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\begin{document}

\title{Targeting Monoacylglycerol Lipase in Triple-Negative Breast Cancer
Reduced Tumor-Associated Inflammation and Decreased Colonization in the
Brain}



\author[1]{Shalom Avraham}%
\author[2]{Hava Avraham}%
\author[3]{Othman Benchama}%
\author[2]{Sergiy Tyukhenko}%
\author[2]{Michael Malamas}%
\author[4]{Mark Williams}%
\author[2]{Alexandandros Makriyannis}%
\affil[1]{Beth Israel Deaconess Medical Center}%
\affil[2]{Northeastern University}%
\affil[3]{Northeastern University College of Science}%
\affil[4]{Minerallogic Inc}%


\vspace{-1em}



  
  \date{May 30, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Background and Purpose: While the prevalence of breast cancer metastasis
in the brain is significantly higher in triple-negative breast cancers
(TNBCs), there is a lack of novel and/or improved therapeutic approaches
for these patients. Here, we explore the role of monoacylglycerol lipase
(MAGL) in tumor growth and colonization in the brain. Experimental
Approach: MAGL inhibitor AM9928 was selected for these studies due to
its high specificity (hMAGL IC50 = 9nM). The effects of AM9928 on TNBC
adhesion and transmigration across a 3D culture with human brain
microvascular endothelial cells (BMECs) and the secretion of
chemokines/cytokines were examined. The effects of AM9928 on TNBC tumor
growth and tumor colonization in vivo in the brain were performed using
TNBC mice models. Key Results: The residence time based on NMR time
course data for AM9928 is 46 hours indicating prolonged pharmacodynamic
effect. AM9928 blocked TNBC cell adhesion and transmigration across
HBMECs in a 3D culture. AM9928 inhibited the secretion of IL-6, IL-8,
and VEGF-A from TNBC cells and from HBMECs cultures exposed to
TNBC-derived exosomes. Using in vivo studies of syngeneic GFP-4T1-BrM5
mammary tumor cells, AM9928 inhibited tumor growth in the mammary fat
pads and attenuated blood-brain barrier (BBB) permeability changes,
resulting in reduced TNBC colonization in the brain. Together, these
results demonstrate inhibition of TNBC tumor growth and brain
colonization by AM9928 and support the potential clinical application of
MAGL inhibitors as a novel treatment for TNBC.%
\end{abstract}%



\sloppy


August 15, 2021

Targeting Monoacylglycerol Lipase in Triple Negative Breast Cancer
Reduced Tumor-Associated Inflammation and Decreased Colonization in the
Brain

Othman Benchama\textsuperscript{1}, Sergiy
Tyukhtenko\textsuperscript{1}, Michael S. Malamas\textsuperscript{1},
Mark K. Williams\textsuperscript{1}, Alexandros
Makriyannis\textsuperscript{1}, Shalom Avraham\textsuperscript{2}, and
Hava Karsenty Avraham\textsuperscript{1++*}

\textsuperscript{1}The Center for Drug Discovery, Northeastern
University, Boston MA

\textsuperscript{2}The Division of Experimental Medicine, Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston, MA

\emph{Running Title: Inhibition of TNBC brain colonization by targeting
MAGL}

\emph{Keywords: TNBC - triple negative breast cancer, tumor-associated
inflammation, blood-brain barrier, monoacylglycerol lipase}

*Corresponding author:

Dr. Hava Karsenty Avraham, Center for Drug Discovery (CDD),

Northeastern University

Boston, MA 02115

Email: h.avraham@northeastern.edu

\textsuperscript{+}Current address:

The Center for Drug Discovery

Northeastern University,

Boston, MA 02115

\textsuperscript{+} Previous address: The Division of Experimental
Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, MA

The authors declare no potential conflicts of interest.

The data that support the fundings of this study are available from the
corresponding author upon reasonable request. Some data may not be made
available because of privacy or ethical restrictions.

Abstract: 247 words

Total number of Figures \& tables: 6 Figures + 1 Table

\emph{ABSTRACT}

\emph{Background and Purpose} : While the prevalence of breast cancer
metastasis in the brain is significantly higher in triple negative
breast cancers (TNBCs), there is a lack of novel and/or improved
therapeutic approaches for these patients. Here, we explore the role of
monoacylglycerol lipase (MAGL) in tumor growth and colonization in
brain.

\emph{Experimental Approach} : MAGL inhibitor AM9928 was selected for
these studies due to its high specificity (hMAGL IC\textsubscript{50} =
9nM). The effects of AM9928 on TNBC adhesion and transmigration across a
3D culture with human brain microvascular endothelial cells (BMECs) and
the secretion of chemokines/cytokines were examined. The effects of
AM9928 on TNBC tumor growth and tumor colonization in vivo in brain were
performed using TNBC mice models.

\emph{Key Results} : The residence time based on NMR time course data
for AM9928 is 46 hours indicating prolonged pharmacodynamic effect.
AM9928 blocked TNBC cell adhesion and transmigration across HBMECs in a
3D culture. AM9928 inhibited the secretion of IL-6, IL-8, and VEGF-A
from TNBC cells and from HBMECs cultures exposed to TNBC-derived
exosomes. Using \emph{in vivo} studies of syngeneic GFP-4T1-BrM5 mammary
tumor cells, AM9928 inhibited tumor growth in the mammary fat pads and
attenuated blood brain barrier (BBB) permeability changes, resulting in
reduced TNBC colonization in brain. Together, these results demonstrate
inhibition of TNBC tumor growth and brain colonization by AM9928 and
support the potential clinical application of MAGL inhibitors as a novel
treatment for TNBC.

\emph{Conclusion and Implications} : MAGL inhibition may represent a
therapeutic approach to reduce TNBC growth and metastasis in brain by
inhibiting the BBB permeability changes.

\emph{What is already known:}

MAGL assigns lipid stores in pro-tumorigenic signaling lipids in cancer
cells.

MAGL inhibition impaired cell migration, invasion and tumorigenicity in
some types of cancers

\emph{What this study adds:}

\begin{enumerate}
\tightlist
\item
  Inhibition of MAGL by AM9928 inhibited TNBC trans-migration in 3D
  cultures with BMECs.
\item
  AM9928 decreases levels of inflammatory cytokines and VEGF secretion
  in TNBC cells.
\item
  AM9928 decreased TNBC tumor growth in vivo and colonization in brain.
\end{enumerate}

\emph{What is the clinical significance?}

Development of MAGL inhibitor-based drugs/compounds is a promising
approach for TNBC therapeutics.

AM9928 may provide survival opportunity for TNBC patients and improve
their living conditions.

\emph{BACKGROUND:}

Breast cancer is a common cause of brain metastases occurring in at
least 10-16\% of patients and reaching 25-35\% of TNBC patients
(Hubalek, Hubalek et al., 2017; Yam, Mani et al., 2017; Yates, Knappskog
et al., 2017; Omabe, Ezeani et al., 2014; Da Silva, Cardoso Nunes et
al., 2020). Unfortunately, patients who are diagnosed with brain
metastases often have poor prognosis with short overall survival times
(J\selectlanguage{ngerman}ézéquel, Kerdraon et al., 2019; Boire, Brastianos et al., 2020;
Lehmann, Jovanovi\selectlanguage{polish}ć \selectlanguage{english}et al., 2016; Hu, Kang et al., 2009; Cacho-D\selectlanguage{ngerman}íaz,
García-Botello et al., 2020; Roma-Rodrigues, Mendes et al., 2019; Belli,
Trapani et al., 2018; Nakamura \& Smyth, 2017; Cruceriu, Baldasici et
al., 2020).

Cancer cells endogenously synthesize 95\% of free fatty acids (FFAs) de
novo (Omabe, Ezeani et al., 2015) and incorporate and remodel exogenous
palmitate into structural and oncogenic glycerophospholipids,
sphingolipids and ether lipids (Omabe, Ezeani et al., 2015). Fatty acid
synthesis (FAS) pathway confers a survival advantage to cancer cells and
especially to tumor cells that are resistant to chemotherapy (Omabe,
Ezeani et al., 2015). Fatty acid-binding protein 5 (FABP5) promoted
lipolysis droplets, de-novo fatty acid synthesis, and coordinated lipid
signaling that promoted prostate cancer metastasis (Senga, Kobayashi et
al., 2018; Carbonetti, Wilpshaar et al., 2019). Increased FFA levels in
tumors lead to enhanced tumor aggressiveness by increasing lipid
synthesis mediated by lipolytic pathways (Omabe, Ezeani et al., 2015;
Senga, Kobayashi et al., 2018; Carbonetti, Wilpshaar et al., 2019; Zhu,
Zhao et al., 2016; Zhang, Guo et al., 2020; Li, Gao et al., 2019;
Nomura, Long et al., 2010). Thus, targeting lipid metabolism may prevent
the gain of survival advantage and improve treatment response in cancer
cells.

MAGL is a serine hydrolase that regulates a fatty acid network that
promotes cancer pathogenesis by enriching pro-tumorigenic signaling
molecules. MAGL is a key hydrolytic enzyme in the FFA tumor network
reported in colorectal cancer, neuroblastoma, nasopharyngeal carcinoma,
and other cancers (Zhu, Zhao et al., 2016; Zhang, Guo et al., 2020; Li,
Gao et al., 2019; Nomura, Long et al., 2010). MAGL primarily hydrolyzes
endocannabinoid 2-AG and other monoacylglycerides and indirectly
controls the levels of free fatty acids derived by their hydrolysis.

A new series of highly potent carbamate MAGL inhibitors were synthesized
and characterized in our lab. In this study, we have investigated the
inhibitor AM9928, which exhibited high potency against recombinant human
MAGL with IC\textsubscript{50} value of 8.9nM and lacked any affinity
for the cannabinoid receptors CB1 and CB2 (Tyukhtenko, Ma et al., 2020;
Tyukhtenko, Karageorgos et al., 2016; Tyukhtenko, Rajarshi et al.,
2018). AM9928 demonstrated: (1) potency and selectivity for the target;
(2) suitable physicochemical properties with low lipophilicity (ClogP
3-4); (3) good microsomal stability (\textgreater{}20 min) and plasma
stability (\textgreater{}120 min); and (4) long target engagement by
assessing the time required for MAGL reactivation (residence time)
following the covalent inhibitory interaction between ligand and
protein.

Since MAGL is important for lipid metabolism and lipid metabolism plays
various roles in tumorigenesis, we investigated MAGL's role in the
process of tumor cell extravasation across the BBB and tumor
colonization in brain. MAGL is highly expressed in TNBCs, which secrete
high levels of inflammatory cytokines and chemokines. We hypothesized
that tumor growth in the mammary fat pads and tumor cell infiltration
across the BBB is facilitated via inflammatory chemokines/cytokines
secreted from TNBC cells, leading to activation of HBMECs and resulting
in TNBC spreading and colonization in brain. We found that TNBC adhesion
to HBMECs and transmigration across HBMECs was inhibited by AM9928. In
addition, AM9928 inhibited TNBC's secretion of inflammatory cytokines
and the angiogenic factor VEGF-A. Notably, AM9928 inhibited TNBC tumor
growth in vivo, decreased i\emph{n vivo} BBB permeability and
significantly reduced TNBC colonization in brain. Taken together, these
results demonstrate novel mechanisms by which MAGL mediates its effects
on brain metastasis by modulating BBB permeability.

\emph{Methods:}

\emph{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\emph{. All animal studies were performed in BIDMC.}

\emph{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.

\emph{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
IC\textsubscript{50} 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).

\emph{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\% CO\textsubscript{2} 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.

\emph{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\% CO\textsubscript{2} 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.

\emph{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 8\selectlanguage{greek}μ\selectlanguage{english}m 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 100\selectlanguage{greek}μ\selectlanguage{english}L
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 Ca\textsuperscript{2+} and
Mg\textsuperscript{2+}) 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.

\emph{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).

\emph{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\selectlanguage{ngerman}°C for 48 h and sectioned using a Vibratome 2000 (Leica,
Germany), while the other hemi\selectlanguage{english}-brain was snap\selectlanguage{english}-frozen and stored at \selectlanguage{english}-70degC
for protein analysis or for BBB permeability analysis. Hippocampal brain
tissues were dissected and post\selectlanguage{english}-fixed, and treated with primary
antibodies overnight at 4deg, 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\selectlanguage{english}-1
(1:500 dilution), claudin\selectlanguage{english}-5 (1:500 dilution; Invitrogen), CD31 (1:500),
Pan\selectlanguage{english}-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\selectlanguage{english}-coded sections were imaged with the
LSCM (MRC1024, Bio-Rad).

\emph{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 \emph{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 (5x10\textsuperscript{4} 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\selectlanguage{english} =\selectlanguage{english} 0.52 x (length) x
(width)\textsuperscript{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~\emph{in vivo} ~imaging, spectral fluorescence images were obtained
using a Maestro\selectlanguage{english}-based imaging system (CRI Inc., Woburn, MA, USA)
(Rodriguez, Jiang et al., 2014; Avraham, Jiang et al., 2014). Five sets
of side\selectlanguage{english}-by\selectlanguage{english}-side whole\selectlanguage{english}-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.

\emph{Statistical analysis:} All values are expressed as the mean\selectlanguage{english} +-\selectlanguage{english} SEM
of the mean and are representative of two to three independent
experiments as indicated. \emph{P} values were calculated using unpaired
two-tailed Student's tests, built in the GraphPad Prism Software for
evaluation of statistical significance.

\emph{Results}

\emph{Assessment of The Residence Time of AM4301 and AM9928} : \emph{In
vitro} binding and unbinding of potent AM9928, as compared to another
MAGL inhibitor AM4301, carbamate-based inhibitors to human MAGL (hMAGL)
was monitored by \textsuperscript{1}H NMR spectroscopy using
downfield\textsuperscript{1}H NMR spectral pattern as a probe of
conformational changes (Zhu, Zhao et al., 2016; Tyukhtenko, Ma et al.,
2020; Tyukhtenko, Karageorgos et al., 2016) associated with binding. The
chemical shift perturbations (CSPs) induced by binding in the
characteristic downfield NMR pattern are shown in Figure 1. Addition of
equimolar amounts of inhibitor in both cases caused significant upfield
chemical shift (from 14.9 to \textasciitilde{}13.9 ppm) for the
resonance peak corresponding to the catalytic His269 and smaller shifts
for other downfield resonances. Following the fast formation of hMAGL
covalent adducts with AM4301 (Figure 1a) and AM9928 (Figure 1b), slow
reactivation of the enzyme due to hydrolytic decomposition of adducts
was monitored by real-time NMR spectroscopy. The residence time
(lifetime) of ligand on a target is equal to the lifetime of covalent
adduct which is a period required for adduct to decrease to 1/e (0.367)
of its original concentration. The estimated residence time based on NMR
time course data for AM4301 is 1.4 hours whereas AM9928 residence time
is 46 hours. Significantly longer \emph{in vitro} residence time of
AM9928 in comparison with AM4301 could be translated to its prolonged
pharmacodynamic effect. Based on these observations we selected AM9928
for our studies.

\emph{The toxicity effects and time kinetics of treatment with
AM9928:}The toxicity of AM9928 on TNBC cells were analyzed. The
following doses were examined: 10, 100, 250, or 500 nM, for durations of
24, 48 or 72 hours. No significant toxicity of AM9928 (Figure 2) was
observed on the tested cells.

\emph{MAGL mediates adhesion and transmigration of breast cancer cells
through human brain microvascular endothelial cells (HBMECs):} To
determine the effects of MAGL on TNBCs co-cultured with HBMECs, \emph{in
vitro} adhesion assay was performed using co-cultures of the
brain-seeking variant MDA-MB-BrM2 cells and HBMECs in the presence of
vehicle control or AM9928. MDA-MB-BrM2 cells adhered to the untreated
monolayer by as early as 10 minutes (Figure 3a). Pre-treatment of HBMECs
monolayers with AM9928 (100 ng/ml) blocked the adhesion of the tumor
cells at time points of 10 and 30 minutes (Figure 3a). Next, we examined
the role of MAGL in transmigration of TNBC cells across HBMECs using
an\emph{in vitro} model where HBMECs were co-cultured with breast tumor
MDA-MB-231 cells or with MDA-MB-BrM2 cells (Avraham, Jiang et al.,
2014). Tumor cell transmigration across HBMECs was significantly
inhibited (about 50\%) by AM9928 (Figure 3b).

\emph{Targeting MAGL in human breast cancer cells:} Cytokines are
produced by both immune cells and cancer cells (Parker, Limebeer et al.,
2016; Nagarsheth, Wicha et al., 2017). To characterize the potential
activity of MAGL inhibitor AM9928 on the inflammatory responses in
breast cancer, we have used TNBC cells, the highly invasive MDA-MB-231
and the brain- seeking variant MDA-MB-BrM2 cells, as compared to the
immortalized normal breast epithelial MCF-10A cells.

First, we examined the effects of AM9928 as compared to the vehicle
control on secretion of cytokines/chemokines in cells treated with
either the vehicle control or with AM9928 (250nM) for 72 hours. The
supernatants were collected and analyzed by specific ELISA assays for
IL-6, IL-4, INF-alpha2, TGF-alpha, TNF-alpha, and IL-8. We observed
significant inhibitory effects of AM9928 on IL-6 and IL-8 (Figure
4a-4b). Both inflammatory cytokines are important in tumor-associated
inflammation (Omabe, Ezeani et al., 2015; King, Mir et al., 2017;
Masjedi, Hashemi et al., 2018; Banerjee \& Resat, 2016; Atretkhany,
Drutskaya et al., 2016).

Second, we analyzed the effects of AM9928 on the angiogenic factor
VEGF-A secretion and observed significant inhibitory effects of AM9928
on VEGF-A secretion (Figure 4c).

Third, to determine if chemokines/cytokines are directly involved in
activation of HBMECs, without the contact of tumor cells, we prepared
TNBC-derived exosomes untreated or treated with AM9928 and tested their
direct effects on HBMECs. MDA-MB-231 and MDA-MB-BrM2 -derived exosomes
caused activation of HBMECs leading to secretion of elevated levels of
the pro-inflammatory IL-8 and VEGF-A. AM9928 treated TNBC-derived
exosomes significantly inhibited activation of HBMECs, as observed by
the secretion levels of IL-8 and VEGF-A (Figure 4d-4e).

\emph{In vivo analysis of tumor growth in mammary fat pads and
colonization of TNBCs in brain:} To examine the \emph{in vivo} effects
of AM9928 on TNBC tumor growth and BBB integrity, GFP-4T1-BrM5 cells
were administered (5 x 10\textsuperscript{4} cells) into the mammary fat
pads of BALB/c mice and were injected with either the vehicle control or
with AM9928 as shown in Figure 5a (\selectlanguage{english}\emph{n =} \selectlanguage{english} 10 per group per
treatment). The GFP-4T1-BrM5 tumor cell growth in mammary fat pads and
tumor cell seeding in the brain was observed by \emph{in vivo} imaging
and immunohistochemistry.

Tumor growth in mammary fat pads was significantly reduced in mice
treated with AM9928 as compared to mice treated with vehicle control
(Figure 5b). Quantitation of GFP--4T1\selectlanguage{english}-BrM5 tumor cell colonization in
brain was determined by~\emph{ex vivo} ~imaging of the brain and by
immunohistochemistry using GFP antibody. The expression of GFP positive
tumor cells was significantly lower in the mice group treated with
AM9928, while the presence of GFP--4T1\selectlanguage{english}-BrM5 tumor cells in brain was
significantly higher in the untreated GFP-4T1BrM5 cells and the vehicle
control treated mice (Figure 5c, 5d).

Increased in BBB permeability was found in mice administered with tumor
cells or with vehicle treated mice, while mice treated with AM9928 had
lower BBB permeability changes as compared to control mice at day 28
(Figure 6a). We then analyzed BMEC-tight junction proteins' expression
of ZO-1 and Claudin-5 \emph{in vivo} in normal brain, as compared to TJs
in tumor brains from syngeneic GFP-4T1-BrM5 mammary tumor cell model.
ZO-1 is the main scaffolding protein and claudin-5 is known as the TJ
sealer protein of the BBB. As shown in Figure 6b, both expression of
ZO-1 and claudin-5 were abundant in the normal brain vasculature.
Following GFP--4T1\selectlanguage{english}-BrM5 cells administration to the mammary fat pads,
the GFP-tagged tumor cells migrated across the BBB after about 4 weeks.
The GFP tagged tumor cells were detected by immunostaining with either
Monoclonal Pan-Cytokeratin (Pan-CK) antibodies or GFP antibodies. No
staining of normal brain sections with Pan-ck antibodies was observed
(data not shown). These tumor cells were usually located around the
brain capillaries and in-close proximity to BMECs (BMECS were detected
with CD31 antibodies) (figure 6c). Both ZO-1 and claudin-5 structures
were damaged in the brain tumor vasculature (Figure 6d) as compared to
the control brain (Figure 6b). In some images, both ZO-1 and claudin-5
structures withing BMECs were seen to colocalized with tumor cells,
indicating the proximity of BMECs to the tumor cells in vivo.
Interestingly, following treatment with AM9928, the expression of both
ZO-1 and claudin-5 was less impaired as compared to the mice treated
with vehicle control (Figure 6d).

The number of CD31\textsuperscript{+} vessels expressing TJ proteins,
ZO-1, and claudin-5, was performed and images were analyzed using Adobe
Photoshop CS2. The expression of ZO-1, as determined by average sum of
intensity was 582,096 and following treatment with AM9928 the average
sum of intensity was 610,920 (Figure 6e). The average sum of intensity
of Claudin-5 expression in untreated group was 518,599 and following
treatment with AM9928 the average sum of intensity was 681,457 (Figure
6f). Although these differences in TJ numbers were not statistically
significant between the untreated mice and the treated AM9928 mice, both
ZO-1 and claudin-5 were observed as less damage's structures in the
AM9928 treated mice, as compared to the vehicle treated mice.

In summary, the number of mice positive with mammary tumors and brain
tumors was significantly higher in the vehicle treated group as compared
to AM9928 treated mice (Table 1). Importantly, the number of alive mice
at day 28 was higher in the AM9928 treated mice as compared to the
vehicle control group (Table 1). Thus, AM9928 significantly inhibited
changes in the BBB permeability and reduced TNBC colonization in brain.

\emph{Discussion}

Breast cancer metastases in brain (BCM/B) show significant morphological
and genomic heterogeneity (Hubalek, Hubalek et al., 2017; Yam, Mani et
al., 2017; Yates, Knappskog et al., 2017; Omabe, Ezeani et al., 2014; Da
Silva, Cardoso Nunes et al., 2020; Jezequel, Kerdraon et al., 2019; Hu,
Kang et al., 2009; Cacho-Diaz, Garcia-Botello et al., 2020;
Roma-Rodrigues, Mendes et al., 2019; Belli, Trapani et al., 2018;
Nakamura \& Smyth, 2017). Patients with BCM/B have high mortality
resulted from the brain lesions and are resistant to chemotherapy
treatments (Hu, Kang et al., 2009; Cacho-Diaz, Garcia-Botello et al.,
2020). Metastatic breast tumor cells transmigrate across the blood-brain
barrier (BBB) and form colonies of tumor cells in brain (Boire,
Brastianos et al., 2020; Hu, Kang et al., 2009; Cruceriu, Baldasici et
al., 2020). Under normal conditions, the BBB is a highly selective
barrier due to existence of tight junctions (TJs) between adjacent brain
microvascular endothelial cells (BMECs). However, in the process of
invasion of tumor cells to the brain, inflammatory cytokines and
chemokines secreted by these infiltrating tumor cells disrupt the BBB
integrity by causing damage to the continuous TJ structures formed by
endothelial cells of the brain parenchyma and astrocytes. Although the
BBB prevents the delivery of most therapeutic drugs into the brain, the
circulating metastatic tumor cells invade the damaged BBB to form
colonies in the brain (Hu, Kang et al., 2009; Cacho-Diaz, Garcia-Botello
et al., 2020).

During tumor progression, cells undergo reprogramming of metabolic
pathways that regulate glycolysis and the production of lipids (Omabe,
Ezeani et al., 2014; Omabe, Ezeani et al., 2015; Zhang, Guo et al.,
2020; Li, Gao et al., 2019; Nomura, Long et al., 2010; Tyukhtenko, Ma et
al., 2020; Wang, Nag et al., 2015; Beloribi-Djefaflia, Vasseur et al.,
2016). Since MAGL is important in assigning the lipid stores in the
direction of pro-tumorigenic signaling lipids in cancer cells, we
studied the mechanisms by which MAGL contributes to promoting TNBC
metastases. The energy supply of lipids comes from \emph{de
novo}synthesis rather than circulating lipids during tumor development,
which are modulated and regulated by MAGL (Wang, Nag et al., 2015;
Beloribi-Djefaflia, Vasseur et al., 2016; Taib, Aboussalah et al., 2019;
Yecies \& Manning, 2010).

The assessment of MAGL inhibitors potency and selectivity was performed
to select compounds for the \emph{in vitro} and \emph{in vivo} studies
using the application of protein-based real-time \textsuperscript{1}H
NMR spectroscopy (Senga, Kobayashi et al., 2018; Carbonetti, Wilpshaar
et al., 2019; Zhu, Zhao et al., 2016), which allowed us to determine the
residence time on target for potent MAGL inhibitor AM9928 (46 h) (Figure
1). The longer inhibitory effect of AM9928 strongly suggest prolonged
pharmacodynamic effect of AM9928 which is required for our \emph{in
vivo} studies.

Acute and chronic inflammation~are regulated by interferons, the
interleukins, the chemokine family, mesenchymal growth factors, the
tumor necrosis factor family and adipokines (Tuo, Leleu-Chavain et al.,
2017; Karki, Man et al., 2017). Key pro-inflammatory cytokines involved
in inflammation include interleukin-1 (IL-1), IL-6, tumor necrosis
factor (TNF\selectlanguage{greek}α\selectlanguage{english}), IL-8, IL-12,~IFN-\selectlanguage{greek}γ \selectlanguage{english}and~IL-18. Here, we analyzed the
anti-inflammatory profile by targeting MAGL. Based on our results,
AM9928 (Figure 4) target tumor-inflammatory molecules IL-6 and IL-8 and
the angiogenic factor VEGF-A. These effects were like the effects
observed by siRNA-MAGL, but not with control siRNA (data not shown).

Several genes were shown to be involved in the development of brain
metastases which include cyclooxygenase COX-2, EGFR ligand HBEGF and
\selectlanguage{greek}α-\selectlanguage{english}2,6-sialyltransferase ST6GALNAC5 (Hu, Kang et al., 2009; Bos, Zhang et
al., 2009), all involved in facilitating cancer cell passage through the
BBB. We have previously reported the roles of the proinflammatory
peptide P in impairing the BBB integrity and the angiogenic factor
Angiopoietin-2 in mediating activation of brain microvascular
endothelial cells and BBB impairment, resulting in infiltration of TNBCs
into the brain (Rodriguez, Jiang et al., 2014; Avraham, Jiang et al.,
2014). The TME cells are the source of proinflammatory cytokines
(Cacho-D\selectlanguage{ngerman}íaz, García-Botello et al., 2020; Omabe, Ezeani et al., 2015),
which are secreted by both autocrine and paracrine manner though
activation of STAT3 (Karki, Man et al., 2017; Bahiraee, Ebrahimi et al.,
2019). Indeed, the strong inhibition of IL-6 by AM9928 indicates the
potential of AM9928 to target inflammation associated with TNBC.
Therefore, MAGL may regulate lipid quality and/or quantity to promote
aggressiveness such as migration and inflammation in breast cancer
cells.

VEGF-A/VEGF-R signaling functions as an important survival pathway in
breast cancer cells \textsuperscript{48-50} (Huang, Ouyang et al., 2018;
Mery, Rowinski et al., 2019; Banys-Paluchowski et al., 2018). VEGF-A
promoted tumor cell self-renewal through VEGF-A/VEGF-R/STAT3 signaling
resulting in activation of Myc, Sox2 and STAT3 (Huang, Ouyang et al.,
2018; Mery, Rowinski et al., 2019; Banys-Paluchowski, Witzel et al.,
2018). High VEGF-A levels strongly correlated with both STAT3 and Myc
expression as well as with tumor metastatic potential. We found
significant inhibition of VEGF-A expression by AM9928 (Figure 4c),
suggesting the effectiveness of targeting MAGL in inhibition of
angiogenesis.

Breast cancer-derived exosomes accumulate in the lung, spleen, and bone
marrow of naïve mice. At these sites, the exosomal content rich in IL-6
causes pro-metastatic alterations associated with reduction of T cell
proliferation, NK cell cytotoxicity and modulation of immune responses.
However, the role of TNBC-derived exosomes on HBMECs is not known. Here
we show that HBMECs were activated by TNBC-derived exosomes which
resulted in secretion of IL-8 and VEGF-A, thereby facilitating tumor
transmigration across HBMEC, and promoting tumor colonization. Further,
AM9929 treated TNBC-derived exosomes inhibited HBMECs activation as
indicated by the secretion levels of IL-8 and VEGF-A. Thus, TNBC-derived
exosomes may act as important mediators of intercellular communication
between TNBCs and brain endothelium facilitating the formation of
pre-metastatic niches and contributing to tumor dissemination in brain.

The BCM/B gene signature includes \emph{ANGLT4, MMP1, PTGS2 and
TNC\textsuperscript{16}} . This gene signature facilitates the
metastatic process and changes in endothelial tight junction, cell
migration and invasion. In vivo studies demonstrated that AM9928
significantly inhibited tumor growth in the mammary fat pads (Figure
5b), as compared to untreated mice or mice treated with vehicle control.
Further, AM9928 inhibited tumor cell colonization in the brain as
detected by GFP positive tumor cells ( Figure 5c + 5d). Indeed,
quantitation of GFP--4T1\selectlanguage{english}-BrM5 tumor cell colonization in brain showed
that the expression of GFP-expressing Pan-CK positive tumor cells was
significantly lower in the mice group treated with AM9928, while the
presence of GFP--4T1\selectlanguage{english}-BrM5 tumor cells in brain was significantly higher
in the untreated GFP-4T1BrM5 cells and the vehicle control treated mice
(Figure 5d). Since decrease in tumor colonization in the brain by AM9928
was observed, we then decided to determine BBB permeability. The
permeability of the BBB plays a crucial role in brain metastasis (Boire,
Brastianos et al., 2020; Bos, Zhang et al., 2009; Rodriguez, Jiang et
al., 2014; Avraham, Jiang et al., 2014). The changes in permeability of
the BBB\selectlanguage{english}-BMEC\selectlanguage{english}-TJs are a dynamic process dependent on the interaction
between TNBCs and BMECs. Our results showed that TNBC transmigration
across the BBB resulted in increased BBB permeability and these changes
in BBB permeability were reduced by AM9928 (Figure 6a) . Further, tumor
cells were shown to be in closed proximity to BMECs and the expression
of the tight junctions ZO-1 and Claudin 5 were impaired in the BBB from
mice untreated or treated with vehicle control (figure 6c), while
treatment of mice with AM9928, the expression of both ZO-1 and claudin-5
was observed as less damaged structures. Quantitation of TJs showed
there was lower expression levels of both ZO-1 and claudin-5 in mice
untreated or treated with vehicle control as compared to AM9928 treated
mice (Figure 6e-6f). However, these differences in TJ numbers were not
statistically significant between the untreated mice and the treated
AM9928 mice. Further, importantly, the number of alive mice at day 28
was higher in the AM9928 treated mice as compared to the vehicle control
group (Table 1).

Since AM9928 inhibited tumor growth in the mammary fat pads and
prevented changes in BBB integrity \emph{in vivo,} resulting in reduced
tumor cell colonization in brain, we conclude that MAGL plays a role in
TNBC tumor growth and TNBC transmigration across the BBB. Taken
together, we suggest that targeting MAGL may provide a new approach to
reducing expression of proinflammatory factors as well as inhibiting
tumor growth and tumor cell colonization in brain.

\subsection*{\texorpdfstring{\emph{Acknowledgments}}{Acknowledgments}}

{\label{acknowledgments}}

We thank Shuxian Jiang for her help in the in vivo studies, Yigong Fu
for his technical support and help, Lili Wang for the quantitative
analysis of tight junctions in brain microvascular endothelial cells,
Lauren Graham and Elizabeth C. Ethier for text editing, compilation, and
figures preparations. We thank the following members at the CDD for
providing the MAGL inhibitors: Drs. Shakiru O. Alapafuja, Vidyanand
Shukla, Alexander Zvonok, and Kiran Vemuri.

The funders had no role in study design, data collection and analysis,
decision to publish or preparation of the manuscript. This study was
supported by departmental grants to Hava Karsenty Avraham and Shalom
Avraham.

\subsection*{\texorpdfstring{\emph{Author
contributions}}{Author contributions}}

{\label{author-contributions}}

HKA and SA designed experiments, analyzed data, and wrote the
manuscript; OB performed in vitro experiments and analyzed data; SJ
helped with the in vivo experiments. ST helped with the NMR analysis of
MAGL inhibitors, AM for the synthesis of MAGL inhibitors, MKW, CY and AM
helped with the concept of MAGL as modulator of the BBB breaching.

* All \emph{in vivo} studies were performed in the Division of
Experimental Medicine, BIDMC, Harvard Medical School, Boston and
all\emph{in vitro} studies were performed in CDD at Northeastern
University.

All in vivo studies were approved by BIDMC institutional Animal care and
use committee (IACUC) and the mice were handled in accordance with the
animal care policy of BIDMC, Harvard Medical School and in accordance
with the Declaration of Helsinki.

\emph{Data availability:}

All data generated or analyzed during this study are included in this
published article and its supplemental Information files.

\emph{Competing Interests:} The authors declare no competing financial
interests.

\emph{Funding information:} This study was supported by DOD idea award
BC094909 (HA), DOD idea award BC102246 (HA), DOD idea award
G81XWH-08-1-0625 (SA), departmental grants (to HA and SA). The funders
had no role in study design, data collection and analysis, decision to
publish or preparation of the manuscript.

\par\null

\emph{Abbreviations}

BBB: blood-brain barrier.

BC: breast cancer.

BCM: breast cancer metastasis.

BCM/Brain: breast cancer metastasis in brain.

BMEC: brain microvascular endothelial cells.

ER\textsuperscript{-}: estrogen receptor negative.

ECM: extracellular matrix.

HBMECs: human brain microvascular endothelial cells.

IHC: immunohistochemistry.

IF: immunostaining.

LCM: laser capture microdissection.

RT-PCR: reverse transcription polymerase chain reaction.

TJ: tight junction.

TGL: A triple-modality reporter gene of thymidine kinase (T), GFP (G),
and luciferase (L)

TEER: trans-endothelial electrical resistance.

TME: tumor microenvironment

TNBC: triple negative and basal type breast cancer.

VEGF-A: vascular endothelial growth factor.

WB: western blotting.

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