A novel neuregulin – jagged1 paracrine loop in breast cancer transendothelial migration
© The Author(s). 2018
Received: 29 August 2017
Accepted: 21 March 2018
Published: 10 April 2018
The interaction of breast cancer cells with other cells in the tumor microenvironment plays an important role in metastasis. Invasion and intravasation, two critical steps in the metastatic process, are influenced by these interactions. Macrophages are of particular interest when it comes to studying tumor cell invasiveness. Previous studies have shown that there is paracrine loop signaling between breast cancer cells and macrophages involving colony stimulating factor 1 (CSF-1) produced by tumor cells and epidermal growth factor (EGF) production by macrophages. In this paper, we identify a novel paracrine loop between tumor cells and macrophages involving neuregulin (NRG1) and notch signaling.
The aim of this study was to determine the role of NRG1, a ligand of the ErbB3 receptor, in macrophage stimulation of tumor cell transendothelial migration and intravasation. We used fluorescence-activated cell sorting (FACS) and western blot to determine ErbB3 and NRG1 expression, respectively. An in vitro transendothelial migration (iTEM) assay was used to examine the effects of short hairpin (sh)RNA targeting NRG1 in tumor cells and clustered regularly interspaced short palindromic repeats (CRISPR) knockout of jagged 1 (JAG1) in macrophages. Orthotopic xenograft injections in mice were used to confirm results in vivo.
In our system, macrophages were the primary cells showing expression of ErbB3, and a blocking antibody against ErbB3 resulted in a significant decrease in macrophage-induced transendothelial migration of breast cancer cells. Stimulation of macrophages with NRG1 upregulated mRNA and protein expression of JAG1, a ligand of the Notch receptor, and JAG1 production by macrophages was important for transendothelial migration of tumor cells.
This study demonstrates that stimulation of macrophages by tumor cell NRG1 can enhance transendothelial migration and intravasation. We also demonstrate that this effect is due to induction of macrophage JAG1, an important ligand of the Notch signaling pathway.
In women, breast cancer is the most commonly diagnosed and second leading cause of cancer death . Although there have been significant improvements in screening techniques and information on prevention, breast cancer mortality occurs due to the metastatic spread of cells from the primary tumor to distant sites. The process of metastasis involves many steps, including invading the basement membrane and normal surrounding breast tissue, entering the bloodstream (intravasating), and then extravasating at distant organs to initiate metastasis formation . Although cancer cells have their own inherent invasive properties, interaction with other cell types in the tumor microenvironment can facilitate metastasis. Specifically, tumor associated macrophages (TAMs) can play a role in breast cancer metastasis, and higher TAM density has been associated with worse prognosis [3, 4]. In particular a subset of TAMs form the tumor microenvironment of metastasis (TMEM), the doorway for intravasation in breast tumors, which is directly involved in systemic tumor cell dissemination . TMEM is a clinically validated prognostic marker of metastatic risk in patients with breast cancer [6, 7]. Previous work has shown paracrine loop signaling between tumor cells and macrophages, where epidermal growth factor (EGF) from macrophages induced by colony stimulating factor 1 (CSF-1) from tumor cells contributes to breast cancer cell motility and invasion [8, 9]. Recent work has also indicated that this paracrine signaling is important to sensitize tumor cells to hepatocyte growth factor (HGF) signaling by endothelial cells to attract them towards blood vessels .
In examining the relationship between tumor cells and macrophages, we sought to identify additional signaling pathways that may be working in conjunction with the canonical paracrine loop to contribute to breast cancer metastasis. Studies characterizing macrophages co-invading with breast cancer cells showed higher expression of ErbB3, a member of the epidermal growth factor receptor (EGFR) family, in the invasive macrophages [11, 12]. This along with data showing that expression of ErbB3 in solid tumors is associated with worse prognosis  made it an attractive target for further exploration. ErbB3 has been widely studied in cancer cells, but little is known about its role in macrophages. Utilizing both in vitro and in vivo techniques, we wanted to determine the role of ErbB3 and its ligand neuregulin1 (NRG1) in tumor cell intravasation.
In the studies reported here, we identified a novel paracrine loop for intravasation, in which NRG1 production by tumor cells stimulates macrophages to produce JAG1, resulting in increased transendothelial migration.
Cell culture and generation of stable cell lines
Breast cancer cell lines BT549 and MDA-MB 231 were cultured in Dulbecco’s modified Eagle medium (DMEM) (cat# SH30243, Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (cat# S11550, Altanta Biologicals, Flowery Branch, GA, USA). BAC 1.2F5 macrophages  were cultured in Minimum Essential Medium, Alpha (α-MEM) (cat# 15-012-CV, Corning, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (cat# 100-106, Gemini Bio-Products, Sacramento, CA, USA) and 3000 U/mL CSF-1 (a gift from Chiron Corp, Emeryville, CA, USA). Human umbilical vein endothelial cells (HUVEC) were cultured in EGM-2 medium (cat# CC-3162, Lonza, Allendale, NJ, USA) according to the manufacturer’s instructions and not used beyond passage 4 for any experiments.
Neuregulin (NRG1) short hairpin RNA (shRNA) sequences
NRG1 shRNA vector
Jagged 1 (JAG1) clustered regularly interspaced short palindromic repeats (CRISPR) guide sequences
Cells were washed once with phosphate-buffered saline (PBS), lysed using sample buffer containing 10% SDS and analyzed by SDS-PAGE. Membranes were imaged on a Li-Cor scanner, and processed using ImageJ. To examine the induction of JAG1 protein expression, BAC cells were stimulated with 12 nM NRG1 (cat# 396-HB, R&D Systems, Minneapolis, MN, USA) for 8 h and lysed: antibodies and their dilutions were used as follows: Tubulin (1:5000) (cat# T4026, Sigma-Aldrich, St. Louis, MO, USA), NRG1 (1:1000) (cat# sc-28,916, Santa Cruz Biotechnology, Dallas, TX, USA), Jagged 1 (1:1000) (cat# sc-6011, Santa Cruz Biotechnology).
Fluorescence-activated cell sorting (FACS)
Cells were detached with 2 mM EDTA, centrifuged, and resuspended at a concentration of 5 × 106 cells per mL in 100 μL in a FACS buffer containing PBS, 2 mM EDTA, and 2.5% FBS. Cells were then placed on ice and treated for 5 min with 5 μg/mL Fc Block (cat# 553142, BD Biosciences, San Jose, CA, USA). Then, either the ErbB3 blocking antibody (cat# MS-303-PABX, Thermo Scientific, Fremont, CA, USA) or mouse IgG1 isotype control (cat# 0102-01 Southern Biotech, Birmingham, AL, USA) were added at a concentration of 10 μg/mL for 30 min, with mixing of the tubes by flicking every 10 min to ensure proper labeling. Samples were then centrifuged and washed three times in the FACS buffer to eliminate any unbound antibody. Cells were then labeled with a donkey anti-mouse Alexa-647 conjugated secondary antibody (cat# 715-605-151, Jackson Immunoresearch, West Grove, PA, USA) for 30 min. Samples were then washed and filtered in preparation for FACS analysis. A total of 1 × 104 cells per sample were analyzed using a DXP10 Calibur flow cytometer and sample data were processed using FlowJo.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Gene-specific primer sequences
In vitro transendothelial migration assay (iTEM)
The iTEM assay was performed as previously described . Briefly, transwells from EMD Millipore (cat# MCEP24H48) were coated with 2.5 μg/mL Matrigel (cat# 356230, BD Biosciences, San Jose, CA, USA) in a total volume of 50 μL. Then approximately 1 × 104 human umbilical vein endothelial cells in 50 μL of EGM-2 medium were plated on the inverted transwells previously coated with Matrigel and allowed to adhere for 4 h at 37 °C. Transwells were then placed into a 24-well plate with 1 mL of EGM-2 in the bottom well and 200 μL inside the upper chamber and allowed to grow for 48 h in order to form a monolayer. Breast cancer cells were labeled with cell tracker green dye and macrophages with cell tracker red (Green cat# C7025, Red cat# C34552, Invitrogen, Carlsbad, CA, USA), resuspended in M199 media (cat# SH30253.01, Hyclone) and plated at 15,000 breast cancer cells and 60,000 macrophages per transwell and allowed to transmigrate towards EGM-2 containing 3000 U/mL CSF-1 for 18 h. For treatment with JAG1 or scrambled peptide, tumor cells were serum starved overnight in DMEM and then pre-incubated with 30 uM of either Jagged 1 DSL peptide (AS-61298, AnaSpec) or Jagged 1 Scrambled peptide (AS-64239, AnaSpec) in serum starvation medium for 4 h at 37 °C before labeling and plating in the transwell. Samples were then fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X 100 and stained with rhodamine phalloidin (cat# R415, Invitrogen). Transwell membranes were excised and mounted, with Z-series taken in eight random fields per sample.
All in vivo experiments were conducted in accordance with the National Institutes of Health regulations on the care and use of experimental animals and approved by the Albert Einstein College of Medicine Animal Use Committee. Orthotopic tumor xenografts were generated by injecting a total of 2 × 106 MDA-MB 231 cells suspended in sterile PBS with 20% Collagen I (cat# 354249, Corning, Corning, NY, USA) into the inguinal (4th from top) right mammary fat pad of 5-week-old to 8-week-old female mice with severe combined immunodeficiency (SCID) (NCI). Peripheral blood, primary tumors, and lungs were collected when the tumors reached approximately 1 cm in diameter.
Circulating tumor cells were collected by anesthetizing mice and drawing blood from the right atrium using syringes containing 50 μL of heparin to prevent clotting during collection: 500 μL to 1 mL of blood was collected per mouse. Blood was then placed in 9 mL of 1 × red blood cell lysis buffer for 10 min, centrifuged, and resuspended in 10 mL DMEM/F12 medium in a 10-cm cell culture dish. After 3 days of culture, growth medium was changed to DMEM/F12 containing doxycycline to induce red fluorescent protein (RFP) for tumor cell counting (doxycycline treatment did not affect cell growth). A week after collection, samples were counted under a fluorescence microscope, using turbo RFP expression to identify tumor cells. Intravasation was calculated by dividing the number of colonies per plate by the volume of blood collected and normalizing to 1 mL.
Results are representative of at least three independent experiments for in vitro experiments and at least 11 mice per group in in vivo experiments. Statistical analysis was performed using the unpaired or paired two-tailed Student’s t test, or z test as indicated.
ErbB3 is expressed on macrophages and NRG1 protein is expressed by tumor cells
Blocking ErbB3 reduces macrophage-induced transendothelial migration (iTEM)
Knockdown of NRG1 in tumor cells reduces macrophage-induced transendothelial migration in vitro
Knockdown of NRG1 in tumor cells reduces intravasation in vivo
Jagged 1 (JAG1) is upregulated in NRG1-stimulated macrophages and is important for tumor cell transendothelial migration
Previous work has revealed the importance of breast tumor cell contact with macrophages in TMEM during intravasation  and Notch signaling in macrophage-dependent transendothelial migration and intravasation [15, 16]. Those studies demonstrated a role for macrophages in the stimulation of intravasation of tumor cells involving NOTCH on tumor cells. The work presented here is complementary, identifying JAG1 from macrophages as a NOTCH ligand important for the macrophage-stimulated transendothelial migration. For macrophages, JAG1 expression has been well-documented [18, 19]. JAG1 can be induced in macrophages by a variety of stimuli, including growth factors , toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) [21–24], or hypoxia . In our studies, we have found that NRG1 stimulation of macrophages can lead to increased expression of JAG1. The use of an ErbB3 blocking antibody identified ErbB3 as the receptor for NRG1 on macrophages. ErbB4, the other potential receptor for NRG1, was not detectable by PCR, western blot or FACS (data not shown). ErbB3 expression by macrophages, monocytes, and microglia has been previously reported [26–29], possibly involved in suppressing inflammatory responses via NRG1 stimulation. To the best of our knowledge, this is the first report of induction of JAG1 expression by NRG1 in any cell type, including macrophages.
Macrophage-expressed JAG1 has been reported to have a variety of effects. It can mediate juxtacrine effects on T cells [30–33]. In addition, macrophages themselves express Notch receptors and can be altered in polarization [34–36] by JAG1, possibly by autocrine or juxtacrine stimulation. Because Notch signaling plays a role in angiogenesis , there is also the possibility that macrophage JAG1 may affect endothelial cells directly, affecting cell junctions and blood vessel permeability, potentially allowing tumor cells to enter the bloodstream. With respect to intravasation, macrophages can stimulate the Notch receptor on the tumor cells, causing them to become more invasive, and increasing their capacity to intravasate [15, 16]. Our work would indicate that JAG1 can mediate this effect. In colorectal cancer, DLL4 expression by tumor-associated macrophages has also been reported . In addition to effects on invasion and intravasation, activation of NOTCH on tumor cells has the potential to enhance stemness and resistance to therapy [39–41]. Interestingly, previous work in melanoma has shown that stimulation of the Notch receptor leads to upregulation of NRG1 , and thus it is possible that a positive feedback loop is being activated.
While working with cancer cells in vitro and animal models in vivo serve as valuable tools for studying the behavior of tumor cells and the mechanisms of metastasis, there are a number of limitations that must be considered when analyzing the results and overall impact of our study. Our experiments focused mainly on MDA-MB 231 and BT549 cells, which have been thoroughly characterized. These cells have high NRG1 expression and low surface ErbB3 expression, making autocrine signaling less likely to be a factor in our experiments, even though it has been seen that ErbB3 upregulation may occur in drug-resistant tumors . There are also limitations in the design of our in vitro transendothelial migration assay. While components meant to mimic the extracellular matrix and endothelial cell layer are present, factors such as pressure from fluid flow through vessels, and other cell types, such as fibroblasts, present in the microenvironment are not considered. Our in vitro experiments were performed using mouse macrophages and in vivo experiments with SCID mice. While they do provide a consistent model for what we predict is happening in their human counterparts, differences in species between tumor cells and macrophages must be taken into account. Further experiments to test this model could include macrophage-specific deletion of ErbB3 and JAG1.
Taken as a whole, our data suggest that NRG1 in tumor cells, and ErbB3 and JAG1 in macrophages can play an important role in the metastatic cascade. By inhibiting this signaling pathway between tumor cells and macrophages, intravasation of tumor cells is decreased. A recently published study has shown that conventional chemotherapy may induce metastatic spread , leading to worse outcome. Therapies that block cancer cell intravasation could lessen this effect, and also increase the effectiveness of localized treatments such as radiation and surgical resection. Another alternative could be using NRG, ErbB3, JAG1, or Notch as potential biomarkers for metastasis. Past studies have focused on characterizing ErbB3 expression of tumor cells, but our experiments suggest that JAG1 and ErbB3 expression on macrophages may also play an important role.
These studies demonstrate the potential importance of NRG1 expression by tumor cells in macrophage-enhanced transendothelial migration. We show that NRG1 can stimulate the ErbB3 receptor on macrophages in order to induce JAG1 expression, which acts as a ligand for the tumor cell Notch receptor, increasing their capacity for transendothelial migration and intravasation.
We would like to thank members of the Segall, Cox, Condeelis, and Hodgson laboratories for their helpful suggestions and comments. We also thank Dr Richard Stanley for providing CSF-1.
JES is the Betty and Sheldon Feinberg Senior Faculty Scholar in Cancer Research. Funding was provided by CA100324 (JSC, JES) and T32-GM007288 (RMC and SPHM). Funding sources did not play a role in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript.
Availability of data and materials
Data supporting these results are available from the authors upon request.
RC performed the bulk of the work reported, including writing the manuscript, generating cell lines, performing iTEM assays and animal studies. SM aided with the animal studies. CS and JC performed the studies with soluble JAG1. JS contributed to the animal studies and provided overall direction and oversight to the project. All authors read and approved the final manuscript.
This manuscript does not involve human participants, human data or human tissue.
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