http://www.diva-portal.org This is the published version of a paper published in International Journal of Oncology. Citation for the original published paper (version of record): Lindsten, T., Hedbrant, A., Ramberg, A., Wijkander, J., Solterbeck, A. et al. (2017) Effect of macrophages on breast cancer cell proliferation, and on expression of hormone receptors, uPAR and HER-2 International Journal of Oncology, 51(1): 104-114 https://doi.org/10.3892/ijo.2017.3996 Access to the published version may require subscription. N.B. When citing this work, cite the original published paper. © Lindsten et al. This is an open access article distributed under the terms of Creative Commons Attribution License. Permanent link to this version: http://urn.kb.se/resolve?urn=urn:nbn:se:kau:diva-65678 104 INTERNATIONAL JOURNAL OF ONCOLOGY 51: 104-114, 2017 Effect of macrophages on breast cancer cell proliferation, and on expression of hormone receptors, uPAR and HER-2 THERÉSE LINDSTEN1, ALEXANDER HEDBRANT2, ANNA RAMBERG1,2, JONNy WIJKANDER3, ANJA Solterbeck1, Margareta ERIKSSON1,5, DICK DELBRO2 and ANN ERLANDSSON4,5 1Department of Clinical Pathology and Cytology, Central Hospital Karlstad, SE-651 88 Karlstad; 2School of Medical Sciences, Örebro University, SE-702 81 Örebro; Departments of 3Health Sciences, 4Environmental and Life Sciences/Biology, Karlstad University, SE-651 88 Karlstad; 5Department of Urology, Faculty of Medicine and Health, Örebro University, Örebro University Hospital, SE-701 82 Örebro, Sweden Received February 13, 2017; Accepted March 27, 2017 DOI: 10.3892/ijo.2017.3996 Abstract. Malignant tumors, including breast cancers, capability to decrease the tumor cell expression of ERα and PR are frequently infiltrated with innate immune cells and in vitro. In the tumor environment in vivo macrophages also tumor-associated macrophages (TAMs) represent the major contribute to an increase in tumor cell expression of uPAR and inflammatory component in stroma of many tumors. In this Ki67, suggesting that macrophages are involved in impairing study, we examined the immunoreactivity of the macrophage the prognosis for breast cancer patients. markers CD68 and CD163 as well as the hormone recep- tors estrogen receptor α (ERα), progesterone receptor (PR), Introduction estrogen receptor β1 (ERβ1), human epidermal growth factor receptor 2 (HER-2), matrix metalloproteinase 9 (MMP-9), Breast cancer, the most frequently diagnosed cancer and the urokinase-type plasminogen activator receptor (uPAR) and leading cause of cancer deaths in women worldwide (1), is a the proliferations marker Ki67 in 17 breast cancer biopsies. heterogeneous disease with different biological hallmarks, The quantitative score for CD68+ and CD163+ strongly indi- and thereby varying prognostic and therapeutic characteris- cate M2 phenotype dominance in the currently investigated tics. Tumors are classified into different subtypes based on biopsies. We found that an increasing level of macrophages the immunohistochemical expression of estrogen receptor α was negatively associated with ERα or PR, whereas a positive (ERα), progesterone receptor (PR), human epidermal growth association was observed for Ki-67 or uPAR. No significant factor receptor-2 (HER-2) and Ki67 which also provides prog- association could be seen between the level of macrophage nostic and predictive information on response to hormonal and HER-2, ERβ1 or MMP-9 expression. Effect of conditioned or targeted therapies (2). Approximately 20-30% of all breast media (CM) generated from cultured human M1 and M2 cancers overexpress HER-2 leading to an uncontrolled growth macrophage phenotypes were investigated on the proliferation of cancer cells (3). Estrogen and progesterone are steroid and expression of selected markers in the T47D breast cancer hormones that play an essential role for normal mammary cell line. We found that in contrast to the in vivo situation, gland growth and development as well as for breast cancer in particularly the CM from M1 macrophages decreased progression. Most of their effects are mediated by ERs and PR the growth and Ki67 expression in T47D, and significantly which are intracellular receptors which constitute members increased ERβ1 mRNA levels. Moreover, in accordance to the of the nuclear receptor superfamily of transcription factors. in vivo situation the CM from the macrophages decreased the Approximately 75% of malignant breast tumors are ERα posi- expression of ERα protein as well as ERα or PR mRNA. In tive and more than half of these tumors also express PR (4,5). conclusion our results show that macrophages alone have the For decades, ERα was thought to be the only ER present in mammary epithelial cells until the identification of a second estrogen receptor, ERβ, in 1996 (6). ERβ is the most widely expressed ER in normal, mammary tissue. Five different isoforms (ERβ1-ERβ5) exist in humans, though ERβ1 is Correspondence to: Dr Ann Erlandsson, Department of Environmental and Life Sciences, Karlstad University, SE-651 88 considered the only fully functional isoform. The role for Karlstad, Sweden ERβ1 and other splice variants in breast cancer is still being E-mail: [email protected] investigated and may not be consistent among different breast cancer subtypes (7,8). Key words: breast cancer, breast cancer cell line, T47D, estrogen A solid, malignant tumor consists of cancer cells within receptors, progesterone receptor, M1 macrophages, M2 macrophages, a tumor stroma of essentially fibroblasts, endothelial cells, urokinase-type plasminogen activator receptor smooth muscle cells and immune cells (9). Of the latter, macro- phages appear to play a significant role in carcinogenesis (10). Thus, the association between inflammation and cancer is lindSten et al: EFFECTS OF MACROPHAGES IN BREAST CANCER 105 well established (11,12). An inflammatory microenvironment phenotypes, respectively, may influence the proliferation and influences every hallmark of cancer e.g. cell proliferation, the expression of the markers mentioned above. invasion, angiogenesis and metastasis (9). Macrophages are innate immune cells originating from peripheral blood mono- Materials and methods cytes with important roles in normal tissue homeostasis such as primary response to pathogens, resolution of inflammation Immunohistochemistry. Breast cancer specimens being and wound healing. They also constitute the most abundant analyzed in this study were archival material stored in immune cell present in the tumor stroma and display extensive paraffin blocks, having been taken for diagnostic purpose diversity and plasticity (13-15). Within the tumor microen- prior to any treatment at the Department of Clinical Pathology vironment monocytes differentiate into tumor-associated and Cytology, Karlstad Central Hospital (Karlstad, Sweden). macrophages (TAMs), mainly due to tumor-derived chemo- The study included tumor specimen from all patients (n=19) tactic factors. TAMs are active in the progression of tumors selected for neoadjuvant therapy at Karlstad Central Hospital and may, in response to various signals, exhibit dual roles in between 2009 and 2012, two samples were excluded because the microenvironment such as facilitate tumor growth, or in of too little materials left. All samples were de-identified prior contrast, contribute to destruction of tumors (16,17). Simplified, to analysis. Serial sections of 4 µm were cut from each sample human macrophages can be classified into two phenotypically and were mounted on IHC microscope glass slides (Dako, extremes, the classically activated M1 phenotype considered Glostrup, Denmark). The sections were de-paraffinized to exhibit tumoricidal activities arising from stimulation of followed by antigen retrieval using PT-link at 97˚C for 20 min macrophages with Th1-cytokines such as interferon-γ (IFNγ) in EnVision FLEX Target Retrieval Solution (Dako). The and/or microbial products like lipopolysaccharide (LPS). This sections were incubated for 30 min with either of the following phenotype is characterized by high levels of pro-inflammatory primary antibodies: Monoclonal rabbit anti-human estrogen cytokines such as interleukin (IL)12, IL23, IL6, tumor necrosis receptor (ER)α (clone EP1, ready-to-use), monoclonal mouse factor-α (TNF-α) and reactive oxygen/nitrogen intermediates anti-human progesterone receptor (PR; clone PgR 636, ready- (ROI/RNI) (10,15). to-use), monoclonal mouse anti-human-CD68 antibody (clone On the contrary, the alternatively activated M2 macro- Kp1, ready-to-use), HercepTest™ polyclonal rabbit anti-human phage is polarized by Th2-cytokines such as IL4 and IL13, HER2, monoclonal mouse anti-human estrogen receptor β1 and releases high levels of anti-inflammatory cytokines such (clone PPG5/10, dilution 1:40), monoclonal mouse anti- as IL10 and transforming growth factor-β. In most tumors human Ki-67 (clone MIB1, ready-to-use), polyclonal rabbit the infiltrating macrophages are polarized towards the M2 anti-human MMP-9 (1:50), monoclonal mouse anti-human phenotype and show a pro-tumoral role (10,15,16). CD163 is uPAR (clone R4, 1:50) (all from Dako) and monoclonal mouse a scavenger receptor that is regarded as highly specific for anti-human-CD163 antibody (clone 10D6, 1:200, Novocastra, M2 macrophages, while CD68 is a pan-macrophage marker Leica Microsystems, Newcastle, UK). The monoclonal and stains both M1 and M2 phenotypes (18,19). Furthermore, mouse anti-human estrogen receptor β1 (clone PPG5/10, 1:40) TAMs produce growth promoting and sustaining cytokines,