1
Deciphering the impact of cancer cell´s secretome and its derived-
peptide VGF on breast cancer brain metastasis
Rita Carvalho
1,2,8
, Liliana Santos
3,4,5
, Inês Conde
1,2,8
, Ricardo Leitão
3,4,5
, Hugo R. S. Ferreira
3,4,5
, Célia
Gomes
3,4,5
, Ana Paula Silva
3,4,5
, Fernando Schmitt
2,6,10
, Carina Carvalho-Maia
7
, João Lobo
7,8,9
, Carmen
Jerónimo
7,8
, Joana Paredes
1,2,10
, Ana Sofia Ribeiro
1,2
1
Cancer Metastasis group, i3S – Institute for Research and Innovation in Health, University of Porto, 4200-135 Porto,
Portugal.
2
IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, 4200-465 Porto, Portugal.
3
Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
4
iCBR – Institute for Clinical and Biomedical Research, Faculty of Medicine, University of Coimbra, 3000-
548 Coimbra, Portugal
5
CIBB - Center for Innovation in Biomedicine and Biotechnology, University of Coimbra, 3000-548 Coimbra, Portugal.
6
CINTESIS@RISE, 4200-450 Porto, Portugal
7
Cancer Biology and Epigenetics Group, IPO Porto Research Center (GEBC CI-IPOP), Portuguese Oncology Institute of
Porto (IPO Porto) / Porto Comprehensive Cancer Center Raquel Seruca (P.CCC) & CI-IPOP@RISE (Health Research
Network), R. Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal
8
Department of Pathology and Molecular Immunology, ICBAS - School of Medicine and Biomedical Sciences, University of
Porto, Rua Jorge Viterbo Ferreira 228, 4050-513, Porto, Portugal
9
Department of Pathology, Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center
Raquel Seruca (P.CCC), R. Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal
10
FMUP – Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal.
*Ana Sofia Ribeiro
Adress: R. Alfredo Allen 208, 4200-135 Porto
Phone number: 22 607 4900
To whom correspondence may be addressed. Email: [email protected]
Conflict of interest statement
The authors have no conflicting interests to disclose.
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2
Abstract
Brain metastases (BM) are one of the most serious clinical problems in breast
cancer (BC) progression, associated with lower survival rates and a lack of
effective therapies. Thus, to dissect the early stages of the brain metastatic
process, we have searched for a brain-tropic metastatic signature on BC cells’
secretome, as a promising source for the discovery of new biomarkers involved in
brain metastatic progression.
Therefore, six specifically deregulated peptides were found to be enriched in the
secretome of brain organotropic BC cells. Importantly, these secretomes caused
significant blood-brain barrier (BBB) disruption, as well as microglial activation, in
vitro and in vivo. We identified the VGF nerve growth factor inducible as a brain-
specific peptide, promoting BBB dysfunction similar to the secretome of brain
organotropic BC cells. Concerning microglial activation, a slight increase was also
observed upon VGF treatment.
In a series of human breast tumors, VGF was found to be expressed in both cancer
cells and in the adjacent stroma. VGF-positive tumors showed a significant worse
prognosis and were associated with HER2 overexpression and triple-negative
molecular signatures. Finally, in a cohort including primary breast tumors and their
corresponding metastatic locations to the lung, bone, and brain, we found that VGF
significantly correlates with the brain metastatic site.
In conclusion, we found a specific BC brain metastatic signature, where VGF was
identified as a key mediator in this process. Importantly, its expression was
associated with poor prognosis for BC patients, probably due to its associated
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3
increased risk of developing BM.
Keywords: Breast cancer brain metastasis, brain pre-metastatic niche, brain
signature, VGF
Introduction
Breast cancer (BC) is the most common malignancy and leading cause of cancer
death in women due to distant metastases to the bone, lung, liver, and brain (1).
According to Paget's theory, metastases are a non-random process and are
instead determined by the interaction between cancer cells (the seeds) and the
metastatic microenvironment (the soil) (2-4). For example, BC expresses
chemokine receptors, namely CXCR4 and CCR7, which pair with chemokine
ligands expressed in lymph nodes (CXCL12) and lung (CCL21), thus determining
the destination of metastases (5). Several studies already showed that cancer cells
modulate metastatic niches way before they reach the target organ. Several efforts
are being made to uncover and characterize the molecular factors that are
secreted by cancer cells, aiming to identify metastatic biomarkers. Long before
intravasation, cancer cells communicate with distant metastatic organs, mainly by
secreting soluble factors and/or extracellular vesicles (EVs), that recruit immune
cells, promote resident cells activation, and extracellular matrix (ECM) remodeling,
thereby preparing a supportive and receptive pre-metastatic niche (PMN) to the
growth and survival of cancer cells (6-9). These molecular factors released by
cancer cells collectively compose the “cancer cell secretome”, which is a promising
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source of molecular targets involved in the crosstalk between the primary tumor
and the future metastatic site (8, 10). Importantly, PMNs may influence the success
of metastatic colonization, and understanding its formation and regulation may
help to improve the development of effective therapies.
Although BC survival rates have improved significantly in recent years, owing to
advancements in surveillance and more effective systemic treatments, 10% to
30% of patients with metastatic BC will develop brain metastases (BM) during the
course of their disease (11-14). Breast cancer brain metastasis (BCBM) are a
major cause of poor quality of life and the prognosis of patients remains dismal,
with survival typically measured in months (15-17). Current treatments of BM
include surgery, stereotactic radiotherapy, whole brain radiotherapy and systemic
therapy. However, no effective chemotherapeutic options for BM are available as
opposed to treatments for lung and bone metastases. It is known that BC patients
with HER2 overexpression and triple-negative molecular subtypes metastasize
more frequently to the brain, suggesting that these cancer cells have specific
molecular signatures that award their intrinsic advantage to survive in the brain
tissue (11, 18). The unique composition of the brain, including the blood-brain
barrier (BBB) and specialized resident cells, helps to create a highly protected
environment, which is essential for its proper function and long-term health. Thus,
cancer cell-brain metastatic niche interaction must present distinct properties
compared with other metastatic organs. To prevent and treat BM, it is critical to
identify new molecular drivers responsible for the communication between cancer
cells and the brain microenvironment.
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David Lyden's research have already demonstrated that exosomes secreted by
cancer cells contain specific molecules that allow them to home on target
metastatic organs, a process known as organotropism (9, 19, 20). Particularly, in
the brain, it was recently demonstrated that CEMIP exosomal protein pre-condition
the brain microenvironment, enhancing cancer cell outgrowth (21). In fact,
important efforts have been made to identify players involved in this process and
to understand how cancer cells directly modulate the brain PMN. However, it is still
an unexplored and very challenging area of research.
Therefore, in this work, we aimed to decipher the soluble factors secreted by BC
cells responsible for the remodeling of the brain PMN. Our data demonstrated that
the secretome of brain organotropic BC cells plays an important role in remodeling
the brain PMN, namely by affecting the BBB integrity and microglia activation.
Interestingly, we identified a specific brain secretome signature, being VGF (also
known as VGF nerve growth factor inducible) one of the peptides specifically
deregulated in the secretome of brain organotropic BC cells. Noteworthy, we
validated its impact in the remodeling of the brain PMN and its potential to be a
novel clinical predictive and prognostic biomarker for BM.
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Materials and Methods
Cell Culture
The parental BC cell line MDA-MB-231 (parental 231) was obtained from ATCC
(American Type Culture Collection, Manassas, VA, USA), while its organotropic
BC variants lung (231.lung) (22), bone (231.Bone) (23), brain (231.Brain) (24) and
brain with HER2 overexpression (231.Brain.HER2) (25) clones were obtained from
J Massagué (MSKCC) and P. Steeg (NCI) (Supplementary Figure S1). The
human cerebral microvascular endothelial cell line (hCMEC/D3), was kindly
provided by Pierre-Olivier Couraud (Institute Cochin, Université René Descartes,
Paris, France). The human microglial clone 3 cell line (HMC3) was purchased from
ATCC (ATCC
®
CRL-3304™). Details are provided in Supplementary Materials
and Methods.
Secretomes Preparation
For in vitro assays, we plated BC cells (3x10
5
cells in a 6 well-plate) embedded in
collagen type ӏ (Merck) to mimic the breast ECM.
To study the secretomes impact in vivo, we plated BC cells (6x10
6
cells) embedded
in collagen type I in a T175 flask (Supplementary Figure S2). Details in
Supplementary Materials and Methods.
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In vitro evaluation of brain endothelial cell monolayer integrity
Cell monolayer integrity was determined by measuring the transendothelial flux of
fluorescent 4kDa macromolecule across the endothelial cells (ECs) and the
transendothelial electrical resistance (TEER) as previously described (26). Details
about protocol and treatments are provided in Supplementary Materials and
Methods.
In vitro microglial phagocytosis assay
To evaluate phagocytic capacity, microglia cells were incubated with 0.0025%
(w/w) 1 μm fluorescent latex beads for 75 minutes. After incubation, the total of
microglia cells and the number of ingested beads per cell were counted using
ImageJ (27). Details in Supplementary Materials and Methods.
Mice secretome induced-model
Nude mice were pre-treated with secretomes (140 μg) or TLQP-21 (4.5 mg/kg;
Sigma-Aldrich T1581) via intraperitoneal injection to assess their impact on BBB
integrity and microglia modulation.
Mice follow-up was performed as previously described (28). All the experiments
were conducted with the application of the 3Rs (replacement, reduction, and
refinement) (JP_2016_02 Project, animal ethics committee, and animal welfare
body of i3S) (29). Details are provided in Supplementary Materials and Methods.
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Immunofluorescence assay
Cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes. Brain tissue was
postfixed in 4% PFA for 24 hours. The protocol and antibodies can be found in the
Supplementary Materials and Methods.
Western Blot
Western blot was performed as described previously (30). Details about protocol
and antibodies can be found in the Supplementary Materials and Methods.
Proteomic analysis
100 μg of protein were processed for proteomics analysis following the solid-
phase-enhanced sample-preparation (SP3) protocol as described by Hughes et al
(31). Additional details are provided in Supplementary Materials and Methods.
Kaplan Meier plotter survival analysis for VGF mRNA
Kaplan Meier (KM) plotter online survival analysis tool (https://kmplot.com) was
used to assess the impact of VGF mRNA levels on overall survival (OS) in BC
patients. Additional details are provided in Supplementary Materials and
Methods.
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Primary Breast Cancer Series
A series of 218 primary breast carcinomas diagnosed between 1978–1992, were
retrieved from the Pathology Department, Hospital Xeral-Cíes, Vigo, Spain. Patient
follow-up information was available for the 218 (Supplementary Table S1), with a
maximum follow-up of 120 months after diagnosis. The tumors were characterized
for clinical and pathological features, as previously described (32). All analyses
were performed according to the reporting recommendations for tumor MARKer
prognostic studies (REMARK) recommendations for prognostic and tumor marker
studies. Details are provided in Supplementary Materials and Methods.
Breast Cancer Metastases series
We used two complementary paired primary and metastatic BC series
(Supplementary Table S1). The first one was retrospectively collected from the
archives of the Department of Pathology of the Portuguese Oncology Institute of
Porto (IPO Porto) and includes primary breast tumors that metastasized to the lung
(n=17), bone (n=56), and brain (n=4). The second one is an exclusive BCBM series
with paired primary breast tumors and their corresponding brain metastasis, which
was retrospectively collected from Barretos Cancer Hospital, Brazil.
The present study was conducted with the approval of the Ethical Commission
from both cancer centers, under the national regulative law for the usage of
biological specimens from tumor banks, where the samples are exclusively
available for research purposes in retrospective studies (Ethical approvals:
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Portuguese Oncology Institute of Porto (CES. 64/023) and Barretos Cancer
Hospital/Fundação Pio XII (2-777-372). Details are provided in Supplementary
Materials and Methods.
Immunohistochemistry
Immunohistochemistry for IBA1 and VGF were performed in 3 µm sections. Further
information regarding the protocol and antibodies can be found in the
Supplementary Materials and Methods.
Immunohistochemical evaluation
The expression of VGF was independently evaluated by one pathologist (F.S.)
based on grading systems previously established for other markers (33, 34).
Details are provided in Supplementary Materials and Methods.
Statistical analyses
Prism software (GraphPad,v9.0) and IBM® SPSS® Statistics v.26 were used for
statistical analysis, as detail in Supplementary Materials and Methods.
Results
The secretomes of brain organotropic breast cancer cells affect BBB
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integrity. Our goal was to evaluate the impact of the secretomes produced by
brain organotropic BC cells in the preparation of the brain PMN, with a particular
emphasis on BBB permeability and microglia activation. As models, brain
organotropic BC cells (without or with HER2 overexpression), derived from the
parental 231, were used (supplementary material, Figure S1). To control for
brain metastatic specificity, the results were always compared to data obtained
with 231.Lung and 231.Bone organotropic BC cells.
To investigate the impact of the secretome of brain organotropic BC variants on
the remodeling of the brain PMN, we started by evaluating BBB integrity. We found
that the secretome of both brain organotropic BC variants show a specific and
significant increase in cell permeability (Figure 1A) and decrease TEER (Figure
1B) of hCMEC/D3 monolayers. Interestingly enough, this significant effect was not
seen with the secretome of the parental 231, nor with the ones from the non-brain
organotropic BC variants. By immunofluorescence analyzes, we observed a
significant decrease in β-catenin expression in hCMEC/D3 cells, in the presence
of the secretomes of both brain organotropic BC variants (Figure 1C-D), indicating
an in vitro disruption of intercellular junctions.
These results were further confirmed by the in vivo mice secretome-induced
model. After priming mice with secretomes for 15 days, we showed a significant
and specific impact of the secretome of both brain organotropic BC variants in BBB
permeability, shown by an increased accumulation of a fluorescent dye in the
brain, when compared with the secretome of the parental 231, MCF7 (non-
metastatic BC cells) and serum-free DMEM (control condition) (Figure 1E-F).
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Although the secretome from the non-brain organotropic BC variants also showed
a small impact on the in vivo permeability after region of interest (ROI)
measurement (Figure 1F), this effect was more pronounced and specific when
nude mice were pre-treated with the secretome from both brain organotropic BC
cells. Concomitantly, the brain from these animals showed a decrease in collagen
IV and an increase in albumin expression in the pre-frontal cortex when pre-treated
with the secretome of 231.Brain.HER2 overexpression (Figure 1G), indicating
structural and functional changes in the BBB integrity. Taken together, these data
provide strong evidence that the secretome of brain organotropic BC cells cause
BBB disruption, thus promoting the passage of molecular factors through this
unique brain barrier.
The secretomes of brain organotropic breast cancer cells promote microglia
activation. Some studies demonstrated that microglia can be recruited and
activated during brain metastatic colonization (35-41). However, little is known
about its role in the remodeling of the brain microenvironment in the early stages
of the metastatic cascade, more specifically during the formation of the brain PMN.
In order to understand the impact of the secretomes of brain organotropic BC cells
on microglia activation, HMC3 were pre-treated with the secretomes collected from
the parental 231 and their organotropic BC variants to evaluate their phagocytic
capacity, as well as Stat3 phosphorylation.
Our in vitro data demonstrate that the secretome of both brain organotropic BC
cells promote microglia activation since this treatment significantly increased both
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phagocytic capacity (Figure 2A and supplementary material, Figure S3A), as
well as Stat3 phosphorylation (Figure 2B-C) of microglia cells when compared
with the control condition. Nevertheless, the secretome of the other non-brain
organotropic variants also induced microglia activation, demonstrating that the
previously observed specificity for the BBB is not occurring in this setting.
To validate these findings, we evaluated the role of the several secretomes on
microglia cell population in the cerebral cortex from mouse models. Through
immunohistochemical analysis for IBA1 (a microglia marker) (supplementary
material, Figure S3B), microglia cells were counted in the cerebral cortex, and
the positive area and staining intensity by each cell was quantified, in order to
measure their degree of activation. Interestingly, we demonstrated that the
secretome of parental 231 significantly increased the positive area; however, the
results were more evident with the secretome of 231.Brain.HER2 (Figure 2D). In
addition, this secretome significantly increased the staining intensity per cell
(Figure 2E), an effect not observed with the secretome of parental 231 and the
other organotropic BC cells. These data suggest that factors present in the
secretome of 231.Brain.HER2 trigger a more prominent response by microglial
cells during the brain PMN formation.
The identification of a brain-specific protein secretome signature. In order to
identify a specific brain secretome signature, high-throughput proteomic Label-
Free quantitation analysis of the secretome of parental 231 and their organotropic
BC variants was performed. We identified a total of 9064 peptides, but only 341
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peptides were differentially expressed in the secretome of all organotropic BC cells
compared with the secretome of the parental 231. In detail, within these peptides,
120 were differentially expressed in the secretome of 231.Lung, 75 in 231.Bone,
76 in 231.Brain and 70 in 231.Brain.HER2. We could observe a specific secretome
signature for each metastatic location, as observed by unsupervised hierarchical
clustering analysis (supplementary material, Figure S4A). Interestingly, by
principal component analysis (PCA), we observed that the secretome of parental
231 and the secretome of 231.Lung clusters close to each other, whether the
secretome of both brain organotropic BC variants are more similar to the
secretome of 231.Bone (supplementary material, Figure S4B). To obtain the
specific deregulated peptides present in the secretome of both brain organotropic
BC variants, we intersected the lists of the significantly deregulated peptides. From
this analysis, we identified 6 common peptides differentially expressed in both
brain organotropic BC variants (Figure 3A). Out of these, we focus on VGF due to
its role on brain-related disorders. We further validated that VGF (full-length) was
significantly enriched both at the cell level and in the secretome of both brain
organotropic BC cells (Fig. 3C-D and supplementary material, Figure S4 C).
VGF promotes BBB disruption and microglia activation. We next investigated
the role of VGF on the remodeling of the brain PMN, starting by its impact on BBB
structure and function. We pre-treated hCMEC/D3 cell monolayer with TLQP-21,
a bioactive C-terminal VGF-derived peptide for 24 hours. Interestingly, we found
similar permeability and TEER values as observed with the secretome of
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231.Brain.HER2 when compared with the control condition (Figure 4A-B). Since
TLQP-21 binds to the complement component 3a (C3a) receptor-1 (C3aR1), which
is expressed in brain ECs (42), we further used a C3aR antagonist in the following
experiments. Thus, we co-exposed hCMEC/D3 to the endogenous VGF secreted
by 231.Brain.HER2 or to the exogenous VGF in combination with the selective
antagonist for C3aR. Interestingly, we verified that the increase of endothelial
permeability and the decrease of TEER values induced by exogenous and
endogenous VGF were significantly prevented, proving that C3aR is mediating
VGF-induced BBB hyperpermeability. Furthermore, we observed a significant
decrease in β-catenin and ZO-1 (Figure 4C-E) protein levels in EC monolayers
after treatment with the exogenous TLQP-21 peptide similar to what was observed
with the secretome of 231.Brain.HER2. Accordingly, this result was also blocked
in the presence of the C3aR antagonist.
The same specific effect was also observed for microglia activation. Pre-treatment
with TLQP-21 significantly increased the phagocytic capacity (Figure 5A and
supplementary material, Figure S5A) and Stat3 phosphorylation (Figure 5B-C)
of microglia cells, when compared with control condition. Interestingly, this effect
was abolished when microglia cells were pre-treated with the C3aR antagonist,
inhibiting VGF induced effects (Figure 5A-C and supplementary material,
Figure S5A).
Importantly, these results were validated in vivo. After priming mice with the TLQP-
21 for 3 consecutive days, we showed a significant and specific impact on BBB
permeability, shown by a significant increase in the accumulation of the fluorescent
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dye in the brain, when compared with the control condition (Figure 4F-G).
Immunohistochemical analyzes of the mice' brains also showed that TLQP-21
significantly increased IBA1 positive area of microglia cells (supplementary
material, Figure S5B and Figure 5D), as well as the staining intensity per
microglia cell (supplementary material, Figure S5B and Figure 5E).
Collectively, these findings support the functional role of VGF in impacting BBB
stability, as well as in the activation of microglia cells during early stages of the
brain metastatic cascade, preparing a permissive brain PMN.
VGF expression is a poor prognostic marker for breast cancer patients and
a predictive factor for brain metastases. Further, we decided to evaluate the
clinical impact of VGF on BC prognosis and on BM prediction. For that, we started
to study VGF mRNA expression using the Kaplan Meier plotter and VGF protein
expression using a large series of primary breast carcinomas. Kaplan-Meier
survival analysis revealed that VGF mRNA did not correlate with prognosis (10-
year overall survival (OS): hazard ratio (HR)=1.16; p=0.12), when all molecular
subtypes of BC were considered. However, VGF mRNA expression was
significantly associated with a worse prognosis specifically in triple-negative (10-
years OS: HR=1.84, p=0.0019) and in HER2 overexpressing (10-years OS:
HR=1.93, p=0.022) molecular subtypes (supplementary material, Figure S6),
which correspond to the molecular subtypes that more frequently metastasize to
the brain. Further, we validate this data in a series of primary breast carcinomas,
previously characterized by our group (32, 43). Immunohistochemical staining
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revealed that VGF was preferentially expressed at the cytoplasm of cancer cells
and in some cases in the tumor-adjacent stroma, as shown in representative
images (supplementary material, Figure S7). Since we were studying the soluble
factors secreted by BC cells that could lead to brain PMN remodeling, we
considered VGF positivity when there was concomitant expression in cancer cells
but also in the tumor-adjacent stroma. We found that BC patients with VGF positive
tumors showed significantly lower disease-free survival (DFS) and OS (10-year
DFS: p=0.009; 10-year OS: p=0.011) (Figure 6A). In addition, VGF positivity
significantly correlated with poor prognostic markers in BC, such as estrogen
receptor (ER) negativity (p=0.010), and positive expression for epidermal growth
factor receptor (EGFR) (p=0.021), metabolic proteins ((CAIX (p=0.013) and
GLUT1 (p=0.004)). More importantly, VGF positive tumors were significantly
associated with HER2 overexpression and triple-negative (p=0.029) molecular
subtypes (Table 1).
Finally, the impact of VGF expression was evaluated on the prediction of the
metastatic site. BM series were significantly associated with poor prognostic
features, such as high histological grade, age, and molecular subtype (p<0.0001)
(supplementary material, Figure S8A-C and Table S2). We found that primary
breast tumors that metastasized to the brain were associated with poor prognostic
factors, such as ER (p<0.0001) and progesterone receptor (PR) (p=0.002)
negative expression, as well as higher proliferative index (assessed by Ki67
labelling) (p=0.005), when compared with primary breast tumors that metastasized
to other locations (lung and bone) (supplementary material, Figure S9A). As
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expected, primary breast tumors that metastasized to the brain were mainly from
the HER2 overexpression and triple-negative molecular subtypes (p<0.0001)
(supplementary material, Figure S9B). More importantly, we found that primary
tumors with VGF positive expression were significantly associated with an
increased and specific propensity to metastasize to the brain (60%), when
compared with the metastatic propensity to the lung (20%) or bone (25%)
(p=0.003) (Figure 6B). Moreover, correlating VGF gain or loss of expression in
metastases vs. matched primary breast tumors, we found a significant enrichment
of the maintenance of VGF expression in BM (Figure 6C) when compared with the
other metastatic sites (bone and lung) (p=0.001).
Discussion
BC is the most common cancer among women worldwide, which can metastasize
to several parts of the body, including the brain, strongly contributing to cancer-
related mortality. A major breakthrough to study the brain metastatic process was
the establishment of BC models with a specific tropism to the brain (24).
In this study, we used organotropic MDA-MB-231 cell models to the lung, bone,
and brain, which were embedded in collagen type-I, to better mimic the breast
microenvironment. Additionally, another brain organotropic cell line transfected
with HER2 (231.Brain.HER2) was also included, since its overexpression induces
the occurrence of extensive BM in mice (25). Interestingly, we demonstrated that
the secretome of both brain organotropic BC cells affect BBB integrity when
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compared to the secretome of non-brain organotropic cells, which suggest that
brain organotropic cells secrete specific factors that play an important role in the
remodeling of the brain microenvironment, creating a more permissive site for
cancer cell brain colonization. Importantly, the results were always compared to
data from non-brain organotropic BC secretomes to control for brain metastatic
specificity. Indeed, there are evidence suggesting that factors secreted by cancer
cells promote BBB dysfunction by altering the expression and localization of key
BBB proteins, namely tight junction proteins. This can lead to increased BBB
permeability, allowing cancer cells to enter the brain and potentially form
metastases (21, 44-46). In particular, Lu et al. demonstrated that exosomal lncRNA
GS1-600G8.5 secreted by brain organotropic BC cells increased in vitro BBB
permeability, promoting the passage of BC cells across the EC monolayer (45).
Moreover, it was recently demonstrated that brain organotropic BC cells secrete
CEMIP+ exosomes that can be uptaken by ECs and microglial cells, inducing brain
vascular remodeling and inflammation (21).
In addition to a restrictive vascular barrier, the brain is composed of unique resident
cells, such as microglia, that play an important role in the immune response after
injury. Accordingly, considering cancer progression, microglia cells were described
as being responsible for identifying and attacking cancer cells that have invaded
the brain (47, 48). Nevertheless, some studies suggest that microglia can also
promote the growth and spread of metastatic cells in the brain, contrasting with its
tumor surveillance function (35-41). EI Chen et al. demonstrated that NT-3
promote the growth of metastasis by decreasing microglia cell activation (36). In
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opposition, another study demonstrated that XIST loss in BC cells increased the
release of exosomal microRNA-503, which promote in vitro anti-inflammatory
microglia phenotype (37). Therefore, using the secretome from brain organotropic
BC cells, we looked for microglia modifications and found that, in contrast with the
specific impact of brain derived secretome on BBB integrity, all secretomes from
lung, bone and both brain organotropic cell lines were able to promote microglia
activation, as measured by its in vitro phagocytic capacity and Stat3
phosphorylation. Considering that in vitro microglial cell culture is frequently
performed under artificial conditions, which can alter microglial behavior and limit
the relevance of experimental findings (49, 50), we also evaluated the impact of
the secretomes in microglia using in vivo studies. Importantly, we observed that
only the secretome of brain organotropic BC cells with HER2 overexpression could
promote modifications in microglia of the cerebral cortex, which is the brain region
where metastases occur most frequently (3, 51).
Based on these results, we sought to determine the soluble factors responsible for
these modifications in the brain PMN. The proteomic analysis revealed that 6
peptides were specifically deregulated in the secretome of both brain organotropic
BC models compared to the secretome from lung and bone organotropic cells.
VGF emerged as prominent peptide, since it has been suggested to have a role in
the brain, both under normal and pathological conditions (43, 52). VGF can be
proteolytically processed into multiple bioactive peptides that are involved in brain
biological processes related to neuronal growth, differentiation, synaptogenesis,
and synaptic plasticity, being extensively studied in several brain disorders (43,
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21
53-57). TLQP-21 is the most common VGF derived peptide, which has been
shown to be specifically involved in obesity, diabetes, and neurodegenerative
disorders (55, 58-61). In cancer, VGF impact has been underexplored, although it
has been regarded as a marker associated with poor prognosis in patients with
glioblastoma (62), lung cancer (63), BC featuring a neuroendocrine component
(64). However, nothing is known about its role in the context of BM.
Therefore, we went further to evaluate its impact on the brain pre-metastatic
remodeling. We demonstrated that TLQP-21 affected BBB integrity and promoted
microglia modulation, not only in vitro but, most importantly, in vivo. Still, the effect
of TLQP-21 on microglia modulation was not as pronounced as the secretome of
brain organotropic BC cells, suggesting that other factors may also play a role on
microglia activation. Interestingly enough, a previous study in Alzheimer’s disease
had also shown that TLQP-21 affected the microglia phagocytic capacity (65).
Taken together, our findings suggest that VGF is one of the peptides involved in
brain remodeling, thus creating a brain pre-metastatic microenvironment,
permissive to the success of BMs.
Lastly, the clinical impact of VGF was evaluated in BC samples. We found that
VGF expression (both at tumor and stroma) was significantly associated with a
poor prognosis for BC patients. Actually, a recent study found that VGF plays a
role in conferring resistance to EGFR kinase inhibitors, triggering epithelial-to-
mesenchymal transition (EMT) and being associated with a poor prognosis in
patients with lung adenocarcinoma (63). It is interesting to note that both lung and
BC frequently metastasize to the brain. Furthermore, it has also been shown that
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22
VGF have a dual role in glioblastoma, promoting both the survival and self-renewal
of glioblastoma stem cells, as well as the proliferation of differentiated glioblastoma
cells (62). More importantly, we demonstrated that VGF positive breast tumors
specifically correlated with BM, but not to lung or bone metastases, suggesting
VGF as a potential predictive biomarker for BM in the BC context.
In summary, we revealed a novel function for VGF in brain remodeling, by
promoting BBB disruption and microglia activation. Our data also provide new
insights for the role of VGF as a promising prognostic biomarker and therapeutic
target for BC patients with an increased risk of developing BM.
Acknowledgments
MDA-MB-231 BC organotropic variants were kindly provided by Joan Massagué
laboratory. The MDA-MB-231.Brain.HER2 overexpressing cells were kindly
provided by Patricia S. Steeg´s laboratory (Center for Cancer Research, National
Cancer Institute, Bethesda, MD, USA). We still thank all patients and clinicians for
their participation in this study. Dr. Cristiano Souza, Márcia Marques, Vinícius
Duval da Silva, Alison Barroso and Daniel Preto compiled patient data from
Barretos Cancer Hospital. Jorge Dr. Cameselle-Teijeiro compiled patient data from
the Pathology Department, Hospital Xeral-Cíes, Vigo, Spain. The authors
acknowledge the i3S Proteomics Scientific Platform. The mass spectrometry
technique was performed by Hugo Osório, and the work had the support from the
Portuguese Mass Spectrometry Network, integrated in the National Roadmap of
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23
Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-
01-0145-FEDER-022125). The authors acknowledge the assistance of Nuno
Mendes from the HEMS core facility at i3S. The i3S Scientific Platform HEMS, is
a member of the national infrastructure PPBI—Portuguese Platform of Bioimaging
(PPBI-POCI-01-0145-FEDER-022122).
Funding
This work was funded (in part) by Programa Operacional Regional do Norte and
co-funded by European Regional Development Fund under the project "The Porto
Comprehensive Cancer Center Raquel Seruca" with the reference NORTE-01-
0145-FEDER-072678 - Consórcio PORTO.CCC – Porto.Comprehensive Cancer
Center and by FCT - Fundação para a Ciência e a Tecnologia/ Ministério da
Ciência, Tecnologia e Ensino Superior under the project POCI-01-0145-FEDER-
030625. FCT funded the research grants of RC (SFRH/BD/135831/2018) and IC
(SFRH/BD/14381/2022).
Statement of author contributions
ASR and RC conceived the structure of the work. RC was involved in all
experimental work. RC and IC prepared the secretomes. LS and RL performed the
brain EC culture experiments and APS and CG performed the respective data
analyses. RC, LS, HF and ASR performed the in vivo experiments. CC-M, JL, and
CJ compiled patient data from IPOP. FS performed pathology analysis. RC and
ASR performed all experimental data analyses. RC and ASR wrote the manuscript.
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24
ASR and JP supervised all the work and final-edited the manuscript. All authors
have read and agreed to the published version of the manuscript.
Data Availability
The mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium via the PRIDE (66) partner repository with the
dataset identifier PXD044100. All study data are included in the article and/or
Supplementary Materials and Methods Appendix.
Abbreviations
BBB: blood-brain barrier
BC: breast cancer;
BCBM: breast cancer brain metastasis;
BM: brain metastasis;
BSA: bovine serum albumin;
C3aRA: complement component 3a-receptor antagonist;
C3aR1: complement component 3a receptor-1;
CSC: cancer stem cell;
DFS: disease free survival;
DMEM: Dulbecco’s minimal essential media;
EC: Endothelial cell;
ECM: extracellular matrix;
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25
EGFR: epidermal growth factor receptor;
EMT: epithelial-to-mesenchymal transition;
ER: estrogen receptor;
EVs: extracellular vesicles;
FBS: fetal bovine serum;
hCM3C/D3: human cerebral microvascular endothelial cell line;
HER2: human epidermal growth factor receptor 2;
HMC3: human microglial clone 3 cell line;
HR: hazard ratio;
IFN-y: interferon-y;
IP: intraperitoneal injection;
IV: intravenous injection;
KM: kaplan meier;
NaHCO3: sodium bicarbonate;
NaOH: sodium hydroxide;
NH4Cl: ammonium chloride;
OS: overall survival;
PBS: phosphate buffered saline;
PCA: principal component analysis;
PFA: paraformaldehyde;
PMN: pre-metastatic niche;
PR: progesterone receptor;
ROI: region of interest;
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26
RT: room temperature;
SEM: standard error of the mean;
TEER: transendothelial electrical resistance.
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27
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Tables and Figures legends
Table 1. Association analysis of VGF expression in primary breast tumors with
classical breast cancer poor prognostic factors such as ER (p=0.010), EGFR
(p=0.021), CAIX (p=0.013), GLUT1 (p=0.004) and breast cancer molecular
subtypes (p=0.029). Chi-square test was used to determine associations between
groups and the results were considered statistically significant when the p-value
was lower than 0.05. ER - Estrogen receptor, PR - Progesterone receptor; EGFR
- Epidermal growth factor receptor.
Figure 1. The secretome of brain organotropic breast cancer cells promotes
BBB disruption. (A-D) Human cerebral microvascular endothelial cells (ECs)
(hCMEC/D3 cells) monolayers were treated with serum-free medium (control
condition) and secretomes collected from the parental 231 and their organotropic
breast cancer variants. (A) In vitro permeability using a 4kDa FITC-dextran and (B)
transendothelial electrical resistance (TEER) assay after 24 hours of treatment.
(n=4 biological replicates; the results are expressed as mean ± SEM). (C)
Representative confocal images of β-catenin. β-catenin: green, DAPI: Blue. (Scale
bar corresponds to 20 μm). (D) Quantification of β-catenin. (n=3 biological
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31
replicates; the results are expressed as mean ± SEM). (E) In vivo near-infrared
fluorescent brain imaging after 15 days of treating the mice with serum-free
medium (control condition), the secretome of parental 231 and their organotropic
breast cancer cells, as well as the secretome of MCF7 (a non-metastatic breast
cancer cell line as a negative control). Representative images of mice brains
acquired at 30 minutes post-injection of Cy7.5-dextran 5 kDa. (F) Quantification of
radiant efficiency in equal-sized regions of interest (ROI) corresponding to the top
of the skull (2/3 animals/group; the results are expressed as mean ± SEM). (G)
Representative confocal images of collagen ІV (a marker of the basal membrane
that gives support to brain vessels) and albumin (a marker of BBB disruption)
staining after treatment with serum-free medium (control condition), as well as with
the secretome of 231.Brain.HER2. Collagen ІV: green, albumin: red, DAPI: blue.
(Scale bar corresponds to 20 μm). Statistical significance was assessed using one-
way ANOVA.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 2. The secretome of brain organotropic breast cancer cells impacts
microglia activation. (A) Quantification of microglia phagocytic capacity after
treatment with serum-free DMEM (control condition), IFN-y (positive control), as
well as with the secretomes of parental 231 and their organotropic breast cancer
variants for 8 hours. (n=4 biological replicates; the results are expressed as mean
± SEM). (B) Western blot of Stat3 phosphorylation (pStat3 Tyr705) and total Stat3
(tStat3) in HMC3 cell lysates after 8 hours of treatment with the serum-free DMEM
(control condition), IFN-y (positive control), as well as with the secretomes of
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parental 231 and their organotropic breast cancer variants. (C) Quantification of
Stat3 phosphorylation over total Stat3 normalized to tubulin. (n=3 biological
replicates; the results are expressed as mean ± SEM). (D) Quantification of
microglia positive area and (E) staining intensity per microglia cell in the cerebral
cortex of mice after 15 days of treatment with serum-free DMEM (control
condition), as well as with the secretomes from the parental 231, and their
organotropic breast cancer variants (IBA1- a marker of microglia cells). (2 mice/per
group, 6-8 cerebral cortex independent areas; the results are expressed as mean
± SEM). Statistical significance was assessed using one-way ANOVA .*P < 0.05,
**P < 0.01, ****P < 0.0001.
Figure 3. Brain organotropic breast cancer cells show a specific secretome
signature. (A) Venny diagram to obtain the specific deregulated peptides in the
secretome of both brain organotropic breast cancer variants. (B) Western Blot
analysis to evaluate the expression and the presence of VGF in cell lysates and in
the secretomes. (C) Western blot quantification. (n=4 and 8 biological replicates;
the results are expressed as mean ± SEM). Statistical significance was assessed
using one-way ANOVA .*P < 0.05, ***P < 0.001.
Figure 4. VGF promotes blood-brain barrier disruption. (A-B) Endothelial cells
(ECs) monolayers were treated with TLQP-21 (100nM), a VGF derived-peptide,
and with a secretome of 231.Brain.HER2 in the absence and presence of the C3a
receptor antagonist (10µM) in order to evaluate (A) ECs monolayer permeability
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33
using a 4kDa FITC-dextran and (B) TEER after 24 hours of exposure. (n=4
biological replicates; the results are expressed as mean ± SEM). (C)
Representative confocal images of β-catenin and ZO-1 immunoreactivity. β-
catenin/ZO-1: green, DAPI: Blue. (Scale bar corresponds to 20 μm). (D-E)
Quantification of β-catenin and ZO-1 expression. (F-G) In vivo dynamic near-
infrared fluorescent imaging after 3 days of treatment with serum-free DMEM
(control condition) and TLQP-21 (4.5 mg/kg). (F) Representative images of mice
brains acquired at 30 minutes post-injection of Cy7.5-dextran 5 kDa. (G)
Quantification of radiant efficiency in equal-sized ROI corresponding to the top of
the skull. (4 animals/group; the results are expressed as mean ± SEM). Statistical
significance was assessed using one-way ANOVA.*P < 0.05, **P < 0.01, ***, P <
0.001, ****P<0,0001
Figure 5. VGF impacts microglia activation. (A) Quantification of microglia
phagocytic capacity after treatment with serum-free medium (control condition)
and TLQP-21 (100nM) in the absence and presence of the C3a receptor
antagonist (10µM) for 8 hours. (n=3 biological replicates; the results are expressed
as mean ± SEM). (B) Western blot of Stat3 phosphorylation (pStat3 Tyr705) and
total Stat3 (tStat3) in microglia cell lysates (HMC3) after 8 hours of treatment with
the serum-free medium (control condition) and TLQP-21 in the presence or
absence of C3a receptor antagonist. (C) Quantification of Stat3 phosphorylation
over total Stat3 normalized to tubulin. (n=3 biological replicates; the results are
expressed as mean ± SEM). (E) Quantification of IBA1 microglia positive area and
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34
(G) intensity per microglia cell in the cerebral cortex of mice after 3 days of
treatment with serum-free medium (control condition) and TLQP-21 (4.5 mg/kg).
(2 mice/per group, 6-8 cerebral cortex independent areas; the results are
expressed as mean ± SEM). Statistical significance was assessed using one-way
ANOVA.*P < 0.05, **P < 0.01, ***, P < 0.001, ****P<0,0001.
Figure 6. VGF expression associates with a worse prognosis for breast
cancer patients and is a predictive factor for brain metastases. (A) Kaplan-
Meier plot analysis for overall survival (OS) and disease-free survival (DFS)
(OS:p=0.011 and DFS:p=0.009). (black lines indicate patients with VGF negative
tumors; gray lines indicate patients with VGF positive tumors). Survival analyses
were compared using the log-rank test. (B) Correlation analysis of VGF expression
in primary breast tumors with their corresponding metastatic location. (C) VGF
expression in primary breast tumor and its paired metastasis. Chi-square test was
used to determine associations between groups and the results were considered
statistically significant when the p-value was lower than 0.05.
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Table 1. Association analysis of VGF expression in primary breast tumors
VGF Negative
VGF Positive
frequency
%
frequency
%
p-value
ER
Negative
15
22,4
54
38,0
0,01
Positive
52
77,6
88
62,0
PR
Negative
36
52,9
88
62,0
ns
Positive
32
47,1
54
38,0
HER2
Negative
64
94,1
118
84,9
ns
Positive
4
5,9
21
15,1
EGFR
Negative
67
98,5
128
89,5
0,021
Positive
1
1,5
15
10,5
CAIX
Negative
34
53,1
49
34,8
0,013
Positive
30
46,9
92
65,2
GLUT1
Negative
60
92,3
105
75,5
0,004
Positive
5
7,7
34
24,5
Molecular
subtype
Luminal A-like
27
40,9
44
32,6
0.029
Luminal B-like
26
39,4
46
34,1
HER 2 overexpressing
1
1,5
12
8,9
Triple-Negative
12
18,2
33
24,4
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