Cellular and Molecular Neurobiology

https://doi.org/10.1007/s10571-020-00988-y

ORIGINAL RESEARCH

Exploration of Pericyte‑Derived Factors Implicated in Lung Cancer

Brain Metastasis Protection: A Pilot Messenger RNA Sequencing Using
the Blood–Brain Barrier In Vitro Model
Kenta Ujifuku1 · Takashi Fujimoto2 · Kei Sato2 · Yoichi Morofuji2 · Hideki Muto3 · Hiroshi Masumoto3 ·
Shinsuke Nakagawa4 · Masami Niwa5 · Takayuki Matsuo1

Received: 9 June 2020 / Accepted: 23 October 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Metastatic brain tumors have poor prognoses and pose unmet clinical problems for the patients. The blood–brain barrier
(BBB) implication is supposed to play a major role in brain metastasis. However, the role of pericytes remains to be eluci-
dated in the brain metastasis. This pilot study described the expression profile of interactions between pericytes, endothelial
cells, and cancer cells. We applied an in vitro BBB model with rat primary cultured BBB-related cells (endothelial cells
and pericytes), and performed the gene expression analyses of pericytes under the lung cancer cells coculture conditions.
Pericytes demonstrated inhibition of the cancer cell proliferation significantly (p < 0.05). RNA was extracted from the peri-
cytes, complementary DNA library was prepared, and RNA-seq was performed. The sequence read data were analyzed on
the Management and Analysis System for Enormous Reads and Tag Count Comparison-Graphical User Interface platforms.
No statistically or biologically significant differentially expressed genes (DEGs) were detected in the explanatory analyses.
Lot-specific DEG detection demonstrated significant decreases in the expression of two genes (Wwtr1 and Acin1), and
enrichment analyses using Metascape software revealed the inhibition of apoptotic processes in fibroblasts. Our results sug-
gest that the expression profiles of brain pericytes are partially implicated in the prevention of lung cancer metastasis to the
brain. Pericytes exerted an anti-metastatic effect in the BBB model, and their neurohumoral factors remain to be elucidated.

Keywords Blood–brain barrier · Pericytes · Brain metastasis · Lung cancer · RNA-seq

Electronic supplementary material The online version of this

Introduction
article (https​://doi.org/10.1007/s1057​1-020-00988​-y) contains
supplementary material, which is available to authorized users. Metastatic brain tumors have poor prognoses and pose
unmet clinical problems for the patients (Bray et al. 2018).
* Kenta Ujifuku
Although the overall prognosis of cancer has greatly
kujifuku‑nag@umin.ac.jp
improved, brain metastasis remains to be an important issue.
1
Department of Neurosurgery, Nagasaki University A surveillance system showed the development of metastatic
Graduate School of Biomedical Sciences, 1‑7‑1 Sakamoto, brain tumors in 9.6% of 170,000 cancer patients. (Barnholtz-
Nagasaki 852‑8501, Japan
Sloan et al. 2004) The median overall survival for metastatic
2
Department of Neurosurgery, Nagasaki University Hospital, brain tumors remains at approximately 6 months. Accord-
1‑7‑1 Sakamoto, Nagasaki 852‑8501, Japan ing to a National Survey Report on Brain Tumors, about
3
Biomedical Research Support Center, Nagasaki University 30% of cancer patients who had brain metastasis died due
School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852‑8523, to brain metastasis (Committee of Brain Tumor Registry of
Japan
4
Japan 2017). Furthermore, brain metastasis could cause the
Department of Medical Pharmacology, Nagasaki University patients’ quality of life to decline because of neurological
Graduate School of Biomedical Sciences, 1‑12‑4 Sakamoto,
Nagasaki 852‑8523, Japan deficits and might well limit the treatment strategy. Because
5 standard treatment for the prevention of brain metasta-
BBB Laboratory, PharmaCo-Cell Company Ltd.,
Dai‑ichi‑senshu bldg. 2nd floor, 6‑19 Chitose‑machi, sis remains to be established, it is crucial to elucidate its
Nagasaki 852‑8135, Japan mechanism.

13
Vol.:(0123456789)

The blood–brain barrier (BBB) is supposed to play an
important role in brain metastasis protection. Endothelial
cells, astrocytes, and pericytes are the main components
of the BBB, and they facilitate BBB functions induced by
their crosstalk. Although the BBB is seemed to be resistant
to hematogenous metastasis, cancer cells invade the BBB
and brain parenchyma via unknown mechanisms. Some
authors have reported the interactions between astrocytes
and metastatic cancer cells (Wasilewski et al. 2018). In con-
trast, the role of pericytes remains to be elucidated in the
brain metastasis. In terms of in vitro BBB models, primary
cultured cells have the advantage of preserving the in vivo
physiological characteristics, especially tight junction func-
tions (Hayashi et al. 2004; Nakagawa et al. 2007, 2009).
Fujimoto and colleagues reported their original in vitro BBB
model, consisting of primary cultures of rat brain endothelial
cells, astrocytes, pericytes, and two lung cancer cell lines, to
demonstrate the interaction between lung cancer cells and
BBB-related cells (Fujimoto et al. 2019). The present study
examined the differentially expressed gene analysis of peri-
cytes in the in vitro BBB and lung metastasis model.
Materials and Methods
Materials
All reagents were purchased from Sigma (St. Louis, MO,
USA) otherwise described. Wistar rats were obtained
from Japan SLC, Inc. (Hamamatsu, Japan). All animals
were treated in strict accordance with the National Insti-
tutes of Health Guide for the Care and Use of Labora-
tory Animals (NIH Publication No. 80–23). The insti-
tutional review boards approved the animal experiment
Fig. 1  The scheme of this
report. A549 is a cell line that
has low metastatic potential.
KNS-62 is a cell line that has
high metastatic potential
13
Cellular and Molecular Neurobiology
(number 0704190570) and the RNA-seq experiment (num-
ber 18070906-2).
The human NSCLC cell line A549 was purchased from
the American Type Culture Collection (Manassas, VA, USA)
and the KNS-62 cell line was obtained from the Japanese
Collection of Research Bioresources (JCRB, Japan). A549
human lung adenocarcinoma cells, which were derived from
a human alveolar cell carcinoma, were used to establish vari-
ant cell lines with low metastatic potentials. KNS-62 cells
were derived from brain metastases of a human squamous
cell carcinoma of the lung. Cancer cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM)/F12 sup-
plemented with 10% fetal bovine serum (FBS).
Lung Cancer Brain Metastasis Analysis Using In Vitro
BBB Models
We established in vitro BBB models with rat primary cul-
tured BBB-related cells (endothelial cells and pericytes) and
investigated the relationship between BBB-related cells and
two metastatic cancer cell lines.
Briefly, primary cultures of rat brain endothelial cells and
pericytes were prepared from 3-week-old rats as previously
described, and the BBB model of endothelial cells cocul-
tured with pericytes (E0P) was established (Nakagawa et al.
2009). Pericytes (1.0 × 105 cells/well) were seeded on the
bottom side of the collagen-coated 24-well plate, and let
to adhere firmly for overnight. Rat brain endothelial cells
(1.0 × 105 cells/well) were seeded on the upper side of the
collagen and fibronectin-coated polyester membrane inserts
(0.4-µm pore size) placed in the well of the 24-well culture
plates as shown in Fig. 1. We used three biologically dif-
ferent lots of pericytes. The day when the endothelial cells
were plated was defined as day 0. From day 0, BBB models
Cellular and Molecular Neurobiology
were maintained in DMEM/F12 supplemented with 10%
fetal bovine plasma derived from serum (Animal Technolo-
gies, Inc., Tyler, TX, USA), basic fibroblast growth factor
(1.5 ng/mL; Roche Applied Sciences, Penzberg, Germany),
heparin (100 μg/mL), insulin (5 μg/mL), transferrin (5 μg/
mL), sodium selenite (5 ng/mL) (insulin–transferrin–sodium
selenite medium supplement), gentamycin (50 μg/mL), and
hydrocortisone (500 nM). Under these conditions, in vitro
BBB models were established within 3 days after seeding the
cells. On day 3, the medium was replaced by DMEM/F12
supplemented with 10% FBS, and cancer cells (1.0 × 104
cells/well) were seeded on the luminal membrane. BBB
models with cancer cells were incubated for 12–24 h in a
humidified atmosphere with 5% C ­ O2 (Fujimoto et al. 2019).
Nine batches of rat cerebral pericytes were obtained from
each well of culture plates (Fig. 1).
Functional evaluation of tight junctions in BBBs and cell
proliferation assays were performed, as previously reported
(Fujimoto et al. 2019).
RNA Purification and Library Generation
Total RNA was extracted from Rat cerebral pericytes from
the BBB model using the Qiagen miRNeasy mini kit (cat#
217004, Qiagen) according to the manufacturer’s instruc-
tions (Fig. 1). Samples were lysed in QIAzol lysis reagent,
chloroform was added, and the extract was shaken and then
centrifuged at 12,000×g to separate the organic and aque-
ous phases. Total RNA was purified from the aqueous phase
using the kit spin column. The RNA concentration was
measured using the NanoDrop (Thermo Fisher Scientific)
and RNA integrity was measured with an Agilent 2100 bio-
analyzer. All samples had RIN values above 8.
RNA-seq libraries were prepared with the QuantSeq
3′mRNA-Seq Library Prep Kit-FWD (cat #15, Lexogen,
Vienna, Austria) following the manufacturer’s instructions
using more than 250 ng of total RNA per library (Ma et al.
2019).
Sequencing/Data Analysis of RNA‑seq Reads
The pooled libraries were sequenced on an Illumina MiSeq
instrument (Illumina K.K., Tokyo, Japan), and sequence
data quality was evaluated by FastQC (https​://www.bioin​
forma​tics.babra​ham.ac.uk/proje​cts/fastq ​c/). Sequencing
reads were mapped to rn5 with TopHat (v.2.0.6). Samtools
(v.0.1.18) was used to convert files with the TopHat2 pro-
gram. An estimation of transcript abundance was calculated,
and the count values were normalized to the upper quartile
of the fragments per kilobase of exon per million fragments
mapped reads (FPKM) using CuffLinks (v.2.0.21). Gene
lists for the following enrichment analysis were generated
using the output from the program gffcompare (v.2.0.2).
Mapped reads were counted with HTSeq (v.0.5.3p9) for the
explanatory and differentially expressed gene (DEG) analy-
sis between pericytes and lung cancer cell lines. All of these
analyses were performed on the Management and Analysis
System for Enormous Reads (Maser) platform (Kinjo et al.
2018). For performing explanatory analysis and DEG, the
Tag Count Comparison-Graphical User Interface (TCC-
GUI) platform was utilized (https​://infin​itylo​op.shiny​apps.
io/TCC-GUI/), which uses R, R Studio, and the R packages
EdgeR, DEseq2, and baySeq (R Core Team 2019; Su et al.
2019; Hardcastle and Kelly 2010; Love et al. 2014; Rob-
inson et al. 2009). Metascape (https​://metas​cape.org/) was
used for the gene set enrichment analysis (Huang da et al.
2009; Zhou et al. 2019).
The original datasets are available in DNA Data Bank of
Japan Sequence Read Archive under accession number from
DRR219358 to DRR219366.
Results
The scheme of this study is described in Fig. 1. From the
BBB model experiments, cell proliferation was inhibited
during pericytes preparation only in KNS-62, a highly inva-
sive lung cancer cell line. This effect was not significantly
found in A549, a lower infiltrating lung cancer cell line. This
effect was presumed to be a neurohumoral effect from the
characteristics of the model, as previously described (Fuji-
moto et al. 2019).
Tophat2-HTSeq was performed on Maser and the DEG
tag-count-list was created (List 1). This spreadsheet was
uploaded to TCC-GUI, and EdgeR data analyses were per-
formed on the platform. MA plots and volcano plots were
made to compare the differential expression profile between
the control group and tumor burden groups.
Upregulated 68 genes and downregulated 138 genes
in the KNS-62 group, and 82 upregulated genes and 191
downregulated genes in the A549 group were detected. More
downregulated genes were found than upregulated genes
(Fig. 2).
Cytokine expressions were drawn but no significant dif-
ferences were found (Fig. S1).
The explanatory analysis was performed on TCC-GUI.
From the results of clustering and multi-dimensional scal-
ing, the clusters were divided depending not based on the
tumor burden but on the lot number of pericytes, and the
average silhouette score was − 0.088 (Fig. 3) (Sun et al.
2013). Therefore, it seems likely that a biologically mean-
ingful comparison cannot be made even if some DEGs are
detected in the tumor burden treatment.
Lot-specific DEG analyses were also performed. Using
Tophat2-CuffLinks2-CummRbund on Maser, both WW
domain-containing transcription regulator 1 (Wwtr1) and
13
 Cellular and Molecular Neurobiology

Fig. 2  MA plots (a KNS-62 group, b A549 group) and Volcano plots 68 upregulated genes and 138 downregulated genes in the KNS-62
(c KNS-62 group, d A549 group) compared with the control group group, and 82 upregulated genes and 191 downregulated genes in the
from TCC analyses (EdgeR). Comparing to the control pericytes, A549 group are detected

Fig. 3  Hierarchical clustering (a) and multi-dimensional scaling (b) inappropriate. X, lot number. C, control. K, KNS-62. A, A549. The
of exploratory analysis on TCC-GUI. The cluster separation depends numbers indicate their batches of pericytes. For example, X1C indi-
not on cell line preparation but on the lot number of pericytes. The cates lot number 1 from the control well, and X2K represents lot
average silhouette score is − 0.088. This clustering appears to be number 2 from KNS-62 loaded well

13
Cellular and Molecular Neurobiology
apoptotic chromatin condensation inducer 1 (Acin1) showed
significant decreases of p < 0.05 and q < 0.1 (List 2). Gene
lists for enrichment analyses were created by extracting
genes showing a difference of p < 0.05, and q values were
not considered in these lists. Based on the results of BBB
model experiments, we assumed that DEGs of KNS-62 cells,
not including DEGs of A549 cells, were involved in cell
growth suppression. Upregulated 142, 259, and 392 genes
and downregulated 252, 134, and 251 genes were shared
between KNS-62-related DEGs and A549-related DEGs in
the lot numbers 1, 2, and 3, respectively. These were manu-
ally excluded in the list 3A and list 3B. The gene lists were
merged using spreadsheet software, and enrichment analy-
ses were performed in Metascape based on the gene lists
(List 3A and 3 B). The enrichment analyses revealed the
downregulation of fibroblast apoptotic process induced by
pericytes (Fig. 4).
Discussion
In a previous study, we demonstrated that pericytes exert
an anti-metastatic effect (Fujimoto et al. 2019), which led
to the present study. KNS-62 was used as a higher meta-
static cancer cell line and A549 was selected as a control
cancer cell line (Fig. 1). At first, we hypothesized that some
cytokines might be implicated in the metastatic process
through the BBB. In respect of pericyte implication, this
hypothesis should be declined (Fig. S1). Especially, we
considered fibroblast growth factor (FGF) 2 as a provoca-
tive factor of the BBB based on our previous glioma model
study but seemed to be less relevant (Toyoda et al. 2013).
In the present BBB model, it is probable to assume some
neurohumoral factors other than cytokines (Figs. 1 and 5).
Other candidates, such as exosome-related mechanisms, are
supposed but remain to be detected.
Existing pipeline analysis tools, such as Maser, tended to
be prioritized for major model organisms, such as humans,
mice, and C. elegance. At the time of the first analysis of
this research, the basic data processing had been completed,
but the detailed analysis stopped halfway due to unknown
reasons. So we decided to use TCC-GUI together.
We used three lots of biologically different pericytes
derived from three rat individuals. When examining cluster
analyses and silhouette scores, the transcriptome differences
between the lots of pericytes are larger than those of tumor
burden treatments, and a meaningful analysis cannot be cre-
ated by direct comparison of DEG analysis (Sun et al. 2013).
Heterogeneity of pericytes may influence on the result
(Holm et al. 2018). When performing similar NGS analyses
in the future, researchers must consider whether individual
differences and/or heterogeneity of pericytes should be con-
cerned about each pericyte experiment.
We tried to salvage the RNA-seq data to detect biologi-
cally precise DEGs. The implication of Wwtr1 and Acin1
was found in DEG analysis, and the fibroblast apoptotic
regulation process was considered as a decreased biological
process in the enrichment analysis (Zhou et al. 2019).
Acin1 is a caspase-3-activated protein required for apop-
totic chromatin condensation (Ishikawa et al. 1998; Sahara
et al. 1999). It may function as a part of fibroblast apop-
totic process regulation, but its functional details remain
unknown. Cancer-associated fibroblasts (CAFs) are known
to facilitate tumor invasion and metastasis. The normal brain
parenchyma generally lacks fibroblasts, whereas metastatic
lesions contain connective tissue and extracellular matrix
(ECM). Platelet-derived growth factor-β/Platelet-derived
growth factor receptor β signaling induces pericyte-to-
fibroblast transition (PFT) and promotes tumor invasion
and metastasis. The PFT cells obtain stromal fibroblast and
myofibroblast markers, and co-implantation of PFT and
tumor cells increases tumor dissemination and invasion
(Hosaka et al. 2016). Perivascular cells (vascular smooth
muscle cells and pericytes) lose their traditional pericyte
markers in response to tumor-secreted factors and exhibit
increased proliferation, migration, and ECM production.
Kruppel-like factor 4 (Klf4) promotes the less differenti-
ated state and fibronectin-rich pro-metastatic niche forma-
tion (Murgai et al. 2017). Thus, pericytes are an important
source of fibroblasts in metastatic brain tumors. CAFs pro-
vide a metastasis-promoting microenvironment for cancer
cells, and cancer-fibroblast-vascular crosstalk activates
tumor angiogenesis (De Palma et al. 2017). In this context,
the downregulation of the fibroblast apoptotic process can
be a candidate of the pericyte response to the brain metasta-
sis of tumor cells. However, FGF1 and FGF2 did not seem
to implicate in this process (Fig. S1), and it is unknown
whether this process inhibits tumor cell proliferation or not.
Wwtr1 is also called as transcriptional coactivator with
PDZ-binding motif (TAZ) and is a transcriptional coacti-
vator (Kanai et al. 2000). Yes-associated protein (YAP)/
TAZ association was reported to be important for mecha-
notransduction (Dupont et al. 2011; Zanconato et al. 2016).
The pericyte-like spread of cancer cell lines, including lung
cancer, was reported to activate YAP and myocardin-related
transcription factor via cell adhesion molecule L1 (L1CAM)
mechanotransduction signaling for metastatic colonization.
L1CAM activated YAP by engaging β1 integrin and inte-
grin-linked kinase. Metastatic cell-initiated outgrowth has
been reported to occur via L1CAM-YAP signaling in the
perivascular microenvironment (Er et al. 2018). Since peri-
cytes also used L1CAM for perivascular spreading, pericyte
function during the process can be considered to competi-
tively inhibit metastasis (Lugassy et al. 2019). Wwtr1/TAZ
also can be a candidate for anti-metastatic pericyte response.
Our data support the competitive inhibition theory. However,
13

Fig. 4  Metascape enrichment analysis. Wwtr1 and Acin1 show sig-
nificant decreases of p < 0.05 and q < 0.1 on the original data, so these
are suspected as differentially downregulated genes in this model
(List 2). Another gene lists are created from differentially expressed
genes in KNS-62 only (a, List 3A, and 3B). Upregulated 142, 259,
and 392 genes and downregulated 252, 134, and 251 genes are shared
13
Cellular and Molecular Neurobiology
between DEGs in KNS-62 model and A549 model in the lot num-
bers 1, 2, and 3 respectively. These are excluded in the list 3A and
list 3B. Q-values are not considered in list 3A and 3B. b The analy-
sis obtained from upregulated genes and c the analysis derived from
downregulated genes. The fibroblast apoptotic process may be down-
regulated in pericytes
Cellular and Molecular Neurobiology
③
① performed on the NIG supercomputer at ROIS National Institute of
② the English language review.
Endothelial cells Pericytes Cancer cells
Fig. 5  Scheme of possible interactions between BBB and metastatic
cancer cells. Direct stimulation (1) and/or indirect stimulation via
endothelia (2 and 3) can be considered in this model. It is probable to
assume some unknown neurohumoral factors
the definitive neurohumoral factor remains unknown in the
present BBB model study.
The burden of RNA-seq analysis continues to decrease.
Even a wet-lab specialist could perform the pipeline analysis
described in this study using Maser and TCC-GUI platform,
without using a character user interface, and without proper
bioinformaticians (Kinjo et al. 2018; R Core Team 2019;
Su et al. 2019). Of course, there are some limitations to the
current study. Only pericyte DEGs were considered in this
study and indirect stimulation via endothelia can be impli-
cated in this anti-metastatic process (Fig. 5). The source data
of enrichment analysis of the pericyte transcriptome were
subjectively selected by the authors based on the pericyte
lot number. Statistical power may not be sufficient because
DEGs were selected based on p-values only, and q-values
(i.e., false discovery rates) were not considered in this analy-
sis. The sample size may be insufficient for the detection of
DEGs. We applied a unique cDNA library preparation kit
for 3′ RNA sequencing (Ma et al. 2019), so DNase was not
used in RNA purification in the present study. The depth
of reading on the next-generation sequencer was 5 million
leads, and this might be insufficient to detect DEGs.
Conclusion
We reported RNA-seq transcriptome analysis of pericytes in
our in vitro BBB and lung cancer metastasis models. Indi-
vidual differences and/or heterogeneity of pericytes can have
strong influences on experimental biology. The results of our
RNA-seq enrichment analysis may reflect the implementa-
tion of pericyte-induced competitive inhibition of cancer
cells. Some pericyte-related tumor-resistant neurohumoral
factors, maybe excluding inflammatory and well-known
major cytokines, are expected and remain to be detected.
Acknowledgements Portions of this work were presented at the 37th
Annual Meeting of the Japan Society for Neuro-Oncology, Nanao-
City, Ishikawa, Japan, December 2, 2019 (Ujifuku et al. 2019). We
referred to Online Mendelian Inheritance in Man (OMIM) (Amberger
et al. 2011) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
(Kanehisa and Goto 2000) in this study. Computations were partially
Genetics. The authors would like to thank Enago (www.enago​.jp) for
Author Contributions KU, TF, YM, and SN contributed to conception
and design. KU, TF, and YM contributed to development of methodol-
ogy. KU, TF, KS, and HM contributed to acquisition of data. KU and
HM are involved in analysis and interpretation of data (e.g., statistical
analysis, biostatistics, computational analysis). KU, YM, and SN are
involved in writing, review, and/or revision of the manuscript. MN and
TM contributed to study supervision.
Funding This work was supported by JSPS and HAS under the Japan-
Hungary Research Cooperative Program (to Y.M.) and, in part, Grants-
in-Aid for Scientific Research from JSPS KAKENHI (C)17K10839 (to
K.U.), (C)17K10869 (to T.M.), and (C) 17K10840 (to Y.M.), and Plat-
form Project for Supporting Drug Discovery and Life Science Research
(Basis for Supporting Innovative Drug Discovery and Life Science
Research; BINDS) from AMED under Grant Number JP17am0101001.
Compliance with Ethical Standards
Conflict of interest The authors declare no conflict of interest and fi-
nancial disclosure of this study.
Ethical Approval All applicable international, national, and/or institu-
tional guidelines for the care and use of animals were followed.
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