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Macrophage traits in cancer cells are induced by macrophage-cancer cell fusion and cannot be explained by cellular interaction
© Shabo et al. 2015
Received: 29 November 2014
Accepted: 16 November 2015
Published: 20 November 2015
Cell fusion is a natural process in normal development and tissue regeneration. Fusion between cancer cells and macrophages generates metastatic hybrids with genetic and phenotypic characteristics from both maternal cells. However, there are no clinical markers for detecting cell fusion in clinical context. Macrophage-specific antigen CD163 expression in tumor cells is reported in breast and colorectal cancers and proposed being caused by macrophages-cancer cell fusion in tumor stroma. The purpose of this study is to examine the cell fusion process as a biological explanation for macrophage phenotype in breast.
Monocytes, harvested from male blood donor, were activated to M2 macrophages and co-cultured in ThinCert transwell system with GFP-labeled MCF-7 cancer cells. MCF7/macrophage hybrids were generated by spontaneous cell fusion, isolated by fluorescence-activated cell sorting and confirmed by fluorescence microscopy, short tandem repeats analysis and flow cytometry. CD163 expression was evaluated in breast tumor samples material from 127 women by immunohistochemistry.
MCF-7/macrophage hybrids were generated spontaneously at average rate of 2 % and showed phenotypic and genetic traits from both maternal cells. CD163 expression in MCF-7 cells could not be induced by paracrine interaction with M2-activated macrophages. CD163 positive cancer cells in tumor sections grew in clonal collection and a cutoff point >25 % of positive cancer cells was significantly correlated to disease free and overall survival.
In conclusion, macrophage traits in breast cancer might be caused by cell fusion rather than explained by paracrine cellular interaction. These data provide new insights into the role of cell fusion in breast cancer and contributes to the development of clinical markers to identify cell fusion.
The theory of cell fusion in cancer states that cancer cells may produce hybrids with metastatic phenotype due to spontaneous fusion with migratory leukocytes. The hybrids acquire genetic and phenotypic characteristics from both maternal cells [1, 2]. Somatic cells acquire nuclear reprogramming and epigenetic modifications to form pluripotent hybrid cells without any changes occurring to their nuclear DNA . The direction of nuclear reprogramming is decided by the ratio of genetic material contributed by the maternal cells . Thus, cell fusion is an efficient process of rapid phenotypic and functional evolution that produces cells with new properties at a much higher rate than random mutagenesis.
Several reports present evidence that macrophages are an important partner in this process. Fusion between macrophages and cancer cells generates hybrids with increased metastatic potential [5, 6]. Powell et al. in an experimental animal model with parabiosis, showed in vivo evidence of fusion between circulating bone-marrow-derived cells (BMDCs) and tumor epithelium during tumorigenesis, demonstrating that macrophages were a cellular partner in this process . Silk et al. (2013) provided evidence that transplanted cells of the BMDCs incorporate into human intestinal epithelium through cell fusion . Circulating hybrids are also reported in colorectal and pancreatic cancer patients .
Based on cell fusion theory and the assumption that the macrophage–cancer cell fusion creates hybrids expressing phenotypic characteristics of macrophages, we reported in previous studies that the macrophage-specific marker, CD163, was expressed in breast and colorectal cancers. CD163 expression in cancer cells was significantly related to advanced tumor stages and poor survival [10, 11]. Fusion events in human cancers are difficult to detect in a clinical context. Clinically, it is difficult to confirm that CD163 expression in tumor tissue is caused by cell fusion because the genetic content of macrophages, cancer cells and any hybrids have the same origin. Further, the expression of CD163 in cancer cells could be explained by other biological processes like abnormal phenotypic expression in cancer cells and paracrine cellular interaction between cancer cells and macrophages [12, 13]. To study the clinical significance of cell fusion in breast cancer, it is important to identify specific markers for this process in clinical tumor material.
In the present study, we have designed an experimental model where the presence of macrophage phenotype in breast cancer cells is examined on the basis of the previously mentioned arguments. Here we review data that CD163 expression is caused by cell fusion and not induced by paracrine cellular interaction.
MCF-7/GFP breast cancer cell line (Cell Biolabs, INC. San Diego, USA) was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 1 % PEST, 10 % FBS, 2.5 % HEPES and 1 % L-glutamine (Gibco®, Life Technologies, USA) in a T-75 tissue culture flasks (Sigma-Aldrich Co, ST. Louis, USA) and incubated at 37 °C in humidified air 5 % CO2 atmosphere. Cell medium was changed every 2–3 days, and the cells were passaged at 95 % confluence.
Monocytes were isolated from buffy coat obtained from male healthy blood donors at the department of Transfusion Medicine, County Council of Östergötland, in Linköping, Sweden. All the blood donors had given their informed consent according to the local guidelines (University Hospital in Linköping) and the Swedish National Law on ethical review of research involving humans (2003:460: 3–4 §). The buffy coat was mixed with 70 ml NaCl, layered onto Lymphoprep (Axis-Shield, Oslo) and centrifuged at 480 g in room temperature for 40 minutes. The mononuclear cell layer was collected into new tubes and washed twice with PBS-Heparin for 5 min and centrifuged at 220 g in 4 °C. The white blood cells were seeded to T-75 tissue culture flasks with RPMI 1640 medium, supplemented with 1 U/ml penicillin, 10 μg/ml streptomycin and incubated for 1–2 h to allow monocyte adhesion. The non-adherent cells were eliminated by washing 2–3 times using PBS. The adherent monocytes were allowed to differentiate to macrophages with 40 ng/ml of macrophage colony-stimulating factor, M-CSF (Nordic Biosite, Sweden) for 5–7 days. To induce M2 macrophages, the M-CSF differentiated macrophages were stimulated with 20 ng/ml human interleukin-4, IL-4 (Nordic Biosite, Sweden) for 18–24 h.
Cell fusion and cellular interaction model
To induce spontaneous cell fusion, macrophages and GFP-labeled MCF-7 cancer cells were co-cultured in the same cell culture vial in RPMI 1640 medium during 2–3 days. The cells were seeded at a ratio of about 3–5:1 (macrophages: MCF-7). Cell fusion experiments were repeated several times, and approximately 5x105 macrophages were used in each trial. We estimated the size of the population of hybrids on the basis of the number of macrophages cultured with MCF-7 cancer cells. We did so for a number of reasons, viz. MCF-7 cancer cells proliferate rapidly, macrophages do not undergo cell division, and we assumed that a hybrid cell is generated by fusion between a macrophage and a cancer cell (Fig. 1b).
Fluorescence-activated cell sorting (FACS)
Cells were washed once with PBS and harvested with a 0.05 % trypsin-EDTA solution. Detached cells were washed with PBS and resuspended in 95 μl Cell Staining Buffer (Biolegend, San Diego, USA) at a concentration of about 5x106 cells/ml. The cell suspension was incubated on ice for 10 min with 5 μl TrueStain FcX solution (BioLegend, San Diego, USA) per 1x106 cells. Combinations of direct conjugated monoclonal anti-human CD163 (APC Anti-human CD163 (IgG1 k), clone GHI/61, con 100 μg/ml) and anti-human CD45 (PerCP/Cy5.5 anti-human CD45 (IgG1 k), clone HI30, 50 μg/ml) antibodies or their respective isotype controls (APC and PerCP/Cy5.5 mouse IgG1 k, clone MOPC-21, con 200 μg/ml) (Biolegend, San Diego, USA) were added to the cell suspension at concentrations recommended by the manufacturer and incubated at 4 °C in the dark for 30 min. The labeled cells were washed twice and diluted in 1 ml PBS and filtrated in pre-separation filter 30 μm (Miltenyi Biotech, Lund, Sweden) before flow cytometry analysis. Cells in both the ThinCert culture system and co-culture were examined initially with a Gallios flow cytometer (Beckman Coulter, Inc.) and cells were sorted with BD FACSAria™ III (BD Bioscience, USA). The cells were examined in relation to GFP, CD163 and CD45 expression. Cells were initially sorted by GFP expression (positive selection of MCF-7/GFP origin) and subsequently by CD163 and CD45 expression (positive selection of cancer cells with macrophage phenotype).
Macrophages, MCF-7 cells and hybrids (1x105 cells) were seeded on coverslips and incubated 24 h in RPMI + 10 % FBS. Cells were fixed with 4 % paraformaldehyde for 30 min at 37 °C, washed once in PBS followed by permeabilization/blocking for 30 min in 2 % BSA/0.1 % Saponin in PBS. Cells were then incubated with a mouse monoclonal α-CD163 antibody (Abcam) in PBS/0.5 % BSA for 2 h at room temperature and washed three times with PBS. A secondary antibody goat anti-mouse IgG Alexa Fluor 546 (Invitrogen) was added in PBS/0.5 % BSA for 45 min, followed by three washes with PBS. The cover slips were mounted on microscope slides in Dako fluorescence mount media containg DAPI. Fluorescence images were taken with a Zeiss Axiovert 200 M fluorescence microscope with a Zeiss Plan-APOCHROMAT 63x/1.4 oil DIC objective.
Immunostaining and expression levels of CD163 in relation to survival data
To investigate whether the proportions of CD163 positive breast cancer cells have been correlated to clinical data, we re-evaluated breast cancer specimens from 127 women, a well controlled patient material that was reported in previous studies [11, 14, 15]. Written informed consent for participation in research was obtained from participants in connection with previous studies. Ethical approval from the Regional Ethics Committee in Linköping obtained according to Swedish Biobank Law (Reference number: 2010/311–31). The patients were diagnosed and treated using conventional methods at surgical departments in southeastern Sweden. All patients were in Stage II according to the UICC, and all received adjuvant tamoxifen therapy. These specimens had previously been collected in a tissue microarray and originated from a Swedish randomized trial of 2 versus 5 years of tamoxifen treatment. Serial sections of 5 mm were cut from tissue array blocks, deparaffinized in xylene, and hydrated in a series of graded alcohols (100 %, 95 %, and 70 %). Heat-induced antigen retrieval was carried out using a water bath pretreatment in Tris Ethylenediaminetetraacetic acid (1 mM, pH 9) for 50 min before staining for CD163. Detection was carried out using the DAKO Envision system. The immunoreactivity of CD163 was characterized by granular cytoplasmic, or cytoplasmatic and membrane staining patterns. In negative control samples, the primary antibody was replaced by an isotype-antimouse immunoglobulin G1 antibody. All immunostaining was evaluated by two of the authors (HO and IS) and scored on a 5-tiered score as follows: 0 %, 1–25 %, 26–50 %, 51–75 %, and 76–100 % of the cancer cells. Macrophages and cancer cells could be distinguished on morphological basis. Macrophage nuclei were small and regular, whereas the cancer cells were enlarged and atypical with pleomorphic hypertrophic and darker nuclei. Moreover, cancer cells show a decreased cytoplasmic - nuclear ratio.
To investigate the significance of CD163 expression levels in relation to survival data, we used four different cut-off points 1–25 %, 25–50 %, 50–75 % and 57–100 % of CD163 positive cancer cells in tumor sections. The correlation of CD163 expression levels and survival rates, both disease specific survival (DSS) and distant recurrence free survival (DRFS), was estimated using Kaplan-Meier analyses and the log rank test.
STR analysis/Quantitative fluorescent PCR
Size and number of alleles from short tandem repeat (STR) analysis
323, 327, 331/3
205, 209, 224/3
351, 355, 388, 392/4
355, 388 /2
475, 483, 487/3
251, 254, 262/3
293, 297, 318/3
203, 210, 214/3
375, 383, 387/3
PCR products were separated by capillary electrophoresis on an ABI 3130xl Genetic Analyzer (Applied Biosystems). For each well, 1 μl PCR product was mixed with 12 μl HiDi formamide and 0.3 μl GeneScan-500ROX size marker, followed by denaturation at 95 °C for 2 min before loading. The POP7 polymer was used in the electrophoresis, and results were analyzed using GeneMapper software version 4 (Applied Biosystems).
MCF-7/macrophage hybrid cell generation and transwell co-culture of macrophages and GFP-labeled MCF-7 cancer cells
MCF-7/macrophage hybrids were generated spontaneously after three days by co-culturing MCF-7 cancer cells with macrophages. The hybrids were defined as GFP+/CD163+/CD45+ positive cells and were separated by FACS. Cells that expressed only GFP were sorted as MCF-7 cancer cells, and GFP-negative cells were defined as macrophages. This experiment was repeated several times, and the proportion of hybrids averaged about 2 % in each experiment. Flow cytometry analysis showed that the GFP+ hybrids expressed both the macrophage-specific marker, CD163, and the leukocyte common antigen, CD45 (Fig. 2e-f).
As experimental controls MCF-7 cancer cells and macrophages co-cultured for three days in the same medium in a transwell chamber system were also analyzed by flow cytometry for GFP, CD163 and CD45 expression. Macrophages expressed both CD163 and CD45, but showed no GFP expression (Fig. 2c). The MCF-7 cancer cells clearly expressed GFP, but neither CD45 nor CD163 despite repeated transwell chamber system experiments (Fig. 2d). Even when the experiments were repeated with different durations (3, 5 and 7 days) of co-culture, the outcome remained the same (data not shown).
STR analysis of macrophages and MCF-7 cells grown in a transwell chamber system showed no shared STR loci. MCF-7 cells did not show any Y chromosome, confirming that cell fusion had not occurred between the macrophages and MCF-7 cancer cells in the transwell culture chamber system (Fig. 4b).
Demographics of the MCF-7/macrophage hybrids
Approximately 2 % of hybrids were sorted after each MCF-7/macrophage hybridization experiment. The hybrids were isolated and cultured for several weeks. We observed that the proliferation rate of the hybrids was slower than that of the parent MCF-7 cells.
Immunohistochemistry and CD163 expression levels in patient material
The genesis of CD163 expression as a macrophage trait in cancer cells reported in previous clinicohistopathological studies is unclear. It is proposed to be caused by fusion between macrophages and cancer cells. Paracrine cellular interaction in the tumor microenvironment has been suggested as an alternative explanation of macrophage traits in cancer cells. In the present study, macrophage traits in MCF-7 cancer cells are only generated by fusion with macrophages, proving they are not induced by cellular interaction between the macrophages and cancer cells.
Many reports present evidence that fusion between cancer cells and BMDCs, both in vivo and in vitro, may occur in cancer [7, 16, 17], but evidence of cell fusion and its clinical significance in human cancer remains controversial. Fusion events in human cancers are difficult to detect in a clinical context due to the lack of clinically safe tracing methods. The expression of tissue-specific markers, such as macrophage-specific antigen CD163, by cancer cells can be a reliable means of detecting the presence and significance of fusion in tumor tissue from clinical patient material. Several clinicopathological studies reported CD163 expression by cancer cells in breast tumors [11, 14], colorectal , and urinary bladder cancers . CD163 expression was associated with advanced tumor stages and poor prognosis. However, these observations in cancer cell phenotype can be caused by other mechanisms, such as intercellular genetic exchange and paracrine interaction .
Transwell experimental in vitro models are well established methods of investigating cellular interaction. Such models have been used to show that breast cancer cells alter the nature of their surrounding cells, such as fibroblasts and macrophages, to support their own progression through paracrine signaling [20, 21]. Yang et al. reported that macrophages stimulated by IL-4 regulated the invasiveness of breast cancer cells through exosome-mediated delivery of the oncogenic miR-223 . In this study, the MCF-7 cancer cells did not acquire macrophage phenotype by in vitro interaction with macrophages. MCF-7 cancer cells obtained CD163 and CD45 expression only by hybridization between MCF-7 cancer cells and macrophages. These findings indicate that CD163 expression in cancer cells can be used as a surrogate marker to detect cell fusion generally in human solid tumors, and specifically in breast cancer.
Cell fusion is a common biological process that produces viable cells and plays a major role in mammalian development and differentiation . Spontaneous cancer-stromal cell fusion is a rare, but active, stepwise process that requires the participation of both cell types . In the present study, the hybrids were generated spontaneously at an average rate of 2 % and were able to survive cultured in RPMI 1640 medium for several weeks. Thus, although the proportion of hybrids may be small in relation to the total tumor mass, the spontaneity of cell fusion, and the survival and growth of the hybrids may cause the development of derivative clones that might have important clinical implications. It has been postulated that 1 gram of tumor mass contains approximately 1 x 108 tumor cells [25, 26]. Based on this calculation, the hybrid rate of 2 % means that each gram of cancer may contain approximately 2 million hybrids. This observation is consistent with the fact that tumor size is a prognostic factors in breast cancer . Furthermore, fusion efficiency can be proportional to the malignant level of tumor cells . In this study, the proportions of CD163 positive cancer cells were not associated to survival rates. On the other hand, a cutoff point of >25 % was significantly related to both disease free and recurrence free survival. These data indicate that CD163 might be useful in clinical context as histopathological marker for detection of fusion between macrophages and tumor cells in breast cancer.
Cancer is a Darwinian adaptive system where rare genetically unstable cells thwart biological selective pressure . Clonal expansion is traditionally thought to be driven by genetic and epigenetic changes inherited by cell division . Cell-fusion-mediated nuclear reprogramming results in genetic and epigenetic alterations . The histopathological analysis in this study clearly shows that CD163-positive cancer cells are organized in a growth pattern of one or more collections. Thus, tumor cells with macrophage traits may acquire competitive advantages over the cells in tumor stroma. In the light of these observations and previous arguments, we believe that cell fusion might contribute to clonal expansion and the heterogeneity of cancer cells.
Macrophage traits, represented by CD163 and CD45 expression in cancer cells, are due to fusion between cancer cells and macrophages, and cannot be explained by cellular interaction between these cells. Cell fusion might contribute to clonal expansion of cancer and generate considerable numbers of hybrids in tumor stroma. The cutoff point >25 % of tumor cells expressing CD163 in tumor samples is correlated to DSS and DRFS rates suggesting that CD163 might be useful as macrophage/cancer cell fusion marker in clinical context.
Thanks are due to Mrs. Birgitta Frånlund for help with the immunohistochemistry and Dr. Florence Sjögren for valuable expert advice and assistance with flow cytometry. This study was sponsored by County Council of Östergötland (Sweden), National Organization of Breast Cancer Associations (Sweden) and Bengt Ihre Foundation (Swedish Surgical Society).
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