Introduction
Metastasis is often explained with the ‘seed and soil’ theory. Conceptually, it implies that the cancer cell (seed) undergoes epithelial to mesenchymal transition (EMT), invades vessels, becomes a circulating tumor cell (CTC), migrates, extravasates, undergoes mesenchymal to epithelial transition, and eventually colonizes distant sites as a disseminating tumor cell (DTC). ‘Soil’ relates to tumor microenvironment elements which contribute to these processes, making the distant sites permissive to colonization by CTCs or DTCs [
1].
The immune system is a major player in the cancer cell/tumor microenvironment crosstalk. In solid tumors, 5−40 % of the tumor mass consists of tumor-associated macrophages (TAMs). Approximately 80 % of the publications in this field report an association between TAMs and poor prognosis [
2,
3]. In humans macrophage polarization is a continuum that spans two extremes from the classically activated M1 macrophages to the alternatively activated M2 macrophages. M1 macrophages derive from interferon γ (IFN-γ) or lipopolysaccharide (LPS) stimuli and secrete inflammatory cytokines (e.g., IL-6, IL-12, reactive oxygen species (ROS), reactive nitrogen species (RN) and TNF-α). The validated surface-markers of human M1 macrophages include high levels of CD14 and CD16, CD64, CD86 and HLA-DRα [
4,
5]. M2 macrophages, can be further divided into M2a, M2b and M2c macrophages. M2a macrophages arise from IL-4 or IL-13 stimuli and release matrix-remodeling cytokines. Elevated expression of CD200R and CD86 is a validated phenotypic marker of M2a macrophages [
4,
5]. M2b macrophages result from the recognition of immune complexes in combination with IL-1β or LPS stimuli and like M2a macrophages, they are involved in wound healing. The immunosuppressive M2c-macrophages are the outcome of IL-10, TGF-β (transforming growth factor β), glucocorticoids or immune complex rich environments. M2c macrophages generate further IL-10 and matrix-remodeling factors such as matrix metalloproteinases (MMPs) [
4,
5]. Elevated CD163 expression is a validated marker of M2c polarization [
5].
TAMs, a macrophage population recruited and educated by tumor cells, which are therefore exposed to IL-10, TGF-β, M-CSF (monocyte colony stimulating factor) [
6] and other immunosuppressive stimuli [
7], are more closely related to the M2 type [
8]. In the tumor microenvironment, TAMs will preferentially perform trophic and immunosuppressive rather than immune effector tasks [
3,
9,
10]. Hence, TAMs promote epithelial outgrowth and invasion, which are common features of development and cancer [
3,
9]. Wickoff et al. have shown that mammary tumors exhibit a paracrine loop between TAMs and cancer cells. TAMs express monocyte colony stimulating factor receptor (M-CSFR, also known as CSF-1R or cFMS), which binds monocyte colony stimulating factor (M-CSF, also known as CSF-1) secreted by cancer cells. Conversely, TAMs secrete epidermal growth factor (EGF) and activate the EGF receptor (EGFR) on the cancer cells. This allows co-migration of the two cell types, thus, enhancing motility and subsequent invasion of healthy surrounding tissue and intravasation [
11,
12]. Also, breast cancer cell leucocyte receptor, vascular cell adhesion molecule 1 (VCAM1) binding to TAM α4-integrin explains the increased survival of VCAM1
+ tumor cells in leucocyte-rich environments [
13].
Like their phenotype and interactions with tumor cells, the location of TAMs in relation to hypoxic areas is a key parameter controlling tumor growth. In addition to the perivascular TAMs, which take part in cancer cell invasion [
11,
12], TAMs are also recruited into hypoxic areas [
14]. Within these avascular areas TAMs alter their gene expression profile, favoring a pro-tumor M2 phenotype [
15]. This may explain why in the early stages [
16] of cancers of the lung [
17], colon [
18] and stomach [
19], the macrophages in the normoxic milieu display an M1 phenotype and are associated with good prognosis.
Immunohistopathological breast carcinoma studies with restricted numbers of samples (n = 53 and 120, respectively) reveal a gradual increase in the amount of infiltrating macrophages (CD68
+) from normal breast tissue to benign proliferative breast disease, ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma [
20,
21]. Two larger studies (n = 1,322 and 168, respectively) confirmed that CD68
+ macrophages were associated with higher tumor grade, estrogen receptor (ER) and progesterone receptor (PR) negativity, human epithelial growth factor receptor 2 (HER-2) positivity and a basal phenotype, but led to the conclusion that CD68 expression was not an independent prognostic factor [
22,
23]. Another breast cancer cohort study (n = 144), looking at total macrophage number (CD68
+) and M2 macrophages (CD163
+) found that CD163 was also associated with other prognostic markers [
24]. It showed that CD68
+ cells in the tumor stroma but not in the tumor nest were an independent prognostic factor for decreased cancer-specific survival, accounting for the localization of TAMs in the tumors more than their mere presence. Triple-negative/basal-like breast tumor stroma had more CD163
+ and CD68
+ cells and a higher proportion of CD163 relative to CD68 when compared to the stroma of luminal A tumors. This indicates a predominance of mature M2 macrophages and possibly immature myeloid-derived cells (MDCs, also CD163
+) in triple-negative disease [
24].
Several clinical studies have found an association between macrophage infiltration and angiogenesis in breast cancer [
22,
25‐
28]. However, in relation to prognosis it is unanimous that larger studies of macrophage subpopulations are needed. This study intends to fill that gap. Focusing on the expression of M1 and M2 markers in samples from a large cohort of patients with breast cancer (n = 562), we looked for possible associations with tumor progression. Additionally, by studying the ex vivo differentiation of human macrophages in the presence of breast cancer conditioned media (CM), we aimed to find possible mechanisms of TAM education. To achieve these aims, we revisited tissue microarrays from a large cohort [
29] of early human breast tumors of different subtypes, grades and aggressiveness and used different breast cancer cell lines.
Methods
Human samples
TMA samples (n = 562 out of 1,199 patients from the FinXX study, NCT00114816 [
29]) were studied retrospectively. Clinicopathological characteristics of the sub-cohort are described in Table
1. Formalin-fixed, paraffin-embedded tumor samples were used for TMA. Blocks were made using a 1.0-mm tissue cylinder through a histologically representative area of each donor tumor block. From each donor block, 2–4 cores were cut and 15 TMA blocks were prepared, each containing 61–84 tumor samples plus 2–3 liver samples as positive controls.
Table 1
Patient demographics and relevant clinical characteristics
Age, years | | | | | | | | | | |
≤50 | 213 | 95 | 113 | | 105 | 98 | | 112 | 100 | |
>50 | 349 | 182 | 161 | 0.093 | 165 | 169 | 0.602 | 168 | 175 | 0.378 |
Tumor size median | | | | | | | | | | |
≤22 mm | 283 | 144 | 132 | | 137 | 129 | | 140 | 138 | |
>22 mm | 278 | 132 | 142 | 0.348 | 132 | 138 | 0.545 | 139 | 137 | 1.000 |
N.A. | 1 | | | | | | | | | |
Nodal status | | | | | | | | | | |
pN0 | 65 | 27 | 37 | | 28 | 33 | | 37 | 28 | |
pN+ | 497 | 250 | 237 | 0.169 | 242 | 234 | 0.468 | 243 | 247 | 0.267 |
Histological type | | | | | | | | | | |
Ductal | 399 | 181 | 211 | | 171 | 213 | | 204 | 192 | |
Lobular | 110 | 66 | 43 | | 69 | 33 | | 47 | 60 | |
Other | 53 | 30 | 20 | 0.010 | 30 | 21 | <0.001 | 29 | 23 | 0.274 |
Histological grade | | | | | | | | | | |
Grade 1 | 46 | 33 | 13 | | 26 | 16 | | 20 | 25 | |
Grade 2 | 264 | 138 | 119 | | 153 | 97 | | 127 | 132 | |
Grade 3 | 250 | 106 | 140 | 0.001 | 91 | 152 | <0.001 | 131 | 118 | 0.518 |
N.A. | 2 | | | | | | | | | |
ER status | | | | | | | | | | |
Positive | 405 | 215 | 181 | | 214 | 171 | | 191 | 207 | |
Negative | 157 | 62 | 93 | 0.003 | 56 | 96 | <0.001 | 89 | 68 | 0.065 |
HER-2 status | | | | | | | | | | |
Positive | 170 | 85 | 83 | | 72 | 93 | | 93 | 77 | |
Negative | 392 | 192 | 191 | 0.920 | 198 | 174 | 0.040 | 187 | 198 | 0.183 |
Biological group | | | | | | | | | | |
ER+, HER-2− | 314 | 165 | 142 | | 173 | 123 | | 139 | 168 | |
ER+, HER2+ | 91 | 50 | 39 | | 41 | 48 | | 52 | 39 | |
ER-, HER2+ | 79 | 35 | 44 | | 31 | 45 | | 41 | 38 | |
ER-, HER2− | 78 | 27 | 49 | 0.015 | 25 | 51 | <0.001 | 48 | 30 | 0.032 |
Ki67 | | | | | | | | | | |
≤20 % | 271 | 149 | 116 | | 158 | 96 | | 124 | 140 | |
>20 % | 242 | 104 | 134 | 0.005 | 92 | 144 | <0.001 | 126 | 116 | 0.252 |
N.A. | 49 | | | | | | | | | |
Immunohistochemical analysis
Sections (4-μm) of the TMA blocks were stained using standard immunohistochemical techniques for the expression of CD68 (anti-SA2 antibody clone 3C6, Abcam, Cambridge, UK), CD163 (clone 10D6, Novocastra, Newcastle, UK) and HLA-DRα (Dako, Glostrup, Denmark) [
30,
31] (detailed information provided in Additional file
1). All the stained TMA slides were scanned using an Olympus virtual microscope equipped with Dotslide using the 10× objective (Olympus BX51, Olympus, Munich, Germany), and AxioCam camera (Zeiss, Jena, Germany). Positively stained cells were counted using Fiji equipment version 1.48s (Wayne Rasband, NIH). After color deconvolution for hematoxylin and 3,3’-Diaminobenzidine (DAB), the threshold was set for macrophage visualization. The size limit for particle analysis was carefully chosen to include only macrophages. Damaged samples were excluded from the analysis. The data were analyzed in a double-blinded fashion. The investigators were blinded to the identity and clinical pathological characteristics of each sample while analyzing/scoring the macrophage content and differentiation status. The final numbers of positive cells per marker, per sample were passed on to hypothesis-naïve investigators who performed the statistical analysis of the cohort.
Cell culture
Human breast cancer cell lines MCF-7, MDA-MB231 and T47D, obtained from American Type Culture Collection (ATCC), were grown in Roswell Park Memorial Institure (RPMI)-1640 medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10 % fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 100 IU/ml penicillin and streptomycin (Gibco, Bleiswijk, Netherlands) at 37 °C in a 5 % CO2 atmosphere. After reaching confluence, cell culture medium was changed to medium containing only 1 % FBS and kept in culture for 72 h. At the end of the culture period, the CM were collected from at least three independent cell line batches from each cell type. The CM were centrifuged for 5 minutes at 2,800 g, aliquoted and frozen at −20 °C. CM were used as 50 % supplement of the macrophage differentiation culture medium together with 10 % FBS. The cell lines were recently authenticated by STR (Short tandem repeat) profiling by a certified cell line authentication service (DDC Medical, Fisher Scientific, London, UK). Mycoplasma detection was performed on a routine basis by 4’,6-Diamidino-2-phenylindole (DAPI) staining of cultured cells.
Peripheral blood mononuclear cell (PBMC) isolation
PBMCs from five different donors were isolated by centrifugation over Ficoll gradient (Sigma-Aldrich, St Louis, MO, USA). CD14+ cells were magnetically labeled with α-CD14 microbeads and positively selected by MACS technology (Miltenyi Biotec, Cologne, Germany).
Macrophage differentiation
To obtain M1, M2a and M2c macrophages, CD14
+ monocytes were cultured in MEM (Lonza, Basel, Switzerland) supplemented with 10 % FBS (Gibco, Grand Island, NY, USA) (control, CTR), with IFN-γ (50 ng/ml; M1), or IL-4 (50 ng/ml; M2a), or IL-10 (50 ng/ml; M2c) for 5 days with replacement of half of the culture media at day 3 [
32]. To assess the effect of breast cancer cell-line-secreted factors, the same differentiation protocol was carried out in the presence or absence of 50 % CM from MDA-MB231, MCF-7 or T47D cells. For activation status experiments (ELISA), cells were treated with LPS (10 ng/ml, Sigma-Aldrich, St Louis, MO, USA) for one additional day. Unless otherwise stated, all the used cytokines were from R&D Systems (Minneapolis, MN, USA). Supernatants were collected, centrifuged for 5 minutes at 2,800 g, aliquoted and stored at −20 °C until further analysis. Cells were harvested with Accutase (Invitrogen, Paisley, UK), debris were removed by centrifugation (5 minutes at 400 g), and cells were used for flow cytometry analysis. Supernatants were used for ELISA and zymography.
Flow cytometry
Ex vivo polarized macrophages were analyzed by validated flow cytometry methods [
5], with the BD LSR II flow cytometer (BD Biosciences, Erembodegem-Dorp, Belgium). In brief, cells were washed with PBS 0.1 % BSA (Sigma-Aldrich, St Louis, MO, USA) and before staining, Fc receptors were blocked with FcR blocking reagent (BD Biosciences, Erembodegem-Dorp, Belgium): 0.2 × 10
6 cells were incubated with adequate antibody mixes and washed prior to analysis. Surface-marker expression was analyzed with flow cytometry using the following fluorochrome-labeled monoclonal antibodies: CD14-APC-Cy7 (clone61D3; eBioscience, Paris, France), CD16-PE-Cy7 (clone DJ130c; AbD Serotec, Kidlington, UK), CD64-AF488 (clone 10.1; BioLegend, San Diego, CA, USA), CD200R-PE (clone OX108; AbD Serotec, Kidlington, UK), CD163-AF647 (clone GHI/61; BD Pharmingen, Erembodegem-Dorp, Belgium), and CD86-AF488 (clone IT2.2, BD Pharmingen, Erembodegem-Dorp, Belgium). Equivalent amounts of isotype-matched control antibodies and unstained cells were included in all experiments as negative and autofluorescence controls. Data were analyzed with BD FACSDiva software, after gating on the myeloid population in the FSC/SSC plot. Values were expressed as the percent ratio of the median fluorescence intensity (MedFI) of the marker of interest over the MedFI of the unstained cells.
ELISA
LPS-activated macrophage culture supernatants were used in ELISA for quantification of h-IL-10, h-IL-8, and h-IL-6 according to the manufacturer’s instructions (R&D systems). h-M-CSF was quantified in breast cancer cell line CM (Duo set, R&D systems, Minneapolis, MN, USA).
Zymography
The potential proteolytic activity of MMPs in the supernatants of the obtained macrophages was determined by zymography as previously described [
33]. The stained polyacrylamide-gelatin gels were observed with the Image Quant RT ECL imager. Densitometry of the bands corresponding to pro-MMP-9 activity (92 kDa) was performed using Fiji equipment version 1.48s (Wayne Rasband, NIH). Presented values are the optical densities of pro-MMP-9-digested bands normalized to the total protein content of the corresponding total cell lysate compared with the density of the equivalent background area.
Statistics
TMA results were analyzed with SAS version 8.2 for Windows (SAS Institute, Cary, NC, USA) using the median values of the numbers of positive cells in the entire series as the cutoff value. Frequency tables were analyzed using the chi-square (χ
2
) test. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. The log-rank test was used to confirm the robustness of the analysis. The subgroup analyses were performed including the macrophage markers, the subgroup variable, and their interaction in the Cox model. The Mann-Whitney or Kruskal-Wallis tests were applied when suitable. All P values are two-sided and are not adjusted for multiple testing. Experimental data were expressed as median ± SD, unless otherwise indicated. The Kruskal-Wallis test followed by Dunn’s post hoc test was employed to calculate statistically significant differences between the CTR and the various conditions, using GraphPad Prism software.
Study approval
Permission to use the tissues from the FinXX study for research purposes was provided by the Finnish Ministry of Social Affairs and Health. The ethics committee at the Helsinki University Central Hospital (Helsinki, Finland) approved the FinXX study and the current study (permission HUS 35/13/03/02/2015). Ethical approval for the use of peripheral blood from healthy donors was obtained from the Nantes University Hospital Ethics Committee. Samples were obtained from the Établissement Français du Sang with informed consent (agreement reference NTS 2000–24, Avenant n°10).
Online supplemental material
A supplemental table (Additional file
1) and supplemental figures (Additional files
2,
3,
4,
5 and
6) are available online.
Conclusions
This study combines several lines of evidence for the importance of TAM polarization status in breast cancer progression. For the first time, it is clear that CD163
+ TAMs associate with other known prognostic factors like fast proliferation, poor differentiation and ER-negativity. CD163
+ TAMs may be associated with a decrease in RFS according to the multivariate Cox model. The presented ex vivo results are to our knowledge the first demonstrating the modulation of macrophage differentiation solely by breast cancer cell-secreted factors, providing evidence for the mechanisms of breast cancer macrophage education behind clinical findings. Particularly, the mesenchymal-type cell line MDA-MB231 polarizes macrophages toward a mixed M2a/M2c status. It is therefore rational to venture that the screening of TAM activation in breast cancer patients could be useful in predicting patients with a high metastatic risk. The knowledge of TAM activation status may allow the therapeutic targeting of TAMs, once TAMs targeting/modulating agents pass clinical trials and become widely available. These include bisphosphonates [
56]; M-CSF and M-CSFR inhibitors and targeting antibodies [
57], NCT01316822, NCT01444404; anti-macrophage migration inhibitory factor, NCT01765790 and L-MTP-PE, NCT00631631. There is a scarcity of therapeutic options for patients with triple-negative metastatic breast cancer, and growing resistance to the available options biased by a continuous focus on cancer cell targets, which are by nature genetically unstable and prone to mutations. Approaches such as ours fuel a necessary paradigm change, contributing to the notion that the immunological tumor microenvironment should be taken into account in the development of new multi-target cancer therapies.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SS conceived and designed the study, developed the methodology, acquired, analyzed and interpreted the data and wrote and revised the manuscript. RB acquired the flow cytometry data. ML, PK and JS provided technical support in the TMA analysis. SL and OT participated in the TMA study and performed its statistical analysis. HJ participated in the TMA study, acquiring, analyzing, and interpreting the data. DH participated in the study conception and design particularly in supervising the ex vivo macrophage differentiation studies. JuM supervised and coordinated the study. JM conceived, designed, supervised and participated in the manuscript writing and revision. All authors read, revised critically for important intellectual content and approved the final manuscript.