ANTICANCER RESEARCH 33: 1525-1536 (2013)
Normal Mammary Fibroblasts Induce Reversion of the Malignant Phenotype in Human Primary Breast Cancer ANNA-MARIA RÖMER1, INKE LÜHR1, ANDREAS KLEIN2, ANDREAS FRIEDL3, SUSANNE SEBENS4, FRANK RÖSEL1, NORBERT ARNOLD1, ALEXANDER STRAUSS1, WALTER JONAT1 and MARET BAUER1 1Department
of Gynecology and Obstetrics, University Medical Center Schleswig-Holstein, Kiel, Germany; 2Institute of Biochemistry, Charité, Virchow Campus Clinic, Berlin, Germany; 3Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, USA; 4Institute of Experimental Medicine, Christian Albrechts University, Kiel, Germany
Abstract. Background/Aim: The tumor microenvironment plays a major role in tumor growth and progression. Its manipulation can lead to a reversion of the malignant phenotype. Here we explored the ability of normal mammary fibroblasts (HMFs) to induce reversion of the malignant phenotype of primary breast carcinoma cells (PBCs) in a three-dimensional (3D) context. Materials and Methods: PBCs were isolated from 13 primary breast carcinomas and cultured in 3D collagen-I gels as mono- or co-culture with HMFs. Results: In five co-cultures, PBCs exhibited reversion of their malignant phenotype, whereas PBCs in matched monocultures exhibited disorganized growth. Reversion, defined as the restoration of the complete baso-apical polarity axis, was confirmed with established polarity markers. Secretion of the tissue-specific glycoprotein MAM6 into the acinar lumens and deposition of basement membrane indicated functional differentiation. Gene expression analysis revealed a set of differentially regulated genes which possibly affect the reversion process. These included MAL, ELF5, MAP6, ZMYND11 and SQLE. Conclusion: These findings highlight the significant role of fibroblasts in regulating the carcinoma phenotype. The mammary gland consists of various cell types that interact in a complex network that is required for proper development. A bi-layered epithelium is composed of luminal secretory cells and basal myoepithelial cells lines, ducts and acini. The myoepithelial cells produce the basement membrane (BM) as barrier between the epithelial
Correspondence to: Maret Bauer, Department of Gynecology and Obstetrics, University Medical Center Schleswig-Holstein, ArnoldHeller-Str. 3, 24105 Kiel, Germany. E-mail:
[email protected] Key Words: Breast cancer, phenotypic reversion, microenvironment, mammary fibroblasts, primary cells.
0250-7005/2013 $2.00+.40
and stromal compartment (1). Milk ducts and acini are surrounded by the stroma comprising of a variety of different cell types including adipocytes, fibroblasts, endothelial cells, immune cells and the extracellular matrix (ECM) (2). The epithelium and its surrounding microenvironment should be viewed as the functional unit of the mammary gland (3). Abundant evidence indicates that interactions between the epithelium and the microenvironment modulate epithelial differentiation and polarity, as well as proliferation, survival, migration and invasion (1). The mammary epithelium can also undergo malignant transformation, proliferate and eventually become invasive. However, the mammary stroma contributes to both tumor promoting and permissive signals (4, 5). It is well-established that dramatic changes occur in the surrounding stroma during breast cancer development. The tumor microenvironment is characterized by an increased number of fibroblasts expressing alpha-smooth muscle actin, so-called cancerassociated fibroblasts (CAF), and fibrosis, collectively referred to as desmoplasia (6). The tumor microenvironment is one of the main regulators of tumor growth and invasiveness which can also provide protection from the human immune system attacking the cancer cells (3, 7, 8). Experimental evidence from a variety of investigators has demonstrated that it is possible to restore a normal epithelial phenotype in carcinoma cells by adjusting their microenvironment (9, 10). Furthermore, it was shown that epigenetic changes take place throughout the reversion process (11). Remarkably, these findings demonstrate that malignant behavior is reversible when invasive breast cancer cells are placed in a defined microenvironment and that chromatin organization and adjustments to the intracellular signaling milieu can dominate a mutated genome (12-14). Nevertheless, the underlying mechanism of this reversion process remains incompletely understood. The aim of this study was to investigate the influence of normal human mammary fibroblasts (HMFs) on the growth
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ANTICANCER RESEARCH 33: 1525-1536 (2013) and morphology of primary breast cancer cells (PBCs) and to assess their ability to induce reversion of the malignant phenotype of a pre-determined tumorigenic epithelium in a 3D cell culture model. By using HMFs in co-culture with PBCs we confronted the cancer cells with a normalized, nonmalignant stroma. This approach disables the adverse interactions between the altered tumor microenvironment and the cancer cells and places the PBCs in a context that mimicks normal mammary gland. Additionally, we sought to compare, at the molecular level, carcinomas that could be reverted with those that showed no signs of reversion in our experiments.
Materials and Methods Tissue samples and cell isolation. Tissues were obtained in compliance with the Helsinki Declaration with informed consent approved by the Institutional Review Board of the University Medical Center Schleswig-Holstein, Campus Kiel, Germany. PBCs were isolated from fresh surgical specimen from 13 patients with primary invasive breast carcinoma. The obtained tissue was minced into small pieces and digested in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies GmbH, Darmstadt, Germany) containing 2 mg/ml collagenase-I (BD Biosciences, Heidelberg, Germany) and 2 mg/ml hyaluronidase (SIGMAALDRICH Chemie GmbH, Steinheim, Germany) at 37˚C, with constant agitation for 2 h. Cells were centrifuged, pellets were washed with 10 ml Hank’s Balanced Salt Solution (HBSS, Life Technologies GmbH) and centrifuged again. Cells were resuspended in trypsin/EDTA (0.25%) (SIGMA-ALDRICH Chemie GmbH) and incubated at 37˚C for 10 min with constant agitation. Cells were diluted with ice-cold DMEM (Life Technologies GmbH) and filtered through 100 μm and 40 μm cell strainers (BD Biosciences, Bedford, MA USA). The flow-through was centrifuged and the pellet containing the PBCs was resuspended and grown in H14-medium (15). Contamination of primary cultures with fibroblasts was removed using differential trypsinization (16). The purity of the PBC cultures was confirmed by immunolabeling for the epithelial cell marker pancytokeratin (polyclonal rabbit, 1:100; Abcam, Berlin, Germany) and the mesenchymal marker vimentin (monoclonal mouse, 1:100, Labvision/Thermo Fisher Scientific, Kalamazoo, MI, USA). 3-Dimensional cell culture. Normal human mammary fibroblasts (HMFs) (17) labeled with green fluorescent protein were used in cocultures and cultured as described previously (15). PBCs were cultured in 3D collagen-I gels both as mono- and as coculture with HMFs. 3D cultures were prepared and maintained as described previously (15), with the following modifications: In monocultures, 0.05*106 PBC/ml were used and in co-cultures, PBCs and HMFs were used at a ratio of 1:2 (0.05*106 PBC/ml and 0.1*106 HMFs/ml). Immunofluorescence analysis and image acquisition. For the quantification of cell growth and morphology assessment, collagen gels were fixed and stained as described previously (18). For immunostaining the following primary antibodies were used:
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monoclonal mouse anti-golgin-97 (1:100; Life Technologies GmbH), monoclonal mouse anti-β4-integrin (1:300; BD Biosciences, Heidelberg, Germany ), monoclonal mouse anti-β1integrin (1:200; Labvision/Thermo Fisher), polyclonal rabbit antiβ-catenin (1:100; Labvision/Thermo Fisher), monoclonal mouse anti-carcinoma-associated antigen (MAM-6; 1:50; Hycult Biotechnology, Uden, The Netherlands), polyclonal rabbit anti-ZOI (1:25; New England Biolabs GmbH, Ipswich, MA, USA) and polyclonal rabbit anti-laminin-111 (1:1000; Abcam). Secondary antibodies were used as described previously (15). Immunostaining and immunofluorescent image acquisition was performed according to Lühr et al. (15) with modifications: For quantification of cell growth, 12 visual fields of each sample were analyzed. Cell morphology in co-culture was compared to that in monoculture to determine reversion of the malignant phenotype. Reversion was defined as the formation of polarized acini composed of a single layer of PBCs surrounding a hollow lumen. Polarity was identified by expression of golgin-97 at the apical cell pole and by basallylocalized β4-integrin. Disorganized cell clusters were defined as apolar aggregates with irregular shape (length >2-fold width) without lumen formation. Cell aggregates were enumerated and classified according to their morphology. 3D Cultures with a significant difference in the number of polarized acini in co-culture compared to monoculture were defined as reversed. A minimum of 10 visual fields containing at least 60 cell clusters were evaluated for each sample. Microarray analysis. Gene expression profiles of the original tumors used for five revertible and five non-revertible cell cultures were analyzed using the Affymetrix HG-U133 Plus 2.0 GeneChip. Those for the five non-reversible cell cultures were randomly chosen out of the collective of non-reversible breast cancer samples. RNA of breast carcinoma cells from the fresh frozen surgical specimen was extracted using the RNeasy Micro kit, according to the manufacturer’s instructions (Qiagen, Hilden, Germany) and quantity and quality of the RNA samples were determined using the Agilent RNA 6000 Nano kit (Agilent Technologies, Waldbronn, Germany). cRNA was amplified from 3 μg of total RNA using the MessageAmp™ II-Biotin Enhanced Single Round cRNA Amplification Kit (Applied Biosystems, Darmstadt, Germany). Human genome HG-U133 Plus 2.0 GeneChip arrays were then hybridized with the biotin-labeled cRNA fragments for 16 h at 45˚C. Washing steps for the chip, staining with streptavidin-phycoerythrin, signal-amplification and scanning were performed according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA, USA). Semi-quantitative real-time polymerase chain reaction (PCR). For validation of the results obtained by the microarray analysis, seven genes were chosen for RT-PCR based on their expression levels found in the microarray analysis and on biologically relevant information given in recent literature. Levels of three up-regulated E74-like factor5 (ets domain transcription factor, ELF5), Mucin-like 1 (MUCL1), Prolactin-induced protein (PIP) and four down-regulated Microtubule-associated protein-6 (MAP6), Zinc finger, MYNDdomain containing 11 (ZMYND11), Leucine-rich repeat and Ig domain containing-1 (LINGO1), Squalene epoxidase (SQLE) genes were determinated by qRT-PCR. cDNA was prepared from 1 μg RNA extracted from the tumors as described before using the QuantiTect Rev. Transcription Kit (Qiagen) according to the manufacturer’s instructions. Primers were used at 20 μmol/μl stock concentration (all
Römer et al: Phenotypic Reversion of Primary Breast Cancer Cells
Table I. Patients’ characteristics. Tissues for cell isolation were obtained from thirteen patients with primary invasive breast cancer without prior treatment. Patient ID
1 2 3 4 5 6 7 8 9 10 11 12 13
Age (years)
Histological subtype
Tumor stage (T)
Lymph nodes status (N)
Grade
ER*
81 84 71 84 45 63 45 71 61 72 73 43 48
Invasive ductal Ductulo-lobular Invasive ductal Invasive ductal Invasive ductal Invasive ductal Invasive ductal Invasive ductal Invasive lobular Neuroendocrine Invasive lobular Invasive ductal Invasive ductal
pT4b pT4b pT1c pT1b pT1c pT2 pT1b pT4b pT2 pT3 pT3 pT1c pT3
x x pN0 (0/4) pN0 (0/2) pN0 (0/24) pN1mic (1/18) pN1a (1/24) pN2a (8/10) pN0 (0/5) pN0 (0/7) pN2a (8/17) pN0 (0/4) pN3a (12/12)
G3 G2 G3 G1 G3 G2 G3 G2 G2 G2 G2 G3 G1
12 12 6 12 8 12 0 12 12 12 12 8 12
PR*
2 6 0 12 4 12 0 9 8 12 6 6 12
HER2 status
Phenotypic reversion
Negative Negative Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
+ + + + + − − − − − − − −
x, No information available; ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor-2; +, phenotypic reversion; − no phenotypic reversion. *Expression of ER and PR was assessed using the immunoreactive score of Remmele and Stegner (27).
Qiagen; reference # PIP: QT01006026; ELF5: QT00004046; MUCL1: QT00051065; ZMYND11: QT00030961; SQLE: QT00012243; MAP6: QT00034524; LINGO1: QT01027950). Quantification of mRNA was carried out using the SYBR Green method on an iCycler iQ™ Real-Time PCR Detection System (both Bio-Rad Laboratories, Inc., Hercules, CA, USA). PCR mix (25 μl) was used for the real-time PCR assay that consisted of 12.5 μl SYBR Green Super Mix (Bio-Rad Laboratories), 2.5 μl primer from each stock, 8 μl of water or 4 μl and 4 μl of MgCl2, and 2 μl of template. PCR for PIP, LINGO1, ZMYND11 and MAP6 were carried out without MgCl2, and that for MUCL1, SQLE and ELF5 were carried out with MgCl2. The thermal conditions consisted of an initial denaturation at 95˚C for 3 min, followed by 40 repeats of 95˚C for 30 sec, 64˚C for 7 sec and 72˚C for 7 sec. Then PCR was run for 1 min at 95˚C and 1 min at 64˚C followed by a cycle of 62-times 10 sec at 64˚C with an increase of set point temperature after cycle 2 by 0.5˚C in order to collect the melting curve data. The PCR reaction was put on hold by lowering the temperature to 4.0˚C. 28S-rRNA was used as housekeeping gene. To reduce variation, all experiments were determined in duplicate, and each experiment was repeated at least twice. cT Values of target genes were normalized to the cT Values of 28S-rRNA, whose expression levels have been shown to be constant under different experimental conditions (19-21). Differences in gene expression were calculated by fold difference=2(cT target gene R – cT target gene NR)/2(cT housekeeping gene R – cT housekeeping gene NR), NR, nonreversion; R, reversion. Statistical analyses. Statistical significance for differences in cell morphology, cellular growth and gene expression was evaluated using the Student’s t-test. For analysis of microarray data, signal values were exported with the GeneChip operating software (GCOS, Affymetrix). Further analyses were performed with the software CorrXpression, which is described in detail elsewhere (22). For data normalization, the average of each experiment was placed in relation to the overall average calculated for all experiments. Over- and underexpression were defined whenever the expression value for each gene for each
group (e.g. reversion phenotype) was higher or lower by at least a factor of two compared to each gene expression value for the other group (e.g. non-reversion phenotype). Due to the heterogeneity of human tumors, we attenuated the stringent ratio analysis conditions such that only 75% of the comparisons between reverted and nonreverted tumor specimens had to fulfill the criteria. Using the Spearman rank correlation coefficient, the correlation between the Gene-Chip values and the expression data obtained by RT-PCR was statistically validated. In all analyses, p-values≤0.05 were considered significant.
Results Patients’ characteristics. To analyze the impact of normal fibroblasts on growth and differentiation of breast carcinoma cells, PBCs were cultured as either monoculture or in coculture with HMFs in a 3D-collagen-I matrix. PBCs were isolated from tumors of a total of 13 patients with primary invasive breast cancer who had not received any neoadjuvant treatment. The histology of the tumor was validated by frozen-section analysis of tissue taken from the tumor before isolating the PBCs. Nine invasive ductal, three invasive lobular and one neuroendocrine carcinoma with diverse tumor stage, grade, HER2 status, and lymph node status were obtained. All tumors except one were estrogenand/or progesterone receptor-positive (Table I). Morphological analysis of 3D cell cultures. To explore whether PBCs are able to reverse their phenotype so as to resemble a normal polarized acinar architecture, PBCs were co-cultured with HMFs in 3D collagen-I gels and growth and morphology were compared to these for PBC monocultures. Phenotypic reversion was defined by the formation of apico-basally polarized acinus-like structures of PBCs with
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ANTICANCER RESEARCH 33: 1525-1536 (2013) Table II. Analysis of cell morphology and growth of primary breast cancer cells (PBCs) in 3D cultures. PBCs from 13 breast carcinomas were grown in 3D mono- and co-cultures with human mammary fibroblasts (HMFs). The percentage of multicellular structures with polarization of the cellular axis and lumen formation are shown. In each 3D culture, a minimum of 60 cell clusters was analyzed (inverted microscope; magnification: ×250). Growth of PBCs in 3D cultures is expressed as the total area of pancytokeratin-positive cells. Breast cancer samples 1-5 exhibited phenotypic reversion in 3D co-culture, breast cancer samples 6-13 did not show phenotypic reversion in 3D co-culture. Patient ID
1 2 3 4 5 6 7 8 9 10 11 12 13
Polarized acini (%) (SD) Monoculture
Co-culture
4.65 (0.55) 13.68 (0.66) 1.75 (0.21) 15.13 (0.39) 5.34 (0.35) 4.62 (0.66) 13.83 (0.88) 15.63 (1.13) 23.44 (1.06) 18.33 (0.80) 7.69 (0.74) 14.29 (1.00) 20.63 (0.98)
22.37 (0.92) 24.78 (0.78) 12.63 (0.81) 25.81 (1.02) 15.15 (0.72) 4.48 (0.64) 14.19 (0.64) 20.97 (0.99) 24.29 (0.97) 21.88 (0.78) 11.11 (0.82) 19.15 (1.22) 22.97 (0.78)
p-Value
Total area* (SD) Monoculture
