Shirmohammadi et al. BMC Cancer (2020) 20:836 https://doi.org/10.1186/s12885-020-07228-y RESEARCH ARTICLE Open Access The efficacy of etanercept as anti-breast cancer treatment is attenuated by residing macrophages Elnaz Shirmohammadi1, Seyed-Esmaeil Sadat Ebrahimi1, Amir Farshchi2 and Mona Salimi3* Abstract Background: Interaction between microenvironment and breast cancer cells often is not considered at the early stages of drug development leading to failure of many drugs at later clinical stages. Etanercept is a TNF-alpha inhibitor that has been investigated for potential antitumor effect in breast cancer with conflicting results. Methods: Secretome data on MDA-MB-231 cancer cell-line were from public repositories and subjected to gene enrichment analyses. Since MDA-MB-231 cells secrete high levels of Granulocyte-Monocyte Colony Stimulating Factor, which activates macrophages to promote tumor growth, the effect of macrophage co-culturing on anticancer efficacy of Etanercept in breast cancer was evaluated using the Boolean network modeling and in vitro experiments including invasion, cell cycle, Annexin PI, and tetrazolium based viability assays and NFKB activity. Results: The secretome profile of MDA-MB-231 cells was similar to the expression of genes following treatment of breast cancer cells with TNF-α. Accordingly, inhibition of TNF-α by Etanercept decreased MDA-MB-231 cell survival, induced apoptosis and cell cycle arrest in vitro and inhibited NFKB activation. The inhibitory effect of Etanercept on cell viability, cell cycle progression, invasion and induction of apoptosis decreased following co-culturing of the cancer cells with macrophages. The Boolean network modeling of the changes in the dynamics of intracellular signaling pathways revealed NFKB activation by secretome of macrophages, leading to a decreased efficacy of Etanercept, suggesting NFKB inhibition as an alternative approach to inhibit cancer cell growth in the presence of macrophage crosstalk. Conclusion: This study indicates that the effect of Etanercept may be influenced by residing macrophages in tumor microenvironment, and suggests a method to predict the effect of drugs in the presence of stromal cells to guide experimental designs in drug development. Keywords: TNF-α, Etanercept, Breast cancer, Macrophages, Systems biology Background hand, drug impacts are usually assayed in cell culture or Crosstalk between cancerous and stromal cells in an immunosuppressed mice models [5], neither of which inflammatory-based microenvironment not only affects provides a clear picture of the complicated interactions the aggressive behavior of cancer cell, but also influences between tumor cells and their surrounding inflammatory the efficacy of anti-cancer drugs [1–4]. On the other stroma, eventually resulting in inconsistencies in the outcome of clinical trials [6]. It is thus naïve to assess the sole effect of inhibiting a drugable target in the ag- * Correspondence: [email protected] 3Physiology and Pharmacology Department, Pasteur Institute of Iran, P.O. gressive cancer cells while ignoring the context of Box: 13164, Tehran, Iran micro-environmental changes [4]. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Shirmohammadi et al. BMC Cancer (2020) 20:836 Page 2 of 10 between cancer cells and their surrounding stroma is factors and cytokines) and displays ligands inducing through secretion by these cells of soluble factors includ- similar gene expression profiles. ing growth factors, chemokines and cytokines [7]. The Gene-set enrichment tests including gene ontology secreted factors reprogram the surrounding stroma with (GO) biological process, transcription factors and signal- the aim of neutralizing the impact of various intruders ing pathways were carried out using the corresponding disrupting the survival of the cancer cells [8, 9]. libraries embedded in Enrichr online web tool. One of the most influential cytokines in tumor micro- environment is Tumor Necrosis Factor-alpha (TNF-α) [10, 11], long known for its dual effects cancer [12]. De- Boolean network simulation pending on the cellular context as well as its concentra- Cellular signaling network was constructed based on the tion, the effects of TNF-α may vary from inducing literature survey [19] and translated into Boolean rules. necrosis to survival benefits, endowing invasive proper- Asynchronous simulation mode was used to predict the ties and driving epithelial-mesenchymal transition effect of macrophages on the anti-cancer efficacy of Eta- (EMT) [13]. nercept. BoolNet R package was used to perform the dy- Considering the pleiotropic and conflicting effects of namic modeling and analysis of attractor states [20]. TNF-α, various efforts have been made to exploit this cytokine as a therapeutic target in cancer. In this regard, Cell culture and condition medium preparation α some approaches have used TNF- administration to MDA-MB-231 human breast cancer cell line and THP-1 tumor tissue to induce cell death due to its necrotizing human monocytic cells were purchased from Pasteur In- α effects [14]. However, using TNF- as a necrotizing stitute of Iran (Tehran, Iran). A total of 2 × 105 THP-1 agent has been associated with significant lethal side ef- cells were seeded in 2 ml RPMI supplemented with 10% α fects limiting the applicability of TNF- administration FBS in the upper chamber of 6-well insert plates (SPL α to patients [15]. On the other side, TNF- activates Life Sciences, Seoul, South Korea). Cells were treated NFKB transcription factor along with JNK and p38 in a with 10 ng/ml of PMA (Phorbol 12-myristate 13-acetate) cellular context-dependent manner [16]. This canonical for 24 h to allow differentiation of THP-1 cells to M0 pathway induces the expression of several downstream macrophages and their subsequent attachment to the in- targets of NFKB leading to the increased invasiveness of serts. After 24 h, 2 × 105 of MDA-MB-231 cells were cancer cells. A different approach that has attracted a seeded in the lower chamber in 2 ml of DMEM. The co- α great of attention is the use of TNF- inhibitors, such as culturing system was kept in 5% CO2 incubator for 72 h. Etanercept, as candidates for cancer biotherapy [17]. To prepare condition medium (CM), the upper insert α Etanercept is a dimeric soluble receptor for TNF- , was withdrawn and MDA-MB-231 cells were washed 3 which competes with the cell surface receptors For TNF times with PBS and starved for 24 h in the serum free binding. A phase II clinical trial evaluating the thera- DMEM. The condition medium was then filtered using peutic efficacy of Etanercept in patients with advanced 0.2 μm filter and kept at − 70 °C until use. CM was di- metastatic breast cancer revealed a reduction in the con- luted with 50% of fresh medium for further experiments. centration of TNF-α related cytokines such as CCL2; however, disease stabilization was detected in only one patient [18]. In order to elucidate the underlying reason Tetrazolium-based viability test for this outcome, we aimed at integrating molecular- Co-culturing of MDA-MB-231 with THP-1 cells was cellular experiments with systems modeling approaches performed as described in the previous section. Co- to compare the effect of Etanercept in breast cancer cells cultured as well as MDA-MB-231 cells were treated with alone and in the context of host immune cells. 1or10μg/ml of Etanercept (Aryogen Pharmed, Tehran, Iran) or control buffer for 72 h. The concentration of Etanercept was selected based on the plasma concentra- Methods tion obtained after weekly administration of the drug to High through-put data analysis and enrichment-based the arthritis rheumatoid patients [7]. After 72 h, THP-1 tests containing inserts were discarded, and 20 μl of 5 mg/ml Proteomics data providing the secretome of MDA-MB- of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- 231 cells were extracted from GSE51938 dataset in zolium bromide) stock solution was added to each well. GEO. The secretion profile was submitted to Enrichr on- Following 3 h of incubation, the supernatants were aspi- line web (http://amp.pharm.mssm.edu/Enrichr/). “LINCS rated, and 200 μl of dimethylsulfoxide (DMSO) were L1000 Ligand Perturbations up” library was used for en- used to dissolve purple formazan in each well. Absorb- richment analysis. This library consists of gene expres- ance was measured at 545 nm using microplate Reader sion changes induced by various ligands (e.g. growth (Stat Fax-2100, ST. Louis, USA). Shirmohammadi et al. BMC Cancer (2020) 20:836 Page 3 of 10 Cell cycle analysis Quantitative real time PCR for the analysis of macrophage MDA-MB-231 cells were harvested after 72 h of co- cytokines culturing with the differentiated macrophages in the A total of 1 × 106 of differentiated THP-1 cells using10 presence and absence of 1 or 10 μg/ml Etanercept.