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. 2017 Jul;51(1):104-114.
doi: 10.3892/ijo.2017.3996. Epub 2017 May 11.

Effect of macrophages on breast cancer cell proliferation, and on expression of hormone receptors, uPAR and HER-2

Therése Lindsten  1 , Alexander Hedbrant  2 , Anna Ramberg  1 , Jonny Wijkander  3 , Anja Solterbeck  1 , Margareta Eriksson  1 , Dick Delbro  2 , Ann Erlandsson  4
Affiliations

Effect of macrophages on breast cancer cell proliferation, and on expression of hormone receptors, uPAR and HER-2

Therése Lindsten et al. Int J Oncol. 2017 Jul.

Abstract

Malignant tumors, including breast cancers, are frequently infiltrated with innate immune cells and tumor-associated macrophages (TAMs) represent the major inflammatory component in stroma of many tumors. In this study, we examined the immunoreactivity of the macrophage markers CD68 and CD163 as well as the hormone receptors estrogen receptor α (ERα), progesterone receptor (PR), estrogen receptor β1 (ERβ1), human epidermal growth factor receptor 2 (HER-2), matrix metalloproteinase 9 (MMP‑9), urokinase-type plasminogen activator receptor (uPAR) and the proliferations marker Ki67 in 17 breast cancer biopsies. The quantitative score for CD68+ and CD163+ strongly indicate M2 phenotype dominance in the currently investigated biopsies. We found that an increasing level of macrophages was negatively associated with ERα or PR, whereas a positive association was observed for Ki-67 or uPAR. No significant association could be seen between the level of macrophage and HER-2, ERβ1 or MMP-9 expression. Effect of conditioned media (CM) generated from cultured human M1 and M2 macrophage phenotypes were investigated on the proliferation and expression of selected markers in the T47D breast cancer cell line. We found that in contrast to the in vivo situation, in particularly the CM from M1 macrophages decreased the growth and Ki67 expression in T47D, and significantly increased ERβ1 mRNA levels. Moreover, in accordance to the in vivo situation the CM from the macrophages decreased the expression of ERα protein as well as ERα or PR mRNA. In conclusion our results show that macrophages alone have the capability to decrease the tumor cell expression of ERα and PR in vitro. In the tumor environment in vivo macrophages also contribute to an increase in tumor cell expression of uPAR and Ki67, suggesting that macrophages are involved in impairing the prognosis for breast cancer patients.

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Figures

Figure 1
Figure 1
Immunohistochemical staining of CD68, CD163, ERβ1 and uPAR in human, breast cancer biopsies. Representative images of CD68 (A) score 1, (B) score 2, (C) score 3; CD163 (D) score 1, (E) score 2, (F) score 3; ERβ1 (G) 30% positive tumor cells, (H) 50% positive tumor cells (I) 100% positive tumor cells; uPAR (J) score 1, (K) score 2, (L) score 3 (×400 magnification, calibration bar is 50 µm in all micrographs).
Figure 2
Figure 2
Infiltration density of CD68+ mononuclear cells in a total of 17 cases ranged from score 1–3; n=4 cases CD68+ score 1 (low), n=6 cases CD68+ score 2 (moderate) and n=7 cases CD68+ score 3 (high) and the expression of estrogen receptor α, estrogen receptor β1, progesterone receptor and Ki67 all denoted as percentage (%) of positive breast carcinoma cells. Results are presented as mean values ± SEM. The Jonckheere-Terpstra test was used to determine significant associations between increasing levels of CD68+ cells and decreasing amount of tumor cells expressing ERα, ERβ1 or PR, or increasing amount of tumor cells expressing Ki67 (*p<0.05, **p<0.01, ***p<0.001).
Figure 3
Figure 3
Infiltration density of CD68+ mononuclear cells in a total of 17 cases ranged from score 1–3; n=4 cases CD68+ score 1 (low), n=6 cases CD68+ score 2 (moderate) (for uPAR one case with CD68 score 2 was not available) and n=7 cases CD68+ score 3 (high) (for uPAR one case with CD68 score 3 was not available). The infiltration density of CD68+ mononuclear cells and the expression of uPAR, HER-2 and MMP-9 in tumor cells all denoted as score 0–3 of positive breast carcinoma cells; 0 = negative, 1 = low, 2 = medium and 3 = high. Each case is represented by a dot and the median value of each group is indicated by a line. The Jonckheere-Terpstra test was used to determine significant associations between increasing levels of CD68+ cells and increasing tumor cell expression of uPAR, HER-2 or MMP9 (*p<0.05, **p<0.01, ***p<0.001).
Figure 4
Figure 4
(A) Effects of conditioned media (CM) from macrophages of M1 and M2 phenotype on the proliferation of T47D breast cancer cells. Results are expressed as percentage of mean value ± standard deviation. Asterisks indicate significant differences compared to the untreated control (100%) (*p<0.05, **p<0.01, ***p<0.001). (B) Immunoreactivity for the proliferation marker Ki67 in T47D breast cancer cell line. Representative examples of the effect from treatment with conditioned media (CM) from macrophages (b) of M1 or (c) M2 phenotype demonstrating a reduced Ki67 protein immunoreactivity compared with (a) untreated T47D cells (×400 magnification, calibration bar is 50 µm in all micrographs).
Figure 5
Figure 5
Relative mRNA expression of estrogen receptor α (ERα), estrogen receptor β1 (ERβ1), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), p21 and p27 in T47D breast cancer cell line treated with CM from M1 (A) and M2 (B) macrophages. Fold changes were calculated using the ΔΔCq method and results are compared with untreated control cells. Results are mean values ± SEM using CM obtained from at least four different macrophage batches from different donors. Asterisks indicate significant differences of ΔCT values between treatment and control (*p<0.05, **p<0.01, ***p<0.001).
Figure 6
Figure 6
Representative immunoreactivity for estrogen receptor α (ERα) in T47D breast cancer cell line (A) untreated T47D control cells (B) T47D cells treated with CM from M1 macrophage phenotype for 48 h (C) T47D cells treated with CM from M2 macrophage phenotype for 48 h (×400 magnification, calibration bar is 50 µm in all micrographs).
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