Methods:
Ethics statement: The animal study protocol was approved by
The Institutional Animal Care and Use Committee of BIDMC Institutional
Animal Care and Use Committee (IACUC), protocol number 086-2011, in
accordance with the Public Health Service (PHS) Policy on Humane Care
and Use of Laboratory Animals and the Guide for the Care and Use of
Laboratory Animals (NIH), for the protection of Vertebrates Used for
Scientific Purposes (Scientific Procedures) Act 1986 as well as the
ARRIVE guidelines. All animal studies were performed in BIDMC.
Breast cancer cell lines: Normal immortalized breast epithelial
cells (MCF-10A) and the human breast cancer cell lines MDA-MB-231 were
purchased from American Type Culture Collection (ATCC, Manassas, VA).
MDA-MB-231 cells are TNBC cells lacking PR, ER and HER-2. The breast
tumor metastatic brain variants include the MDA-231-BrM2 tagged to GFP
as well as the murine mammary tumor cells TGL-4T1-BrM5 which
specifically form metastatic brain tumors (Bos, Zhang et al., 2009;
Rodriguez, Jiang et al., 2014; Avraham, Jiang et al., 2014) and generate
multifocal lesions in the cerebrum, the cerebellum, and the brainstem,
with features typical of brain metastasis in cancer patients.
MAGL inhibitors: MAGL inhibitor AM9928 was synthesized and
characterized at the Center for Drug Discovery (CDD), Northeastern
University. AM9928 inhibits human MAGL (hMAGL) with an
IC50 value of 8.9 nM (Tyukhtenko, Ma et al., 2020;
Tyukhtenko, Karageorgos et al., 2016; Tyukhtenko, Rajarshi et al.,
2018). AM9928 lacked any affinity for the cannabinoid receptors CB1 and
CB2 (Tyukhtenko, Ma et al., 2020).
Human Brain Microvascular Endothelial Cells : Human Brain
Microvascular Endothelial Cells (HBMEC), and all HBMEC culture reagents,
were purchased from Cell Systems (Kirkland, WA). Cells were maintained
according to the manufacturer’s protocol and were cultured in CSC
Complete Media on Attachment Factor pre-coated surfaces. Cells were
grown to confluence and were maintained for 3-5 days for complete tight
junction formation.
HBMEC and TNBC co-culture assay: Approximately 40,000
fluorescent-labeled MDA-MB-231 or MDA-MB-BrM2 cells were seeded over a
confluent layer of HBMEC grown on glass coverslips. Cells were then
allowed to attach at 37 °C in 5% CO2 overnight and
incubation was ended by removing media, rinsing 2-3X with PBS and
immediately adding 2% paraformaldehyde. For HBMEC treatment, the HBMEC
monolayer was pre-incubated with either vehicle control or with MAGL
inhibitor AM9928 (100 ng/mL or otherwise as indicated), or with vehicle
control for 2 hours in media with 0.1% serum. This was followed by
removal of the inhibitor and rinsing with PBS prior to addition of the
breast cancer cells as to observe the effects of the inhibitor on HBMEC
only as compared with controls.
Adhesion assay of MDA-MB-BrM2 cells: Approximately 40,000
fluorescent-labeled MDA-MB-231 or MDA-MB-BrM2 cells were seeded over a
confluent layer of HBMEC grown on glass coverslips. The cells were then
allowed to attach at 37 °C in 5% CO2 overnight and
incubation was stopped by removing media, rinsing 2-3X with PBS and
immediately adding 2% paraformaldehyde. For MAGL inhibitor treatment,
HBMEC monolayer was pre-incubated with either vehicle control or with
the MAGL inhibitor (100 ng/mL) for 2 hours in serum-starving conditions.
This was followed by removal of the inhibitor and rinsing with PBS prior
to addition of the breast cancer cells as to observe the effects of the
inhibitor on HBMEC only. After samples preparation, the average number
of attached labeled tumor cells was calculated across all samples.
Endothelial transmigration assay: HBMECs were dispersed using the
CSC Passage Reagent Group kit (Cell Systems), counted, and resuspended
in the appropriate volume of CSC complete media. Approximately 100,000
HBMECs were seeded into 24-well transwell inserts with 8μm pores (Costar
Corp.) pre-coated with fibronectin and maintained for 5 days to allow
for complete tight junction formation. The media was replaced every 2
days with fresh CSC complete media. For the assay, the apical (upper)
and basal (lower) chamber media was removed. Approximately 40,000
fluorescent-labeled MDA-MB-BrM2 cells or MDA-MB-231 cells in 100μL
aliquots were seeded into the apical chamber in serum-starved conditions
(0.5% serum) in CSC media and allowed 5 hours to transmigrate to the
basal side of the transwell. All basal chambers contained complete media
to provide a chemotactic gradient for the breast cancer cells to follow.
The HBMEC monolayer was pre-treated for 2 hours prior to the addition of
breast cancer cells, either with vehicle control or with the MAGL
inhibitor. After 5 hours, the apical side of the transwell filter was
gently wiped clean with a cotton swab to remove all non-migrating cells
and the basal side was dipped into PBS (with Ca2+ and
Mg2+) and then fixed in 3.7% formaldehyde solution
for 10 minutes. The number of transmigrated cells was determined by
averaging the total number of green, fluorescent cells in 10 random
fields at 200X magnification under a fluorescent microscope.
Isolation of Exosomes: The culture supernatants of MDA-MB-231 and
MDA-MB-BrM2 cells untreated or treated with AM9928 for 24hours, at
approximately 65% confluence and were harvested after 16 hours
conditioning in serum-free media. Cells and debris were cleared from the
supernatants by centrifugation (500 g, 10 min) followed by filtration
using 0.22-micron filters (Millipore Inc.). Exosomes were prepared from
cell-free supernatants using the Exosome Isolation Kit (Kit# EIK-01,
Creative Biolabs, NY). The quantitative and qualitative analysis of
exosomes were performed on double sandwich enzyme-linked immunoassay
using the Total Exosome Capture & Quantification Kit
(Kit#EQK-04Creative Biolabs).
Tissue processing and immunostaining: Methods were detailed
previously in our studies (17,18). Briefly, brains were removed and
divided sagittally. One hemi‐brain was fixed in 4% paraformaldehyde, pH
7.4, at 4°C for 48 h and sectioned using a Vibratome 2000 (Leica,
Germany), while the other hemi‐brain was snap‐frozen and stored at −70°C
for protein analysis or for BBB permeability analysis. Hippocampal brain
tissues were dissected and post‐fixed, and treated with primary
antibodies overnight at 4°, followed by the corresponding fluorescent
secondary antibody (AlexaFluor 488 or Alexa Fluor 594, 1:1000 dilution;
Invitrogen). The following antibodies were used: antibodies for ZO‐1
(1:500 dilution), claudin‐5 (1:500 dilution; Invitrogen), CD31 (1:500),
Pan‐cytokeratin antibodies (1:500 dilution), GFP antibodies (1:50
dilutions; Abcam and Santa Cruz) and their respective controls. All
slides/sections were processed and analyzed under the same standardized
conditions. The immunolabelled blind‐coded sections were imaged with the
LSCM (MRC1024, Bio-Rad).
In vivo effects of MAGL on BBB integrity and TNBC
colonization in brain: Female (6wks) BALB/c mice were purchased from
Jackson Laboratories (Bar Harbor, ME). The mice were housed at an
AAALAC-accredited facility at Beth Israel Deaconess Medical Center,
Boston. The mice were handled in accordance with the animal care policy
of Harvard Medical School. Mice were euthanized humanely by CO2
inhalation in the end of the experiments following treatment and tumor
samples were harvested for further study as described below.
We have selected AM9928 for our in vivo studies based on its
prolonged target engagement (Tyukhtenko, Ma et al., 2020). We expected
that the prolong inhibitory effect of AM9928 would be translatable to a
longer pharmacodynamic effect when compared to other MAGL inhibitors
such as AM4301. To that end, we studied the effects of AM9928 on
BMEC-TJs and tumor colonization in the brain using the spontaneous
breast cancer metastasis mouse model (syngeneic) of mammary tumor cells.
We used GFP-4T1-BrM5 cells which migrate to the brain (Bos, Zhang et
al., 2009; Rodriguez, Jiang et al., 2014; Avraham, Jiang et al., 2014)
and form breast metastasis in the brains of Balb/c mice. We administered
GFP-4T1-BrM5 tumor cells (5x104 cells) into the
mammary fat pads of Balb/c mice. More than 70% of these mice developed
mammary tumors within 3 weeks, while brain microtumors were developed in
about 5 weeks. Here, following administration of GFP–4T1-BrM5 cells,
mice were injected with AM9928 (10 mg/kg, i.v.) or the vehicle control
twice a week for 3 weeks (10 mice/group/treatment/experiment). Tumor
sizes in the mammary fat pads were measured using calipers, and the
volume was calculated, using the formula: V = 0.52 × (length) ×
(width)2. The BBB integrity at the end of the
experiments was analyzed by Evan blue test in control groups and in mice
treated with AM9928 as compared to vehicle control. Briefly, Evans’s
blue (EB) (Sigma Chemical Co. St. Louis, MO. USA.) dye (25% in 0.9%
NaCl solution) was intravenously injected at dose of 25 mg/kg under
anesthesia. One hour after the injection, animals were sacrificed.
Brains were weighed, clipped, and individually placed within formamide
p.a. (2mL/brain). The content of dye extracted from each brain was
determined by spectrophotometer (Photometer 4010, Boehringer) at 620nm
and compared to standard graph created through the recording of optical
densities from serial dilutions of EB in 0.9% NaCl solution.
For in vivo imaging, spectral fluorescence images were obtained
using a Maestro‐based imaging system (CRI Inc., Woburn, MA, USA)
(Rodriguez, Jiang et al., 2014; Avraham, Jiang et al., 2014). Five sets
of side‐by‐side whole‐brain images of animals with tumors from the
GFP–4T1BrM5 cells treated with vehicle control or with AM9928 were
obtained. In the end of the experiment, mice were sacrificed, and brain
and mammary fat pad tissues were collected for further analysis.
Statistical analysis: All values are expressed as the mean ± SEM
of the mean and are representative of two to three independent
experiments as indicated. P values were calculated using unpaired
two-tailed Student’s tests, built in the GraphPad Prism Software for
evaluation of statistical significance.