A novel neuregulin – jagged1 paracrine loop in breast cancer transendothelial migration

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 [14] 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.

The pTRIPZ empty vector or constructs containing NRG1 shRNA (sequences in Table 1) (Dharmacon, Lafayette, CO, USA) were transfected into 293 cells along with the TAT, REV, GAG/POL packaging plasmids and VSV-g pseudotyping plasmid using Lipofectamine 2000 (cat# 11668019, Invitrogen, Carlsbad, CA, USA). MDA-MB 231 cells were then transduced using supernatants containing virus and 8 μg/mL polybrene (TR-1003-G, EMD Millipore, Billerica, MA, USA). Stable transductants were identified by placing the cells under puromycin selection (1 μg/mL) for 1 week. Cells were grown in the presence of 2 μg/mL doxycycline for at least 5 days to induce shRNA expression. For the rescue experiments, cells containing the pTRIPz vector or NRG1 shRNA1 were transfected with either a control vector (VB160226-10013) or NRG1 expression vector (VB160118-10047) both from VectorBuilder (Cyagen Biosciences Inc., Santa Clara, CA, USA).

For the knockout of Jagged 1 in the BAC1.2F5 cells, either a control non-targeted or JAG1 targeted guide RNA (gRNA) (sequences in Table 2) was inserted into the lentiCRISPR v2 construct (Addgene plasmid #52961) and then transformed into Stbl3 bacteria. DNA was then isolated and transfected into 293 cells as described above. Virus-containing supernatants were collected and used to transduce macrophages. Cells were then placed under puromycin selection (2 μg/mL) and western blot was used to screen for gene deletion.

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).

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.

To quantify gene expression, cells were grown under normal culture conditions. Total RNA was isolated using the RNeasy mini kit (cat# 74134, Qiagen, Germantown, MD, USA) and complementary (c)DNA was synthesized and amplified from total RNA using the Superscript II system (cat# 11904-018, Thermofisher Scientific, Fremont, CA, USA). Baseline expression was measured using a SYBR Green Mastermix and gene-specific primers (sequences in Table 3). For the co-culture assay, gene-specific probes and primers from the Taqman system were used to perform real-time PCR, with each reaction being done in triplicate (Human JAG1 cat# Hs00164982_m1, Mouse JAG1 cat# Mm00496902_m1, Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cat# Hs02758991_g1, Mouse GAPDH cat# Mm99999915_g1, Thermofisher Scientific, Fremont, CA, USA). The mean cycle threshold (Ct) values were then used to analyze relative expression. Analysis was done using the ΔΔCt method in which all Ct values were normalized to GAPDH.

The iTEM assay was performed as previously described [15]. 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.

In order to determine surface expression levels of ErbB3, macrophages (BAC), MDA-MB 231 breast cancer cells (231), and endothelial cells (HUVEC) were labeled with an ErbB3 blocking antibody and analyzed by FACS. Of the three cell types, only the macrophages showed significant ErbB3 surface expression (Fig. 1a). After establishing expression of ErbB3 on the macrophages, we then determined expression of the ErbB3 ligand NRG1 in the same cell lines. Using qRT-PCR, we saw that there was very little NRG1 messenger (m)RNA expression in the macrophages, while high levels of mRNA were present in the tumor cells and endothelial cells (Fig. 1b). NRG1 protein expression was evaluated in the three different cell types: western blot analysis of cell lysates showed strong NRG1 protein expression in the MDA-MB 231 cells, and a second breast cancer triple negative cell line BT549, showed less expression in the HUVECs and macrophages (Fig. 1c). In summary, NRG1 was most highly expressed by the tumor cells, while the receptor ErbB3 was mainly present on the surface of macrophages.

To examine the effects of blocking ErbB3 signaling on intravasation, tumor cells and macrophages were placed in a previously established in vitro iTEM assay [15]. This assay models the conditions seen at the interface of the tumor and the blood vessel and allows us to quantify the number of tumor cells that are crossing the endothelial cell layer. It has previously been seen that although breast cancer cells are able to cross the endothelial cell barrier, transendothelial migration is enhanced in the presence of macrophages as shown in Fig. 2 and in previous publications [15, 16]. In the untreated and IgG control conditions the tumor cells alone exhibited a basal level of transendothelial migration, which increased in the presence of macrophages. However, in the presence of an ErbB3 blocking antibody, there was no enhancement of transendothelial migration in the presence of macrophages (Fig. 2a). Similar results were found for the BT549 cells when placed in the iTEM assay (Fig. 2b). Thus inhibition of ErbB3 on macrophages blocked the ability of macrophages to enhance tumor cell transmigration.

After establishing that ErbB3 signaling on macrophages was important for enhancing breast cancer cell migration across the endothelial cell layer, we then tested whether altering expression of the ErbB3 ligand NRG1 by the cancer cells was important. In order to knock down expression of NRG1 in the MDA-MB 231 breast cancer cell line, we used the TRIPZ doxycycline-inducible vector [17] containing either short hairpin (sh)RNAs targeting the NRG1 gene or a control empty vector. In addition to driving shRNA expression, the vector also contains an RFP construct, which leads to fluorescence when the shRNA is expressed. After growing the transduced cell lines in the presence of doxycycline and checking for the expression of RFP, we were able to detect knockdown of NRG1 protein in western blot by two different shRNA sequences (Fig. 3a). After confirming NRG1 knockdown, we then placed the shRNA expressing cells into the iTEM assay to observe the effect of reducing NRG1 expression on transendothelial migration. The cells containing the control vector showed the expected increase in crossing the endothelial cell layer when macrophages were added, but this effect was significantly reduced for the NRG1 shRNA-expressing tumor cells (Fig. 3b). This reduction in transendothelial migration was specifically due to the induction of shRNA expression, since cells carrying the NRG1 shRNA vector but grown without doxycycline still showed macrophage-induced transendothelial migration, compared to cells grown in parallel where the shRNA expression was induced (Fig. 3c). To confirm that the reduction in transendothelial migration seen in the cells expressing NRG1 shRNA was due to loss of NRG1, an NRG1 construct resistant to the shRNA was designed. TRIPZ control and NRG1 shRNA cells were transfected with the rescue NRG1 expression vector or empty vector control and tested in the iTEM assay. Even after induction of the NRG1 shRNA, cells expressing the NRG1 rescue construct showed increased transendothelial migration in the presence of macrophages (Fig. 3d), confirming the role of NRG1.

As a means to validate the results observed in our in vitro studies, we used a xenograft mouse model to study the effects of NRG1 expression on intravasation. MDA-MB 231 cells expressing either the control vector or NRG1 shRNA vectors were orthotopically injected into the mammary fat pads of SCID mice. The mice were then fed with either a control diet or with food containing doxycycline (inducing the shRNA). When tumors reached approximately 1 cm in diameter, peripheral blood was collected from the right atrium. Doxycycline treatment did not affect the growth rate of tumors, but there was a significant decrease in the number of circulating tumor cells in mice containing tumors with the NRG1 shRNA construct, which were fed the diet containing doxycycline (Fig. 4). Rates of metastasis to the lung were relatively low and we did not detect a statistically significant difference in metastasis (data not shown).

To further elucidate the mechanism of macrophage-enhanced transendothelial migration, we examined the effects of NRG1 stimulation on macrophages. We found that treating macrophages with NRG1 led to increased expression of JAG1, a ligand of the Notch signaling pathway. We saw an increase in JAG1 mRNA (not shown) and protein (Fig. 5a). mRNA levels of the other Notch ligands were not increased (data not shown). We were also able to detect an increase in macrophage (mouse) JAG1 mRNA expression when human MDA-MB 231 breast cancer cells were co-cultured with macrophages (Fig. 5b). It has previously been seen that the Notch receptor on breast cancer cells plays an important role in invasion and intravasation in the iTEM assay [16]; therefore we tested whether altering expression of JAG1 in macrophages had an effect on tumor cell transendothelial migration. To achieve this, we used CRISPR to knock out JAG1 in macrophages (Fig. 5c). We then evaluated our clustered regularly interspaced short palindromic repeats (CRISPR) JAG1 knockout (KO) macrophages in the iTEM assay. In comparison to the macrophages transduced with a control non-targeting guide RNA, we found that the macrophages lacking JAG1 expression did not enhance transendothelial migration of MDA-MB 231 cells, indicating an important role for macrophage JAG1 expression (Fig. 5d). This was confirmed with a second breast cancer cell line, BT549, which also showed no enhancement of transendothelial migration when placed in an iTEM assay with macrophages lacking JAG1 expression (Fig. 5e). Addition of an exogenous JAG1 peptide alone can significantly increase transendothelial migration of MDA-MB 231 cells, supporting the conclusion that stimulation of the Notch receptor on tumor cells by JAG1 can enhance intravasation (Fig. 5f).