constituted most of the CD68+ staining, thus demonstrating that the infiltrating macrophages were mainly of the tumor-promoting M2 type. 
There are only a few reports on CD163+ macrophages in solid tumors, and information on their prognostic relevance is as yet limited and controversial. For example, more CD163+ than CD68+ cells were observed in cutaneous melanoma (American Joint Cancer stage I/II) and with double IF staining on six primary melanoma slides, coexpression of both markers was seen in most of the macrophages. 23 In human ovarian tumors, most of the infiltrating TAMs were polarized into the M2 phenotype and the presence of a high number of these CD163+ macrophages was associated with a poor prognosis. 24 In pancreatic cancer the M2-polarized TAMs are associated with a poor prognosis due to accelerated lymphatic metastasis. 25 In rectal cancer, only 10 of 101 cancers carried CD163+ cells, and expression of the macrophage antigen CD163 was associated with reduced survival. 26 Also in breast cancer, the expression of CD163 was related to early distant recurrences and reduced patient survival. 27 However, other studies show the opposite: in non–small-cell lung cancer, M2-macrophages were markedly increased in patients with extended survival. 28 In colorectal cancer, CD163+ macrophages were increased in limited disease compared with advanced disease, and an increased infiltration with CD163+ macrophages indicated a good prognosis. 29 All 40 colorectal cancer samples tested contained cells positive for CD163. In a prior uveal melanoma study, a small population of infiltrating macrophages was identified in nine paraffin-embedded sections and stained strongly for both CD68 and CD163. No correlation with prognosis was made. 30  
In our study, increased CD68+CD163+ staining in uveal melanoma was found to be associated with ciliary body involvement (P = 0.001) and monosomy 3 (P < 0.001), and the absence of such staining was associated with survival. The association between a high density of macrophages and monosomy 3 is remarkable, as loss of one chromosome 3 is very strongly associated with death due to metastases. The reason is not clear, but the association between macrophage density and loss of one chromosome 3 suggests that there must be a pathophysiological mechanism in which inflammation and infiltrating macrophages are related with this genetic aberration. A possible explanation may lie in the role of CD68+CD163+ cells in the early growth and development of the tumor, as they may be involved in the angiogenic switch through perturbing the local balance of proangiogenic factors—for example, stimulating production of vascular endothelial growth factor (VEGF-A). 31 The subsequent angiogenesis can cause invasive tumor growth and metastasis. A recent study by our group showed that patients with uveal melanoma metastasis have increased VEGF levels in their serum. 32 This key role of macrophages is demonstrated in a transgenic mouse model of breast cancer, by showing that the angiogenic switch and the progression to malignancy are regulated by infiltrated macrophages in the primary mammary tumors: inhibition of the macrophages delayed the angiogenic switch and induction of macrophages promoted an early onset of the angiogenic switch. 33 Experimental studies in mice have shown that macrophages may also support intraocular tumor development. 34 As M2-type macrophages are proangiogenic, the high density of especially these cells in uveal melanoma may contribute to the increased vascular density that was previously noticed in tumors with an increased number of CD68 macrophages. 6 Tumors that exhibit higher MVD are thought to behave more aggressively than tumors with low MVD in forming metastases, probably because of the hematogenous, not lymphatic, dissemination of uveal melanoma cells. Interestingly, this hypothesis is also supported by the finding that in our study population, a high number of CD34+ vessels/mm2 was associated with decreased survival (P < 0.01) and we found an association between MVD and M2-macrophage staining (P = 0.004). Another option may be that M2-macrophages stimulate tumor growth in uveal melanoma because of their capacity to suppress cytolytic antitumor CD8+ cells. 35  
Cytokines and microbial products profoundly and differentially affect the function of mononuclear phagocytes. CD163 expression is increased during macrophage differentiation and can be induced by dexamethasone and the anti-inflammatory cytokine interleukin (IL)-10. Transforming growth factor-β, IL-4, and inflammatory mediators have the opposite effect and can suppress CD163 expression. 36 The production of cytokines by uveal melanoma cells may thus affect macrophage polarization and thus their ability to facilitate further tumor progression. We recently showed that anterior chamber fluid from eyes enucleated for uveal melanoma contains increased concentrations of many different cytokines. However, we did not see an association with a high number of macrophages. 37  
We hypothesize that tumors recruit macrophages and create a microenvironment that causes macrophages to suppress immune functions and instead adopt trophic roles found during development and repair. Macrophages are at the center of the proinvasive microenvironment and may be an important drug target for cancer therapy. Studies in our laboratory using the B16 tumor cell line in C57BL/6 mice showed tumor growth curves when cells were implanted in the anterior chamber of young and old mice. Old mice showed a polarization toward the M2-type macrophage population, and removal of these macrophages in old mice led to the prevention of tumor growth. 35 Shifting the macrophage balance from the M2 tumor-promoting phenotype into an M1 tumoricidal phenotype by immune manipulation of the host represents an interesting therapeutic approach, although we have to be careful with macrophage modulation, since in the absence of macrophages, eyes become prone to infection. 38 However, we do not know the role of M1-macrophages with regard to survival. Investigating whether M1- and M2-macrophages potentially play different roles in uveal melanoma necessitates staining for M1-macrophages. The results may provide further evidence that these macrophages represent distinct phenotypes within the uveal melanoma tissue. Unfortunately, there is no good marker for staining M1-macrophages known in humans. 
In summary, the present study shows that most of the infiltrating macrophages in uveal melanoma belong to the M2 type. Tumors with monosomy 3 contained a higher number of M2-macrophages than did tumors with disomy of chromosome 3, and a lack of infiltration of M2-type macrophages can be a protective factor for survival. As M2-type macrophages are proangiogenic, the presence of many of them may play a role in the induction of blood vessel growth in uveal melanoma. 
Supplementary Materials
Footnotes
 Supported by The Netherlands Organization for Scientific Research NWO Mozaiek Grant 017.003.059 (LVL).
Footnotes
 Disclosure: I.H.G. Bronkhorst, None; L.V. Ly, None; E.S. Jordanova, None; H. Vrolijk, None; M. Versluis, None; G.P.M. Luyten, None; M.J. Jager, None
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Figure 1.
 
Immunofluorescence staining of macrophages in uveal melanoma using two antibodies directed against CD68 or CD163. (A) CD68 (green); (B) CD163 (red). (C) Double-positive cells in merged images of both stains (yellow). Images were thresholded to perform digital analysis of the overlay area (D). Magnification: ×250.
Figure 1.
 
Immunofluorescence staining of macrophages in uveal melanoma using two antibodies directed against CD68 or CD163. (A) CD68 (green); (B) CD163 (red). (C) Double-positive cells in merged images of both stains (yellow). Images were thresholded to perform digital analysis of the overlay area (D). Magnification: ×250.
Figure 2.
 
Distribution of CD68 and CD163 IF staining in 43 uveal melanomas. The amount of pixels is the total of 10 scans calculated per square millimeter. Green, CD68+; red, CD163+; yellow, CD68+CD163+ double staining.
Figure 2.
 
Distribution of CD68 and CD163 IF staining in 43 uveal melanomas. The amount of pixels is the total of 10 scans calculated per square millimeter. Green, CD68+; red, CD163+; yellow, CD68+CD163+ double staining.
Figure 3.
 
A comparison of IHC CD68-macrophage staining according to the semiquantitative classification of Mäkitie 17 and CD68 IF staining, expressed in number of pixels, as determined by image-calculation software.
Figure 3.
 
A comparison of IHC CD68-macrophage staining according to the semiquantitative classification of Mäkitie 17 and CD68 IF staining, expressed in number of pixels, as determined by image-calculation software.
Figure 4.
 
Chromosome 3 status by FISH on isolated nuclei of uveal melanoma sections plotted against (A) CD68+ staining (P = 0.0023), (B) CD163+ staining (P = 0.025), and (C) CD163+CD68+ double staining (P = 0.0048) with IF.
Figure 4.
 
Chromosome 3 status by FISH on isolated nuclei of uveal melanoma sections plotted against (A) CD68+ staining (P = 0.0023), (B) CD163+ staining (P = 0.025), and (C) CD163+CD68+ double staining (P = 0.0048) with IF.
Figure 5.
 
Comparison of MVD. (A) MVD was significantly increased in cases with metastasis compared with those with no metastasis. (B) MVD was significantly increased in uveal melanoma with high CD68+CD163+ staining compared with uveal melanoma with a low number of CD68+CD163+ staining.
Figure 5.
 
Comparison of MVD. (A) MVD was significantly increased in cases with metastasis compared with those with no metastasis. (B) MVD was significantly increased in uveal melanoma with high CD68+CD163+ staining compared with uveal melanoma with a low number of CD68+CD163+ staining.
Figure 6.
 
Kaplan-Meier curves showing melanoma-specific survival. Prognosis of mortality due to metastasis was significantly better among patients with low CD68+ staining (P = 0.027) (A) and low CD68+CD163+ staining (P = 0.008) (C), while low CD163+ staining (P = 0.081) was not (B).
Figure 6.
 
Kaplan-Meier curves showing melanoma-specific survival. Prognosis of mortality due to metastasis was significantly better among patients with low CD68+ staining (P = 0.027) (A) and low CD68+CD163+ staining (P = 0.008) (C), while low CD163+ staining (P = 0.081) was not (B).
Table 1.
 
Baseline Characteristics of Patients and Histological Data of Primarily Enucleated Eyes
Table 1.
 
Baseline Characteristics of Patients and Histological Data of Primarily Enucleated Eyes
Categorical Variables Baseline Data (N = 43) Associations (P)
n % CD68+ CD163+ CD68+ CD163+
Sex
    Male 23 53 0.32 0.74 0.74
    Female 20 47
Prognostic groups
    Stage I 3 7 0.011 0.09 0.18
    Stage IIA 9 21
    Stage IIB 13 30
    Stage IIIA 15 35
    Stage IIIB 3 7
Cell type
    Spindle 10 23 0.07 0.07 0.07
    Mixed+epithelioid 33 77
Ciliary body involvement
    Not present 25 58 0.001 0.001 0.001
    Present 18 42
Chromosome 3 status
    Disomy 17 40 <0.001 0.003 <0.001
    Monosomy 26 60
CD68 quantity (IHC)
    Low 14 33 <0.001 0.002 <0.001
    Moderate 16 37
    Many 13 30
Numerical Variables Baseline Data Associations (P)
Mean SD CD68+ CD163+ CD68+ CD163+
Mean age at enucleation 59.9 15.4 0.52 0.25 0.08
Mean diameter (mm) 13 3 0.004 0.023 0.023
Mean prominence (mm) 8 2 0.72 0.86 0.45
Mean MVD (CD34/mm2) 93.7 42.4 0.14 0.026 0.004
Mean CD68+ (pixels × 103 × mm−2) 12.5 7.6
Mean CD163+ 20.3 11.0
Mean CD68+ CD163+ 9.7 5.6
Table 2.
 
Analysis of the Relationship between Different Patient and Tumor Characteristics and Survival, with Death Due to Metastasis as the End Point
Table 2.
 
Analysis of the Relationship between Different Patient and Tumor Characteristics and Survival, with Death Due to Metastasis as the End Point
Cox Univariate Kaplan-Meier
P HR 95% CI
Sex (male*/female) 0.60 1.30 0.49–3.49 0.598
Age at enucleation (for each year) 0.08 1.03 1.00–1.07
Prognostic groups (AJCC-UICC criteria)
    Stage I 0.015
    Stage IIA
    Stage IIB* 1
    Stage IIIA 0.15 2.35 0.73–7.53
    Stage IIIB 0.24 2.77 0.50–15.38
Largest basal diameter (for each mm) <0.01 1.39 1.11–1.70
Prominence (for each mm) 0.40 0.91 0.75–1.15
Ciliary body involvement (not present*/present) <0.01 4.80 1.64–14.00 0.002
Mixed/epithelioid cell type (not present*/present) 0.11 37.31 0.44–3184.09 0.006
Microvascular density (for each CD34/mm2) <0.01 1.02 1.01–1.03
Chromosome 3 status (disomy*/monosomy) <0.01 16.77 2.17–129.43 <0.001
CD68+ macrophages IHC
    Few* 1 0.152
    Moderate 0.15 2.40 0.73–7.89
    Many 0.86 0.87 0.19–3.92
CD68+ (IF) (low*/high staining) 0.03 5.06 1.14–22.50 0.027
CD163+ (IF) (low*/high staining) 0.10 2.95 0.83–10.52 0.081
CD68+CD163+ (IF) (low*/high staining) 0.03 5.29 1.19–23.58 0.008
Table 3.
 
Multivariate Analysis Performed for Several Significant Prognostic Factors, According to the Cox Proportional Hazard Survival Analysis
Table 3.
 
Multivariate Analysis Performed for Several Significant Prognostic Factors, According to the Cox Proportional Hazard Survival Analysis
B P HR 95% CI
Largest basal diameter (for each mm) 0.42 0.015 1.52 1.08–2.14
Ciliary body involvement 1.92 0.011 6.87 1.55–30.52
High microvascular density (for each CD34/mm2) 0.02 0.008 1.03 1.01–1.05
Monosomy 3 3.93 0.012 50.80 2.36–1086.38
Low CD68+ staining (IF) NS
Low CD68+CD163+ (IF) NS
Abstract (Dutch version)
Copyright © Association for Research in Vision and Ophthalmology
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