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Comparative Study
. 2016 Jul;158(1):113-126.
doi: 10.1007/s10549-016-3847-3. Epub 2016 Jun 9.

Breast cancers from black women exhibit higher numbers of immunosuppressive macrophages with proliferative activity and of crown-like structures associated with lower survival compared to non-black Latinas and Caucasians

Tulay Koru-Sengul  1   2 , Ana M Santander  3 , Feng Miao  2 , Lidia G Sanchez  3 , Merce Jorda  4   2 , Stefan Glück  5   2   6 , Tan A Ince  4   2 , Mehrad Nadji  4   2 , Zhibin Chen  3   2 , Manuel L Penichet  7   8   9   10   11 , Margot P Cleary  12 , Marta Torroella-Kouri  13   14   15
Affiliations
Comparative Study

Breast cancers from black women exhibit higher numbers of immunosuppressive macrophages with proliferative activity and of crown-like structures associated with lower survival compared to non-black Latinas and Caucasians

Tulay Koru-Sengul et al. Breast Cancer Res Treat. 2016 Jul.

Abstract

Racial disparities in breast cancer incidence and outcome are a major health care challenge. Patients in the black race group more likely present with an early onset and more aggressive disease. The occurrence of high numbers of macrophages is associated with tumor progression and poor prognosis in solid malignancies. Macrophages are observed in adipose tissues surrounding dead adipocytes in "crown-like structures" (CLS). Here we investigated whether the numbers of CD163+ tumor-associated macrophages (TAMs) and/or CD163+ CLS are associated with patient survival and whether there are significant differences across blacks, non-black Latinas, and Caucasians. Our findings confirm that race is statistically significantly associated with the numbers of TAMs and CLS in breast cancer, and demonstrate that the highest numbers of CD163+ TAM/CLS are found in black breast cancer patients. Our results reveal that the density of CD206 (M2) macrophages is a significant predictor of progression-free survival univariately and is also significant after adjusting for race and for HER2, respectively. We examined whether the high numbers of TAMs detected in tumors from black women were associated with macrophage proliferation, using the Ki-67 nuclear proliferation marker. Our results reveal that TAMs actively divide when in contact with tumor cells. There is a higher ratio of proliferating macrophages in tumors from black patients. These findings suggest that interventions based on targeting TAMs may not only benefit breast cancer patients in general but also serve as an approach to remedy racial disparity resulting in better prognosis patients from minority racial groups.

Keywords: Breast cancer; Crown-like structures; Inflammation; Macrophages; Race/ethnicities.

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Figures

Fig. 1
Fig. 1
TAMs occur at significantly higher densities in breast cancers from blacks. a IHC for CD163 macrophage marker (AbD Serotec, Raleigh, NC, USA) was done in 4 μm sections from FFPE tumor blocks. Sections were deparaffinized in xylene and hydrated in series of graded alcohols (100, 95 and 75 %). Heat-induced antigen retrieval was carried out in water bath (90 °C) in the presence of antigen unmasking solution (Citric Acid Based from Vector Laboratories, Burlingame, CA, USA). Antigen retrieval was followed by one incubation step with Peroxidase blocking reagent (dilution 1:100 of Perdrogen 30 %, Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature, followed by another step of blocking serum (normal horse serum 1.5 %, Vector Laboratories, in PBS) for 20 min at RT. CD163 (1:250) was incubated for 1 h at RT. As a detection system, we used Vectastain Elite ABC kit (Biotin/Avidin System) from Vector Laboratories; slides were counterstained with hematoxylin for 30 s. Pictures (×40) show the different densities of CD163 expression across the three racial groups. b Quantitative (calculated) macrophage density was determined in one tumor section slide per tumor. Density was calculated by dividing the number of CD163+ cells observed using a ×40 lens of a Olympus BX41 microscope by the area of the visual field, calculated as πr2, where the radius of the visual field was determined using a calibration graduated slide. TAMs’ density was calculated for 20 different and randomly selected visual fields in each tissue section, and a final average was determined and reported as the TAMs’ density per each case as cells/mm2. The graph shows TAMs’ density distribution across the three racial groups studied
Fig. 2
Fig. 2
Estimated densities of TAMs and their M1/M2 activation phenotypes in breast cancers exhibit different distributions across races. IHC for CD206 (1:50, M2 macrophage marker) and CD40 (1:200, M1 macrophage marker), both from R&D, Minneapolis, MN, was done as in Fig. 1, except Antigen Unmasking Solution High pH from Vector Laboratories was used. After staining, each histological sample was assessed qualitatively for CD163, CD206, and CD40. These estimated densities were assessed by visually scanning the whole tissue slide with a ×10 lens and arbitrarily assigning 1+ (low), 2+ (medium) or 3+ (high) as follows: 1+ = 1–150 cells/mm2; 2+ = 151–300 cells/mm2, and 3+ = >300 cells/mm2. a Estimated density of CD163+ TAMs expressed in % of total patients with low, medium, or high densities per each race. b Estimated density of CD206+ (M2) expressed in % of total patients with low, medium, or high densities per each race. c Estimated density of CD40+ (M1) expressed in % of total patients with low, medium, or high densities per each race
Fig. 3
Fig. 3
TAMs in breast cancers proliferate when in contact with tumor cells. For the analysis of proliferating macrophages (double staining with Ki-67 and CD163), tumor sections were first incubated with Ki-67 (Dako, Carpinteria, CA, USA) for 180 min (1:25 dilution) at RT and developed in brown. Then, the slides were incubated with CD163 (1:250) for 30 min (RT) and developed in red. The slides were then counterstained with hematoxylin for 30 s. As a detection system, Vectastain Elite ABC and ABC-AP Mouse kits (Biotin/AvidinSystem) from Vector Laboratories were used. In this case, breast biopsies were photographed at ×20 using an Olympus BX41 microscope with an Olympus DP15 digital camera. Images were stored in JPEG format, and density was calculated by dividing the number of proliferating TAMs in one image per case by the area observed in each picture of the analyzed tissue, calculated using the reference of 50 μm divisions. Proliferating macrophages were considered the cells simultaneously expressing CD163 (red) and Ki-67 (brown) staining. a TAMs far from the tumor cells marked negative for proliferative activity (CD163+/Ki67−, see inset with amplification); b TAMs in close contact with tumor cells exhibit proliferative activity (CD163+/Ki-67+, see inset with amplification)
Fig. 4
Fig. 4
Breast cancers from BL patients show the highest numbers of proliferating TAMs. a Double staining (CD163+/Ki-67+) shows proliferative activity in the three racial groups. b Double staining (CD163+/Ki-67+) show proliferative activity with mitotic figures in the three racial groups. c Graph shows the density distribution of total CD163+ TAMs in 23 patients of the three racial groups studied. d Graph shows the density distribution of proliferating CD163+/Ki67+ TAMs in the 23 patients of the three racial groups studied. e TAMs from Black patients exhibit increased proliferating ratios as compared with the other two races
Fig. 5
Fig. 5
Crown-like structure (CLS) occur at significantly higher numbers in breast cancers from Blacks. To calculate the density of CLS, total numbers of CLS present in one slide per tumor sample were counted, and this number was divided by the tumor tissue area (approximately calculated as a rectangle). This was done for CD163, CD206, and CD40 markers in CLS. a IHC results showing CD163 + CLS in the three racial groups (see inset and amplification). b Graph shows density of CD163+ CLS in the three racial groups. c Graph shows density of CD206+ CLS in the three racial groups. d Graph shows density of CD40+ CLS in the three racial groups
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References

    1. Cancer Facts and Figures 2014. American Cancer Society; Atlanta: 2014.
    1. Porter PL, Lund MJ, Lin MG, Yuan X, Liff JM, Flagg EW, Coates RJ, Eley JW. Racial differences in the expression of cell cycle-regulatory proteins in breast carcinoma. Cancer. 2004;100(12):2533–2542. doi:10.1002/cncr.20279. - PubMed
    1. Henderson BE, Lee NH, Seewaldt V, Shen H. The influence of race and ethnicity on the biology of cancer. Nat Rev Cancer. 2012;12(9):648–653. doi:10.1038/nrc3341. - PMC - PubMed
    1. Kolonel LN, Altshuler D, Henderson BE. The multiethnic cohort study: exploring genes, lifestyle and cancer risk. Nat Rev Cancer. 2004;4(7):519–527. doi:10.1038/nrc1389. - PubMed
    1. Amend K, Hicks D, Ambrosone CB. Breast cancer in African-American women: differences in tumor biology from European-American women. Cancer Res. 2006;66(17):8327–8330. doi:10.1158/0008-5472.CAN-06-1927. - PubMed

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