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Reverse engineering of triple-negative breast cancer cells for targeted treatment
Maturitas 108 (2018) 24–30
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Reverse engineering of triple-negative breast cancer cells for targeted treatment Lena Bluemel, Marie-Kristin von Wahlde, Joke Tio, Ludwig Kiesel, Christof Bernemann
T
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Department of Gynecology and Obstetrics, University Hospital, Muenster, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords: Triple-negative breast cancer Personalized therapies HER2 treatment Reverse engineering
Objective: Targeting the human epidermal growth factor receptor HER2 has increased survival in HER2-positive breast cancer patients. In the contrast, for triple-negative breast cancer (TNBC) patients, no targeted agents are available. We hypothesized that artificial overexpression of HER2 in TNBC cells might induce sensitivity to antiHER2 agents in these cells. Methods: TNBC cell lines were transduced using lentiviral HER2 overexpression particles. Functionality of HER2 was determined by protein analysis and localization studies. The tumorigenic potential of HER2 overexpressing cells was assessed by analysis of proliferation, migration and invasion capacity. Response to chemotherapeutic agents and anti-HER2 agents was determined by cell viability assays. Results: We demonstrated functional overexpression of HER2 in TNBC cell lines of different subtypes. Whereas in cell types with more pronounced epithelial features (e.g. MDA-MB-468) HER2 overexpression increases proliferation and migration, in mesenchymal cell lines (MDA-MB-231 and BT-549) HER2 was able to further increase invasive potential. No changes were found in cancer stem cell characteristics or in response to chemotherapy, a trait of TNBC. When treated with anti-HER2 agents, however, HER2 overexpressing TNBC cells showed increased sensitivity to these agents. Conclusion: This proof-of-principle study demonstrates that reverse engineering of TNBC cells might offer a novel targeted treatment strategy for this most aggressive subtype of breast cancer.
1. Introduction The HER2 positive breast cancer subtype is associated with aggressive behavioral traits, including enhanced growth and proliferation, increased invasive and metastatic capability, and stimulation of angiogenesis [1]. This subtype correlates with poor outcome [2]. The development of targeted therapies, however, significantly improved the prognosis of this aggressive subtype of breast cancer. Mainly, development of HER2 targeting drugs, e.g. the HER2 blocking antibody trastuzumab, which binds to the extracellular domain (ECD) of HER2, improved the prognosis of this subtype [3]. Upon binding of trastuzumab, either homodimerization or heterodimerization to EGFR, HER3 or HER4 is blocked. Thus, intracellular phosphorylation of the tyrosine kinase domain is inhibited, thereby blocking both MAPK and PI3K signaling pathways [4]. In 1998, trastuzumab has become the first antiHER2 therapy for HER2 positive breast cancer [5]. Since then, even more treatment strategies for HER2 positive breast cancer have been developed; some of them, which are HER2:HER3 heterodimerization blocking antibodies (pertuzumab), antibody drug conjugates
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(trastuzumab emtansine, T-DM1) or intracellular inhibitors of the tyrosine kinase domain (lapatinib). All these drugs rely on binding to domains of the functional HER2 protein, either extracellularly (trastuzumab, pertuzumab) or intracellularly (lapatinib). In triple negative breast cancer (TNBC), tumor cells express neither growth factor receptor (HER2) nor the hormone receptors estrogen receptor (ER) or progesterone receptor (PR)). Therefore, this cancer subtype cannot be targeted by anti-HER2 treatment or endocrine therapies. Consequently, chemotherapeutic treatment is the most common therapeutic option for this breast cancer subtype. Thus, there is an urgent need for development of novel treatment strategies for this aggressive cancer subtype. As targeted treatment of HER2 protein has significantly improved prognosis and survival of HER2 positive breast cancer patients, we hypothesized that reverse engineering of TNBC cells to HER2 positive cells might positively influence the treatment potential for TNBC. Therefore, we reprogrammed TNBC cells to HER2 positive cells and analysed both molecular changes of the HER2 signaling axis as well as the potential of anti-HER2 treatment in these reverse engineered tumor
Corresponding author. Present address: Department for Urology, University Hospital, Muenster, Germany. E-mail address: [email protected] (C. Bernemann).
https://doi.org/10.1016/j.maturitas.2017.11.010 Received 25 August 2017; Received in revised form 23 October 2017; Accepted 9 November 2017 0378-5122/ © 2017 Elsevier B.V. All rights reserved.
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membranes (Corning, Germany) or BioCoat Matrigel invasion chambers (BD Biosciences, Germany). Membranes were cultured in 24-well plates containing medium with serum. After 48–96 h, cells that passed the membrane were fixed, stained and analysed.
cells. 2. Material and methods 2.1. Cloning of pCDH-EF1-HER2-T2A-Puro
2.6. Immunocytochemistry For cloning, HER2 cDNA was PCR amplified from HER2 positive BT474 breast cancer cell line cDNA using primers containing restriction sites for XbaI at 5′ end and NotI at 3′ end. PCR primers did not amplify the TGA stop codon, since HER2 cds was cloned in frame to a T2A sequence in the cloning vector pCDH-EF1-MCS-T2A-Puro (System Biosciences, USA). The PCR product was ligated into the cloning vector and correct product size was determined by sequencing analysis. Primers are listed in Table S1.
For immunocytochemistry, cells were fixed with phosphate buffered formalin and blocked with 10% Aurion (Dako, USA) in PBS for 1 h. Cells were then washed and incubated with primary antibody (HER2, #2242, Cell Signaling, Netherlands) diluted with Dako REALTM Antibody Diluent (overnight at 4 °C). Fluorescent visualization was carried out using suitable Alexa Fluor-conjugated secondary antibody (1:600) together with 4′,6-diamidino-2-phenylindole (1:400) in Dako REALTM Antibody Diluent for 1 h at RT.
2.2. Cell culture, lentiviral production and transduction 2.7. Chemotherapy sensitivity assay/Dose dependent response to anti-HER2 drugs
All cell lines (MDA-MB-468, MDA-MB-231, BT-549 and BT474) were purchased from ATCC (USA) and cultured under recommended conditions. Medium, trypsin-EDTA, PBS, fetal calf serum and horse serum were received from PAA Laboratories (Germany). STR authentification analysis of cell lines was performed twice a year at DSMZ (Braunschweig, Germany). For lentiviral production, HEK293 cells were transiently transfected with pCDH-EF1-HER2-T2A-Puro along with 2 packaging plasmids. After 72 h, lentiviral particles were sterile filtered from the HEK293 supernatant and subsequently incubated with breast cancer cell lines for 24 h. After 5 washing cycles, HER2 positive breast cancer cell lines were further incubated in the presence of puromycin (Sigma-Aldrich, Germany).
For analysis of chemotherapy sensitivity, cells were incubated with cytotoxic agents (paclitaxel and doxorubicin hydrochloride) using decreasing concentrations. After 72 h, cell viability was determined via MTT (Thiazolyl Blue Tetrazolium Bromide) (all substances were received from Sigma-Aldrich, Germany) according to the manufacturer’s protocol. For analyses of dose dependent response to anti-HER2 drugs, cells were incubated with decreasing concentrations of trastuzumab, pertuzumab, lapatinib and T-DM1. After 72 h, cell viability was determined via MTT according to the manufacturer’s protocol. Measurements were performed in triplicates. Significance was calculated via one-side Welch’s t-test.
2.3. Western blot analysis 2.8. Flow cytometry Cells were incubated with RIPA buffer (10 mM NaF, 1 mM Na3VO4, 10 mM β-Glycerophosphate, 7.6 mM Tris pH 7.4, 52 mM NaCl, 0.4% Triton X-100, 0.8 mM EDTA, proteinase inhibitor (Sigma-Aldrich, Germany). Protein quantification was performed via Pierce BCA assay (Thermo Scientific, Germany) according to the manufacturer’s protocol. SDS page electrophoresis and blotting were performed using standard protocols. Detection was performed using primary antibodies against HER2 (#2242), phospho-HER2 (Tyr1248, #2247), p44/42 MAPK (#9102), phospho-p44/42 MAPK (Thr202/Tyr204, #9101), AKT (#9272) and phospho-AKT (Ser473, #9271) (all Cell Signaling, Netherlands), tubulin (#T5168, Sigma-Aldrich, Germany) and Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Germany). Bands were visualized with AGFA developer and fixer (AGFA, Belgium).
Flow cytometric cell analyses were performed on a FACS Aria (BD FACSAriaTM, BD Biosciences, Germany) as described previously [7]. 3. Results 3.1. Reverse engineering of TNBC cells to HER2 positive cells In order to examine the potential of reverse engineering as a novel therapeutic strategy for TNBC, we stably overexpressed HER2 in the TNBC cell lines MDA-MB-468, MDA-MB-231 and BT-549 using lentiviral particles. Upon lentiviral transduction, all cell lines showed overexpression of HER2 mRNA as well as HER2 protein determined by RT-qPCR and Western Blot analysis using HER2 antibodies against both the extracellular domain and the intracellular phosphorylation domain of HER2 (Fig. 1A, B). Besides, immunocytochemistry analysis revealed strong signals for HER2 protein on the cell membrane (Fig. 1C), suggesting membrane bound localization. These results confirm the correct translation as well as cellular localization of HER2 protein.
2.4. Quantitative real-time PCR RNA isolation was performed using NucleoSpin RNA Kits (Macherey-Nagel, Germany) with on-column DNase digestion. Reverse transcription for real-time quantitative polymerase chain reaction (RTqPCR) was performed using M-MLV reverse transcriptase (USB (Affymetrix), USA) and Oligo-dT15 priming at 42 °C for 1 h and at 60 °C for 10 min. A cDNA equivalent of 50 ng total RNA was used as template in a total reaction volume of 20 μl with Power SYBR Green PCR mix (Applied Biosystems, Germany) on a Step One Plus cycler (Applied Biosystems, Germany). Primers were added at 0.375 μM each. Calculations were based on the ΔΔCt method using two housekeeping genes for normalization. Real-time primer sequences are listed in Table S1.
3.2. HER2 activates MAPK but not PI3K pathway in TNBC cells Next, we analysed the functionality of overexpressed HER2. For this purpose we determined the influence of HER2 on MAPK and PI3K signaling pathways, two known downstream pathways of HER2 [8], by using phospho-specific antibodies for AKT and p42/44 MAPK. No changes were found in levels of phosphorylated AKT, which was present in HER2 positive cells as well as in control cells (Fig. 2A). While levels of MAPK were similar in HER2 positive and control cells, we found induction of phosphorylated MAPK in HER2 positive BT-549 as well as MDA-MB-231 cells, but not in MDA-MB-468 cells (Fig. 2B). These results suggest specific induction of MAPK signaling pathway but not of PI3K signaling pathway by HER2 in MDA-MB-231 and BT-549 cells.
2.5. Cell migration/invasion assay Migration and invasion assays were performed as described elsewhere [6]. Briefly, cells were seeded without serum onto either 8 μm 25
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Fig. 1. Overexpression of HER2 in TNBC cell lines. HER2 overexpression was validated either on the mRNA level (A) as well as on the protein level (B). BT474 cells served as HER2 positive control. Immunofluorescence analysis revealed membrane bound localization of the HER2 protein in TNBC cell lines (C). UTR: untranslated region; cds: coding sequence, OE: overexpression
3.3. HER2 stimulates tumorigenic potential of TNBC ceIls
not mediated via alterations in the cancer stem cell population.
In order to determine the effect of HER2 expression on tumor-associated properties, we performed assays analyzing proliferation, migration and invasion capacity in TNBC cell lines expressing HER2. We found significant increase in proliferation rates in all three cell lines (Fig. 3A). For MDA-MB-468 as well as BT-549 cells, we observed an increase in migratory potential by approximately 2.5 fold and 1.5 fold in MDA-MB-468 and BT-549 cells, respectively (Fig. 3B). For MDA-MB231 and BT-549 cells, an increase in invasive capacity of 1.4 fold in MDA-MB-231 cells and 3.0 fold in BT-549 cells was determined (Fig. 3C). Furthermore, we investigated the influence of HER2 overexpression on the cancer stem cell population. Surprisingly, we were not able to detect a shift in the percentage of breast cancer stem cells, characterized by either CD44/CD24 expression or ALDH expression (Fig. S1, Table S2). These results demonstrate an increase in tumorassociated properties upon HER2 overexpression, which, however, is
3.4. HER2 overexpression does not influence response to chemotherapeutic agents but to anti-HER2 agents Next, we analysed the effect of HER2 overexpression on the response to standard chemotherapeutic agents used in TNBC treatment. For all three cell lines, we were not able to observe an alteration in cell viability when treated with chemotherapeutic agents paclitaxel and doxorubicin (Fig. S2). When treated with standard humanized anti-HER2 antibodies (trastuzumab, pertuzumab), we did not observe any significant change in cell viability in all three cell lines (Fig. S3). As no response to trastuzumab occurred in none of the cell lines, we performed transient transfection of MDA-MB-231 cells with a HER2 overexpression plasmid, resulting in more than 100-fold increase in HER2 expression (Fig. S4A). When these highly HER2 positive MDA-MB-231 cells were treated with Fig. 2. HER2 activates MAPK signaling but not P13K signaling in TNBC cells. A) Western Blot analysis of AKT and phospho-AKT showed no induction of the PI3K signaling pathway. B) Western Blot analysis of MAPK and phospho-MAPK showed induction of MAPK signaling pathway in MDA-MB-231 and BT-549 TNBC cells, whereas no induction was found in MDA-MB-468 cells. OE: overexpression
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Fig. 3. HER2 induces tumor associated characteristics in TNBC cells. A) Increase of proliferative capacity upon HER2 overexpression in TNBC cells. HER2 induces significant increase in migratory potential in MDA-MB-468 and BT-549 cells (B). HER2 significantly activates invasive capacity in MDA-MB-231 and BT-549 cells (C).
Fig. 4. Response of HER2 positive TNBC cells to anti HER2 treatment. HER2 sensitizes TNBC cells to anti HER2 drug T-DM1 in all cell lines (A). Only BT-549 cells showed increased sensitivity to anti HER2 drug lapatinib, whereas no changes were found in MDA-MB-468 and MDA-MB-231 cells.
receptor is one of the most important drug targets at least in the HER2positive breast cancer subtype. In contrast, the TNBC subtype does not express a molecular drug target comparable to HER2. Thus, reverse engineering of TNBC cells to HER2 positive cells might provide a range of targeted therapy options in this hard-to-treat subtype. HER2 is known to activate cellular processes, e.g. proliferation, via two distinct signaling pathways, namely AKT-PI3K or MAPK [8]. The TNBC subtype is characterized by PTEN mutations, leading to increased activation of the PI3K pathway [9]. Indeed, high basal activity shown by phosphorylation of AKT was detected in all three cell lines. This activity, however, seems not to be induced further by HER2 overexpression. For MAPK signaling, we also detected basal expression of p44/42 MAPK in all three cell lines. MDA-MB-468 cells showed
either trastuzumab or lapatinib, we detected a decrease of cell number compared to vector control, suggesting a response to these agents in MDA-MB-231 cells (Fig. S4B). All HER2 positive cell lines showed higher sensitivity to the antibody drug conjugate T-DM1 (Fig. 4A). Additionally, in BT-549 but not MDA-MB-231 and MDA-MB-468 cells, we detected a significant response to lapatinib (Fig. 4B). These results demonstrate an increase of sensitivity towards anti HER2 agents in TNBC cells. 4. Discussion The human epidermal growth factor receptor HER2 plays a pivotal role in stratification of breast cancer patients. Expression of this 27
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effective treatment against the intracellular domain of HER2 more likely [23–25]. This study provides a proof of principle that overexpression of HER2 in TNBC cells makes these cells susceptible to anti-HER2 treatment strategies. We were able to demonstrate functionality of the HER2 protein, determined by both cellular localization as well as induction of a HER2 downstream signaling pathway, resulting in an increased proliferative capacity as well as an increased migratory and invasive potential. We detected a significant decrease of cell viability of TNBC cells overexpressing HER2 in the presence of anti-HER2 agents. An approach of reverse engineering of TNBC cells to express HER2 could therefore provide an opportunity of targeted treatment in TNBC patients. Apparently, however, this approach implicates several limitations. The most crucial point is that overexpression of an oncogene like HER2 leads to an induction of tumorigenic properties, e.g. proliferation as well as migratory and invasive potential. Most notably, this induction would phenotypically lead to an even more aggressive subtype. Interestingly, this increase in tumorigenic potential was found in cell lines, which exhibit molecular characteristics of the basal B subtype of TNBC (MDA-MB-231 and BT-549) described by Neve et al. [26]. These cell types are known to be more invasive presumably due to mesenchymal and stem/progenitor-like characteristics, whereas cells of the basal A subtype of TNBC (MDA-MB-468) display more epithelial characteristics. Therefore, although HER2 overexpression seems to increase the invasive potential of aggressive cells, it is not sufficient to induce a phenotypic switch from an epithelial to a mesenchymal state, reminiscent of an epithelial to mesenchymal transition (EMT). Certainly, the induction of cells to acquire more tumorigenic potential would appear to be counterintuitive for clinical application in its current form. Thus, the goal of the reverse engineering approach would rely on one of the following two considerations: either overexpression of a mutated HER2 variant without intracellular signaling capacity or short term induction of intrinsic HER2 gene expression. There are studies describing a HER2 mRNA variant encoding the extracellular domain without intracellular tyrosine kinase domain, which inhibits growth factor induced proliferation of breast cancer cells [27,28]. Besides, introduction of this variant in trastuzumab resistant, HER2 positive breast cancer cells allows to overcome of this resistance [29]. Treatment in TNBC cells expressing such a variant would presumably lead to response to trastuzumab, pertuzumab and as well as T-DM1 based on ADCC. The most elegant basis for an anti-HER2 treatment, however, would rely on an induction of endogenous HER2 combined with anti-HER2 treatment. Ithimakin et al. were able to demonstrate short term induction of HER2 protein expression in luminal breast cancer cells by RANKL (receptor activator of NF-κB), a member of the TNF family, which is secreted from osteoblasts [30,31]. Studies have already described a discordance in the HER2 status between primary and metastatic tumors [32–34]. Recently, a phenotypic switch from HER2 negative to HER2 positive and vice versa has been described in circulating tumor cells (CTCs) of breast cancer patients [35]. Using cultures of these CTCs the authors demonstrated a spontaneous interconversion rate of 84% between primary tumors and circulating tumor cells. These cells could reversely interconvert leading to heterogeneous populations of daughter cells from one or the other phenotype. Concordant with results from our study, HER2 positive CTCs became more proliferative. When used for in vivo analyses in orthotopic circulating tumor cell-derived tumor models, HER2 positive CTCs generated a mixed population of HER2 positive and negative cells. Paclitaxel treatment results in a dramatic tumor shrinkage, mostly decreasing the number of HER2 positive cells. This would also fit into our model describing that chemotherapeutic efficiency is not altered by overexpression of HER2. Thus, there are likely biological mechanisms which lead to interconversion of the HER2 status in formerly HER2 negative breast cancer cells. Further studies are needed to clarify these mechanisms to
phosphorylated p44/42 MAPK, which was not further induced upon HER2 overexpression. In MDA-MB-231 and BT-549 cells, however, we detected a pronounced increase in phosphorylated MAPK. These results suggest that HER2 overexpression selectively induces activity of the MAPK signaling pathway at least in some TNBC cells. It has been shown that homodimerization of HER2 stimulates MAPK, whereas heterodimerization of HER2 and of other members of the HER family lead to activation of PI3K signaling activity [10]. Overexpression of HER2 protein therefore might lead to homodimerization, thereby activating MAPK rather than PI3K signaling pathway. Furthermore, since induction of MAPK signaling activity was found specifically in the cell lines displaying higher invasive capacity, this pathway activity might directly be linked to increase in metastatic potential. This is in line with studies showing association of MAPK signaling and tumor progression [11,12]. HER2 is a known oncogene, inducing tumor cell proliferation upon overexpression or amplification [8]. The epithelial cell line MDA-MB468 showed increase in proliferation and migratory potential, but not in invasive potential. Thus, overexpression of HER2 alone is not sufficient to induce a switch from epithelial to mesenchymal phenotype in basal A TNBC cell line MDA-MB-468. The other cell lines, MDA-MB-231 and BT-549, displayed increase in invasive potential. These two cell lines exhibit a mesenchymal phenotype, characterized by high invasive capacity due to limited cell–cell contacts [13]. Thus, HER2 increases intrinsic invasive capacity in these basal B TNBC cell lines. Interestingly, this increase does not depend on changes in the cancer stem cell compartment, as no changes in either CD44+/CD24- nor ALDH+ cell populations was observed upon overexpression. This is in contrast to Korkaya et al., showing influence of HER2 on the mammary stem cell pool [14]. Although the TNBC subtype is associated with poor prognosis, several patients showed higher rate of pathologic complete response (pCR) to neoadjuvant chemotherapy compared to patients with nonTNBC. Thus, higher response to chemotherapy is a trait of TNBC [15,16]. Interestingly, we detected no differences in response rates to chemotherapeutic agents upon HER2 overexpression. Thus, intrinsic response to chemotherapy is not altered in none of the cell lines analysed. This is in line with reprogramming reports, describing a kind of epigenetic memory of cells during the process of reprogramming to a pluripotent stem cell state [17,18]. When analyzing the effect of anti-HER2 antibody treatment strategies using trastuzumab or pertuzumab, we detected no significant changes in HER2 overexpressing cells. This might be due to the lack of induction of antibody dependent cellular cytotoxicity (ADCC); a phenomenon, which plays a pivotal role in in vivo response to trastuzumab and pertuzumab [19]. Additionally, PI3K signaling activity is known to inhibit trastuzumab induced apoptosis or cell cycle arrest [20]. Furthermore, heterodimerization of HER2 and insulin-like growth factor 1 receptor (IGF1R) might decrease antibody response [21]. Recently, increased expression of IGF1R in TNBC was found to be associated with loss of PTEN, thereby activating PI3K [22]. When we performed transient overexpression of HER2 in MDA-MB231 cells, leading to > 100-fold higher levels of HER2, we found a response to trastuzumab. Therefore, we cannot rule out that the HER2 levels obtained by lentiviral transduction were not sufficient to prepare TNBC cells for targeted treatment with anti-HER2 antibodies trastuzumab and pertuzumab in the absence of ADCC. The antibody drug conjugate T-DM1 showed significant decrease of cell viability in all cell lines, demonstrated by lower IC50. When treated with the intracellular active agent lapatinib, HER2 overexpressing BT549 cells showed a significant increase in response, whereas no changes in cell viability were observed in HER2 overexpressing MDA-MB-231 and MDA-MB-468 cells. Response to lapatinib does not necessarily depend on the expression of HER2, but also that of EGFR. Indeed, increased EGFR expression is currently being discussed for TNBC cell lines, whereby BT-549 cells showed the lowest EGFR levels, making an 28
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presumably make TNBC patients amenable to targeted therapies, i.e. anti-HER2 therapies. [7]
Contributors [8]
Lena Bluemel conceived, designed and performed the study, analysed and interpreted the data, and drafted the manuscript. Marie-Kristin von Wahlde analysed and interpreted the data. Joke Tio analysed and interpreted the data. Ludwig Kiesel analysed and interpreted the data and drafted the manuscript. Christof Bernemann conceived, designed and performed the study, analysed and interpreted the data, and drafted the manuscript.
[9]
[10]
[11]
[12]
Conflict of interest
[13]
The authors declare that they have no conflict of interest. Funding
[14]
This project was partly funded by the internal funds of the medical faculty of Muenster University IMF (Innovative Medizinische Forschung) – BE 1 1 12 02.
[15]
[16]
Ethical approval The work does not involve the use of human subjects and did not require ethical approval. Ethical statement The work does not involve the use of human subjects.
[17]
[18]
[19]
Provenance and peer review This article has undergone peer review.
[20]
Research data (data sharing and collaboration) [21]
There are no linked research data sets for this paper. Data will be made available on request. [22]
Acknowledgements We thank Dorothea Godulla for technical assistance. Additionally, we thank Dr. Holm Zaehres and Prof. Hans Schöler for support in lentiviral transduction and Dr. Martin Stehling for FACS analyses. This project was partly funded by the internal funds of the medical faculty of Muenster University IMF (Innovative Medizinische Forschung) – BE 1 1 12 02.
[23]
[24]
[25]
Appendix A. Supplementary data
[26]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.maturitas.2017.11.010.
[27]
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Maturitas journal homepage: www.elsevier.com/locate/maturitas
Reverse engineering of triple-negative breast cancer cells for targeted treatment Lena Bluemel, Marie-Kristin von Wahlde, Joke Tio, Ludwig Kiesel, Christof Bernemann
T
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Department of Gynecology and Obstetrics, University Hospital, Muenster, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords: Triple-negative breast cancer Personalized therapies HER2 treatment Reverse engineering
Objective: Targeting the human epidermal growth factor receptor HER2 has increased survival in HER2-positive breast cancer patients. In the contrast, for triple-negative breast cancer (TNBC) patients, no targeted agents are available. We hypothesized that artificial overexpression of HER2 in TNBC cells might induce sensitivity to antiHER2 agents in these cells. Methods: TNBC cell lines were transduced using lentiviral HER2 overexpression particles. Functionality of HER2 was determined by protein analysis and localization studies. The tumorigenic potential of HER2 overexpressing cells was assessed by analysis of proliferation, migration and invasion capacity. Response to chemotherapeutic agents and anti-HER2 agents was determined by cell viability assays. Results: We demonstrated functional overexpression of HER2 in TNBC cell lines of different subtypes. Whereas in cell types with more pronounced epithelial features (e.g. MDA-MB-468) HER2 overexpression increases proliferation and migration, in mesenchymal cell lines (MDA-MB-231 and BT-549) HER2 was able to further increase invasive potential. No changes were found in cancer stem cell characteristics or in response to chemotherapy, a trait of TNBC. When treated with anti-HER2 agents, however, HER2 overexpressing TNBC cells showed increased sensitivity to these agents. Conclusion: This proof-of-principle study demonstrates that reverse engineering of TNBC cells might offer a novel targeted treatment strategy for this most aggressive subtype of breast cancer.
1. Introduction The HER2 positive breast cancer subtype is associated with aggressive behavioral traits, including enhanced growth and proliferation, increased invasive and metastatic capability, and stimulation of angiogenesis [1]. This subtype correlates with poor outcome [2]. The development of targeted therapies, however, significantly improved the prognosis of this aggressive subtype of breast cancer. Mainly, development of HER2 targeting drugs, e.g. the HER2 blocking antibody trastuzumab, which binds to the extracellular domain (ECD) of HER2, improved the prognosis of this subtype [3]. Upon binding of trastuzumab, either homodimerization or heterodimerization to EGFR, HER3 or HER4 is blocked. Thus, intracellular phosphorylation of the tyrosine kinase domain is inhibited, thereby blocking both MAPK and PI3K signaling pathways [4]. In 1998, trastuzumab has become the first antiHER2 therapy for HER2 positive breast cancer [5]. Since then, even more treatment strategies for HER2 positive breast cancer have been developed; some of them, which are HER2:HER3 heterodimerization blocking antibodies (pertuzumab), antibody drug conjugates
⁎
(trastuzumab emtansine, T-DM1) or intracellular inhibitors of the tyrosine kinase domain (lapatinib). All these drugs rely on binding to domains of the functional HER2 protein, either extracellularly (trastuzumab, pertuzumab) or intracellularly (lapatinib). In triple negative breast cancer (TNBC), tumor cells express neither growth factor receptor (HER2) nor the hormone receptors estrogen receptor (ER) or progesterone receptor (PR)). Therefore, this cancer subtype cannot be targeted by anti-HER2 treatment or endocrine therapies. Consequently, chemotherapeutic treatment is the most common therapeutic option for this breast cancer subtype. Thus, there is an urgent need for development of novel treatment strategies for this aggressive cancer subtype. As targeted treatment of HER2 protein has significantly improved prognosis and survival of HER2 positive breast cancer patients, we hypothesized that reverse engineering of TNBC cells to HER2 positive cells might positively influence the treatment potential for TNBC. Therefore, we reprogrammed TNBC cells to HER2 positive cells and analysed both molecular changes of the HER2 signaling axis as well as the potential of anti-HER2 treatment in these reverse engineered tumor
Corresponding author. Present address: Department for Urology, University Hospital, Muenster, Germany. E-mail address: [email protected] (C. Bernemann).
https://doi.org/10.1016/j.maturitas.2017.11.010 Received 25 August 2017; Received in revised form 23 October 2017; Accepted 9 November 2017 0378-5122/ © 2017 Elsevier B.V. All rights reserved.
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membranes (Corning, Germany) or BioCoat Matrigel invasion chambers (BD Biosciences, Germany). Membranes were cultured in 24-well plates containing medium with serum. After 48–96 h, cells that passed the membrane were fixed, stained and analysed.
cells. 2. Material and methods 2.1. Cloning of pCDH-EF1-HER2-T2A-Puro
2.6. Immunocytochemistry For cloning, HER2 cDNA was PCR amplified from HER2 positive BT474 breast cancer cell line cDNA using primers containing restriction sites for XbaI at 5′ end and NotI at 3′ end. PCR primers did not amplify the TGA stop codon, since HER2 cds was cloned in frame to a T2A sequence in the cloning vector pCDH-EF1-MCS-T2A-Puro (System Biosciences, USA). The PCR product was ligated into the cloning vector and correct product size was determined by sequencing analysis. Primers are listed in Table S1.
For immunocytochemistry, cells were fixed with phosphate buffered formalin and blocked with 10% Aurion (Dako, USA) in PBS for 1 h. Cells were then washed and incubated with primary antibody (HER2, #2242, Cell Signaling, Netherlands) diluted with Dako REALTM Antibody Diluent (overnight at 4 °C). Fluorescent visualization was carried out using suitable Alexa Fluor-conjugated secondary antibody (1:600) together with 4′,6-diamidino-2-phenylindole (1:400) in Dako REALTM Antibody Diluent for 1 h at RT.
2.2. Cell culture, lentiviral production and transduction 2.7. Chemotherapy sensitivity assay/Dose dependent response to anti-HER2 drugs
All cell lines (MDA-MB-468, MDA-MB-231, BT-549 and BT474) were purchased from ATCC (USA) and cultured under recommended conditions. Medium, trypsin-EDTA, PBS, fetal calf serum and horse serum were received from PAA Laboratories (Germany). STR authentification analysis of cell lines was performed twice a year at DSMZ (Braunschweig, Germany). For lentiviral production, HEK293 cells were transiently transfected with pCDH-EF1-HER2-T2A-Puro along with 2 packaging plasmids. After 72 h, lentiviral particles were sterile filtered from the HEK293 supernatant and subsequently incubated with breast cancer cell lines for 24 h. After 5 washing cycles, HER2 positive breast cancer cell lines were further incubated in the presence of puromycin (Sigma-Aldrich, Germany).
For analysis of chemotherapy sensitivity, cells were incubated with cytotoxic agents (paclitaxel and doxorubicin hydrochloride) using decreasing concentrations. After 72 h, cell viability was determined via MTT (Thiazolyl Blue Tetrazolium Bromide) (all substances were received from Sigma-Aldrich, Germany) according to the manufacturer’s protocol. For analyses of dose dependent response to anti-HER2 drugs, cells were incubated with decreasing concentrations of trastuzumab, pertuzumab, lapatinib and T-DM1. After 72 h, cell viability was determined via MTT according to the manufacturer’s protocol. Measurements were performed in triplicates. Significance was calculated via one-side Welch’s t-test.
2.3. Western blot analysis 2.8. Flow cytometry Cells were incubated with RIPA buffer (10 mM NaF, 1 mM Na3VO4, 10 mM β-Glycerophosphate, 7.6 mM Tris pH 7.4, 52 mM NaCl, 0.4% Triton X-100, 0.8 mM EDTA, proteinase inhibitor (Sigma-Aldrich, Germany). Protein quantification was performed via Pierce BCA assay (Thermo Scientific, Germany) according to the manufacturer’s protocol. SDS page electrophoresis and blotting were performed using standard protocols. Detection was performed using primary antibodies against HER2 (#2242), phospho-HER2 (Tyr1248, #2247), p44/42 MAPK (#9102), phospho-p44/42 MAPK (Thr202/Tyr204, #9101), AKT (#9272) and phospho-AKT (Ser473, #9271) (all Cell Signaling, Netherlands), tubulin (#T5168, Sigma-Aldrich, Germany) and Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Germany). Bands were visualized with AGFA developer and fixer (AGFA, Belgium).
Flow cytometric cell analyses were performed on a FACS Aria (BD FACSAriaTM, BD Biosciences, Germany) as described previously [7]. 3. Results 3.1. Reverse engineering of TNBC cells to HER2 positive cells In order to examine the potential of reverse engineering as a novel therapeutic strategy for TNBC, we stably overexpressed HER2 in the TNBC cell lines MDA-MB-468, MDA-MB-231 and BT-549 using lentiviral particles. Upon lentiviral transduction, all cell lines showed overexpression of HER2 mRNA as well as HER2 protein determined by RT-qPCR and Western Blot analysis using HER2 antibodies against both the extracellular domain and the intracellular phosphorylation domain of HER2 (Fig. 1A, B). Besides, immunocytochemistry analysis revealed strong signals for HER2 protein on the cell membrane (Fig. 1C), suggesting membrane bound localization. These results confirm the correct translation as well as cellular localization of HER2 protein.
2.4. Quantitative real-time PCR RNA isolation was performed using NucleoSpin RNA Kits (Macherey-Nagel, Germany) with on-column DNase digestion. Reverse transcription for real-time quantitative polymerase chain reaction (RTqPCR) was performed using M-MLV reverse transcriptase (USB (Affymetrix), USA) and Oligo-dT15 priming at 42 °C for 1 h and at 60 °C for 10 min. A cDNA equivalent of 50 ng total RNA was used as template in a total reaction volume of 20 μl with Power SYBR Green PCR mix (Applied Biosystems, Germany) on a Step One Plus cycler (Applied Biosystems, Germany). Primers were added at 0.375 μM each. Calculations were based on the ΔΔCt method using two housekeeping genes for normalization. Real-time primer sequences are listed in Table S1.
3.2. HER2 activates MAPK but not PI3K pathway in TNBC cells Next, we analysed the functionality of overexpressed HER2. For this purpose we determined the influence of HER2 on MAPK and PI3K signaling pathways, two known downstream pathways of HER2 [8], by using phospho-specific antibodies for AKT and p42/44 MAPK. No changes were found in levels of phosphorylated AKT, which was present in HER2 positive cells as well as in control cells (Fig. 2A). While levels of MAPK were similar in HER2 positive and control cells, we found induction of phosphorylated MAPK in HER2 positive BT-549 as well as MDA-MB-231 cells, but not in MDA-MB-468 cells (Fig. 2B). These results suggest specific induction of MAPK signaling pathway but not of PI3K signaling pathway by HER2 in MDA-MB-231 and BT-549 cells.
2.5. Cell migration/invasion assay Migration and invasion assays were performed as described elsewhere [6]. Briefly, cells were seeded without serum onto either 8 μm 25
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Fig. 1. Overexpression of HER2 in TNBC cell lines. HER2 overexpression was validated either on the mRNA level (A) as well as on the protein level (B). BT474 cells served as HER2 positive control. Immunofluorescence analysis revealed membrane bound localization of the HER2 protein in TNBC cell lines (C). UTR: untranslated region; cds: coding sequence, OE: overexpression
3.3. HER2 stimulates tumorigenic potential of TNBC ceIls
not mediated via alterations in the cancer stem cell population.
In order to determine the effect of HER2 expression on tumor-associated properties, we performed assays analyzing proliferation, migration and invasion capacity in TNBC cell lines expressing HER2. We found significant increase in proliferation rates in all three cell lines (Fig. 3A). For MDA-MB-468 as well as BT-549 cells, we observed an increase in migratory potential by approximately 2.5 fold and 1.5 fold in MDA-MB-468 and BT-549 cells, respectively (Fig. 3B). For MDA-MB231 and BT-549 cells, an increase in invasive capacity of 1.4 fold in MDA-MB-231 cells and 3.0 fold in BT-549 cells was determined (Fig. 3C). Furthermore, we investigated the influence of HER2 overexpression on the cancer stem cell population. Surprisingly, we were not able to detect a shift in the percentage of breast cancer stem cells, characterized by either CD44/CD24 expression or ALDH expression (Fig. S1, Table S2). These results demonstrate an increase in tumorassociated properties upon HER2 overexpression, which, however, is
3.4. HER2 overexpression does not influence response to chemotherapeutic agents but to anti-HER2 agents Next, we analysed the effect of HER2 overexpression on the response to standard chemotherapeutic agents used in TNBC treatment. For all three cell lines, we were not able to observe an alteration in cell viability when treated with chemotherapeutic agents paclitaxel and doxorubicin (Fig. S2). When treated with standard humanized anti-HER2 antibodies (trastuzumab, pertuzumab), we did not observe any significant change in cell viability in all three cell lines (Fig. S3). As no response to trastuzumab occurred in none of the cell lines, we performed transient transfection of MDA-MB-231 cells with a HER2 overexpression plasmid, resulting in more than 100-fold increase in HER2 expression (Fig. S4A). When these highly HER2 positive MDA-MB-231 cells were treated with Fig. 2. HER2 activates MAPK signaling but not P13K signaling in TNBC cells. A) Western Blot analysis of AKT and phospho-AKT showed no induction of the PI3K signaling pathway. B) Western Blot analysis of MAPK and phospho-MAPK showed induction of MAPK signaling pathway in MDA-MB-231 and BT-549 TNBC cells, whereas no induction was found in MDA-MB-468 cells. OE: overexpression
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Fig. 3. HER2 induces tumor associated characteristics in TNBC cells. A) Increase of proliferative capacity upon HER2 overexpression in TNBC cells. HER2 induces significant increase in migratory potential in MDA-MB-468 and BT-549 cells (B). HER2 significantly activates invasive capacity in MDA-MB-231 and BT-549 cells (C).
Fig. 4. Response of HER2 positive TNBC cells to anti HER2 treatment. HER2 sensitizes TNBC cells to anti HER2 drug T-DM1 in all cell lines (A). Only BT-549 cells showed increased sensitivity to anti HER2 drug lapatinib, whereas no changes were found in MDA-MB-468 and MDA-MB-231 cells.
receptor is one of the most important drug targets at least in the HER2positive breast cancer subtype. In contrast, the TNBC subtype does not express a molecular drug target comparable to HER2. Thus, reverse engineering of TNBC cells to HER2 positive cells might provide a range of targeted therapy options in this hard-to-treat subtype. HER2 is known to activate cellular processes, e.g. proliferation, via two distinct signaling pathways, namely AKT-PI3K or MAPK [8]. The TNBC subtype is characterized by PTEN mutations, leading to increased activation of the PI3K pathway [9]. Indeed, high basal activity shown by phosphorylation of AKT was detected in all three cell lines. This activity, however, seems not to be induced further by HER2 overexpression. For MAPK signaling, we also detected basal expression of p44/42 MAPK in all three cell lines. MDA-MB-468 cells showed
either trastuzumab or lapatinib, we detected a decrease of cell number compared to vector control, suggesting a response to these agents in MDA-MB-231 cells (Fig. S4B). All HER2 positive cell lines showed higher sensitivity to the antibody drug conjugate T-DM1 (Fig. 4A). Additionally, in BT-549 but not MDA-MB-231 and MDA-MB-468 cells, we detected a significant response to lapatinib (Fig. 4B). These results demonstrate an increase of sensitivity towards anti HER2 agents in TNBC cells. 4. Discussion The human epidermal growth factor receptor HER2 plays a pivotal role in stratification of breast cancer patients. Expression of this 27
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effective treatment against the intracellular domain of HER2 more likely [23–25]. This study provides a proof of principle that overexpression of HER2 in TNBC cells makes these cells susceptible to anti-HER2 treatment strategies. We were able to demonstrate functionality of the HER2 protein, determined by both cellular localization as well as induction of a HER2 downstream signaling pathway, resulting in an increased proliferative capacity as well as an increased migratory and invasive potential. We detected a significant decrease of cell viability of TNBC cells overexpressing HER2 in the presence of anti-HER2 agents. An approach of reverse engineering of TNBC cells to express HER2 could therefore provide an opportunity of targeted treatment in TNBC patients. Apparently, however, this approach implicates several limitations. The most crucial point is that overexpression of an oncogene like HER2 leads to an induction of tumorigenic properties, e.g. proliferation as well as migratory and invasive potential. Most notably, this induction would phenotypically lead to an even more aggressive subtype. Interestingly, this increase in tumorigenic potential was found in cell lines, which exhibit molecular characteristics of the basal B subtype of TNBC (MDA-MB-231 and BT-549) described by Neve et al. [26]. These cell types are known to be more invasive presumably due to mesenchymal and stem/progenitor-like characteristics, whereas cells of the basal A subtype of TNBC (MDA-MB-468) display more epithelial characteristics. Therefore, although HER2 overexpression seems to increase the invasive potential of aggressive cells, it is not sufficient to induce a phenotypic switch from an epithelial to a mesenchymal state, reminiscent of an epithelial to mesenchymal transition (EMT). Certainly, the induction of cells to acquire more tumorigenic potential would appear to be counterintuitive for clinical application in its current form. Thus, the goal of the reverse engineering approach would rely on one of the following two considerations: either overexpression of a mutated HER2 variant without intracellular signaling capacity or short term induction of intrinsic HER2 gene expression. There are studies describing a HER2 mRNA variant encoding the extracellular domain without intracellular tyrosine kinase domain, which inhibits growth factor induced proliferation of breast cancer cells [27,28]. Besides, introduction of this variant in trastuzumab resistant, HER2 positive breast cancer cells allows to overcome of this resistance [29]. Treatment in TNBC cells expressing such a variant would presumably lead to response to trastuzumab, pertuzumab and as well as T-DM1 based on ADCC. The most elegant basis for an anti-HER2 treatment, however, would rely on an induction of endogenous HER2 combined with anti-HER2 treatment. Ithimakin et al. were able to demonstrate short term induction of HER2 protein expression in luminal breast cancer cells by RANKL (receptor activator of NF-κB), a member of the TNF family, which is secreted from osteoblasts [30,31]. Studies have already described a discordance in the HER2 status between primary and metastatic tumors [32–34]. Recently, a phenotypic switch from HER2 negative to HER2 positive and vice versa has been described in circulating tumor cells (CTCs) of breast cancer patients [35]. Using cultures of these CTCs the authors demonstrated a spontaneous interconversion rate of 84% between primary tumors and circulating tumor cells. These cells could reversely interconvert leading to heterogeneous populations of daughter cells from one or the other phenotype. Concordant with results from our study, HER2 positive CTCs became more proliferative. When used for in vivo analyses in orthotopic circulating tumor cell-derived tumor models, HER2 positive CTCs generated a mixed population of HER2 positive and negative cells. Paclitaxel treatment results in a dramatic tumor shrinkage, mostly decreasing the number of HER2 positive cells. This would also fit into our model describing that chemotherapeutic efficiency is not altered by overexpression of HER2. Thus, there are likely biological mechanisms which lead to interconversion of the HER2 status in formerly HER2 negative breast cancer cells. Further studies are needed to clarify these mechanisms to
phosphorylated p44/42 MAPK, which was not further induced upon HER2 overexpression. In MDA-MB-231 and BT-549 cells, however, we detected a pronounced increase in phosphorylated MAPK. These results suggest that HER2 overexpression selectively induces activity of the MAPK signaling pathway at least in some TNBC cells. It has been shown that homodimerization of HER2 stimulates MAPK, whereas heterodimerization of HER2 and of other members of the HER family lead to activation of PI3K signaling activity [10]. Overexpression of HER2 protein therefore might lead to homodimerization, thereby activating MAPK rather than PI3K signaling pathway. Furthermore, since induction of MAPK signaling activity was found specifically in the cell lines displaying higher invasive capacity, this pathway activity might directly be linked to increase in metastatic potential. This is in line with studies showing association of MAPK signaling and tumor progression [11,12]. HER2 is a known oncogene, inducing tumor cell proliferation upon overexpression or amplification [8]. The epithelial cell line MDA-MB468 showed increase in proliferation and migratory potential, but not in invasive potential. Thus, overexpression of HER2 alone is not sufficient to induce a switch from epithelial to mesenchymal phenotype in basal A TNBC cell line MDA-MB-468. The other cell lines, MDA-MB-231 and BT-549, displayed increase in invasive potential. These two cell lines exhibit a mesenchymal phenotype, characterized by high invasive capacity due to limited cell–cell contacts [13]. Thus, HER2 increases intrinsic invasive capacity in these basal B TNBC cell lines. Interestingly, this increase does not depend on changes in the cancer stem cell compartment, as no changes in either CD44+/CD24- nor ALDH+ cell populations was observed upon overexpression. This is in contrast to Korkaya et al., showing influence of HER2 on the mammary stem cell pool [14]. Although the TNBC subtype is associated with poor prognosis, several patients showed higher rate of pathologic complete response (pCR) to neoadjuvant chemotherapy compared to patients with nonTNBC. Thus, higher response to chemotherapy is a trait of TNBC [15,16]. Interestingly, we detected no differences in response rates to chemotherapeutic agents upon HER2 overexpression. Thus, intrinsic response to chemotherapy is not altered in none of the cell lines analysed. This is in line with reprogramming reports, describing a kind of epigenetic memory of cells during the process of reprogramming to a pluripotent stem cell state [17,18]. When analyzing the effect of anti-HER2 antibody treatment strategies using trastuzumab or pertuzumab, we detected no significant changes in HER2 overexpressing cells. This might be due to the lack of induction of antibody dependent cellular cytotoxicity (ADCC); a phenomenon, which plays a pivotal role in in vivo response to trastuzumab and pertuzumab [19]. Additionally, PI3K signaling activity is known to inhibit trastuzumab induced apoptosis or cell cycle arrest [20]. Furthermore, heterodimerization of HER2 and insulin-like growth factor 1 receptor (IGF1R) might decrease antibody response [21]. Recently, increased expression of IGF1R in TNBC was found to be associated with loss of PTEN, thereby activating PI3K [22]. When we performed transient overexpression of HER2 in MDA-MB231 cells, leading to > 100-fold higher levels of HER2, we found a response to trastuzumab. Therefore, we cannot rule out that the HER2 levels obtained by lentiviral transduction were not sufficient to prepare TNBC cells for targeted treatment with anti-HER2 antibodies trastuzumab and pertuzumab in the absence of ADCC. The antibody drug conjugate T-DM1 showed significant decrease of cell viability in all cell lines, demonstrated by lower IC50. When treated with the intracellular active agent lapatinib, HER2 overexpressing BT549 cells showed a significant increase in response, whereas no changes in cell viability were observed in HER2 overexpressing MDA-MB-231 and MDA-MB-468 cells. Response to lapatinib does not necessarily depend on the expression of HER2, but also that of EGFR. Indeed, increased EGFR expression is currently being discussed for TNBC cell lines, whereby BT-549 cells showed the lowest EGFR levels, making an 28
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presumably make TNBC patients amenable to targeted therapies, i.e. anti-HER2 therapies. [7]
Contributors [8]
Lena Bluemel conceived, designed and performed the study, analysed and interpreted the data, and drafted the manuscript. Marie-Kristin von Wahlde analysed and interpreted the data. Joke Tio analysed and interpreted the data. Ludwig Kiesel analysed and interpreted the data and drafted the manuscript. Christof Bernemann conceived, designed and performed the study, analysed and interpreted the data, and drafted the manuscript.
[9]
[10]
[11]
[12]
Conflict of interest
[13]
The authors declare that they have no conflict of interest. Funding
[14]
This project was partly funded by the internal funds of the medical faculty of Muenster University IMF (Innovative Medizinische Forschung) – BE 1 1 12 02.
[15]
[16]
Ethical approval The work does not involve the use of human subjects and did not require ethical approval. Ethical statement The work does not involve the use of human subjects.
[17]
[18]
[19]
Provenance and peer review This article has undergone peer review.
[20]
Research data (data sharing and collaboration) [21]
There are no linked research data sets for this paper. Data will be made available on request. [22]
Acknowledgements We thank Dorothea Godulla for technical assistance. Additionally, we thank Dr. Holm Zaehres and Prof. Hans Schöler for support in lentiviral transduction and Dr. Martin Stehling for FACS analyses. This project was partly funded by the internal funds of the medical faculty of Muenster University IMF (Innovative Medizinische Forschung) – BE 1 1 12 02.
[23]
[24]
[25]
Appendix A. Supplementary data
[26]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.maturitas.2017.11.010.
[27]
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