International Journal of

Molecular Sciences
ArticleDistinct Localization of Mature HGF from its
Precursor Form in Developing and Repairing
the Stomach
Nawaphat Jangphattananont 1,† , Hiroki Sato 1,2,† , Ryu Imamura 1,2 , Katsuya Sakai 1,2 ,Yumi Terakado 3 , Kazuhiro Murakami 3 , Nick Barker 3,4 , Hiroko Oshima 2,5 ,Masanobu Oshima 2,5 , Junichi Takagi 6 , Yukinari Kato 7 , Seiji Yano 2,8and Kunio Matsumoto 1,2,9, *
1

2

3

4
5
6
7
8
9

*
†

Division of Tumor Dynamics and Regulation, Cancer Research Institute, Kanazawa University, Kakuma,
Kanazawa 920-1192, Japan; nawaphatice@gmail.com (N.J.); hiroki.sato@staff.kanazawa-u.ac.jp (H.S.);
imamura@staff.kanazawa-u.ac.jp (R.I.); k_sakai@staff.kanazawa-u.ac.jp (K.S.)
WPI-Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kakuma, Kanazawa 920-1192, Japan;
hoshimam@staff.kanazawa-u.ac.jp (H.O.); oshimam@staff.kanazawa-u.ac.jp (M.O.);
syano@staff.kanazawa-u.ac.jp (S.Y.)
Division of Epithelial Stem Cell Biology, Cancer Research Institute, Kanazawa University, Kakuma,
Kanazawa 920-1192, Japan; y.terakado@staff.kanazawa-u.ac.jp (Y.T.);
kmurakami@staff.kanazawa-u.ac.jp (K.M.); nicholas.barker@imb.a-star.edu.sg (N.B.)
A*STAR Institute of Medical Biology, Singapore 138648, Singapore
Division of Genetics, Cancer Research Institute, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan
Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University,
Osaka 565-0871, Japan; takagi@protein.osaka-u.ac.jp
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine/New Industry
Creation Hatchery Center, Tohoku University, Sendai 980-8575, Japan; yukinarikato@med.tohoku.ac.jp
Division of Medical Oncology, Cancer Research Institute, Kanazawa University, Kakuma,
Kanazawa 920-1192, Japan
Tumor Microenvironment Research Unit, Institute for Frontier Science Initiative, Kanazawa University,
Kanazawa 920-1192, Japan
Correspondence: kmatsu@staff.kanazawa-u.ac.jp; Tel.: +81-76-264-6745; Fax: +81-76-234-4513
These authors contributed equally to this work.

Received: 21 May 2019; Accepted: 13 June 2019; Published: 17 June 2019


Abstract: Hepatocyte growth factor (HGF) is secreted as an inactive single-chain HGF (scHGF);
however, only proteolytically processed two-chain HGF (tcHGF) can activate the MET receptor.
We investigated the localization of tcHGF and activated/phosphorylated MET (pMET) using
a tcHGF-specific antibody. In day 16.5 mouse embryos, total HGF (scHGF + tcHGF) was mainly
localized in smooth muscle cells close to, but separate from, MET-positive epithelial cells in
endodermal organs, including the stomach. In the adult stomach, total HGF was localized in smoothmuscle cells, and tcHGF was mainly localized in the glandular base region. Immunostaining for
pMET and Lgr5-driven green fluorescent protein (GFP) indicated that pMET localization overlappedwith Lgr5+ gastric stem cells. HGF promoted organoid formation similar to EGF, indicating thepotential for HGF to promote the survival and growth of gastric stem cells. pMET and tcHGF
localizations changed during regeneration following gastric injury. These results indicate that
MET is constantly activated in gastric stem cells and that the localization of pMET differs from theprimary localization of precursor HGF but has a close relationship to tcHGF. Our results suggest
the importance of the microenvironmental generation of tcHGF in the regulation of development,
regeneration, and stem cell behavior.

Int. J. Mol. Sci. 2019, 20, 2955; doi:10.3390/ijms20122955

www.mdpi.com/journal/ijms

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Keywords: HGF; MET receptor; regeneration; smooth muscle cell; stem cell

1. Introduction
Hepatocyte growth factor (HGF) and its receptor MET play roles in embryonic development
and the repair of tissues following injury [1–3]. HGF is a fibroblast-derived morphogen that induces
epithelial branching duct formation [4,5]. Scatter factor, a fibroblast-derived cell motility factor for
epithelial cells, is identical to HGF [6]. In previous studies, the secretion of HGF was not notedin epithelial cells, but was in fibroblasts, vascular smooth muscle cells, and hematopoietic cells in
culture [7,8]. Subsequent studies revealed the roles of HGF in the growth and morphogenesis ofdifferent types of epithelial cells and tissues as a mediator of epithelial–mesenchymal interactions [9–13].
Mice lacking HGF were found to exhibit embryonic lethality due to impaired development of
the placenta and liver [14,15]. In addition, MET-deficient mice had no skeletal muscle in the limbs
and diaphragm because of the impaired migration of myogenic precursor cells to the limb buds anddiaphragm [16]. The characterization of conditional MET-deficient mice via analyses on a variety ofcell types revealed that the HGF–MET pathway promotes tissue regeneration and protection, andsuppresses the progression of chronic inflammation and fibrosis [3,17–20].
HGF is a glycosylated protein composed of α- and β-chains linked by a disulfide bridge. It isbiosynthesized and secreted as single-chain HGF (scHGF); the cleavage of scHGF at the Arg494–Val495
generates two-chain HGF (tcHGF) [21,22]. scHGF is a biologically inactive precursor incapable ofactivating the MET receptor, while tcHGF is the only active molecular species that can activate it.
Previous studies indicated that HGF exists as scHGF in normal tissues and that its processing into
tcHGF occurs in injured tissues [23] and the tumor microenvironment [21,24]. Aberrant activationof the HGF–MET pathway, including ligand-dependent MET activation, is strongly associated withinvasive growth, metastasis, and resistance to anticancer drugs [2,22].
These previous findings suggest that the localization of tcHGF is key to understanding how the
HGF–MET pathway participates in tissue development, regeneration, and cancer progression. However,there are currently no molecular tools that specifically recognize tcHGF but not scHGF. Because thestomach is a typical organ composed of epithelial and mesenchymal cells, and stem cells play importantroles in development and regeneration [25], analyses of the localization of sc/tcHGF and its association
with MET activation in the stomach may provide a better understanding of how the HGF–MET
pathway participates in development, regeneration, and the stem cell behavior of endoderm-derived
organs. In a recent study, we obtained several anti-HGF monoclonal antibodies that recognize different
epitopes of human HGF [26], one of which selectively recognizes human tcHGF. In this study, we thus
investigated the localization of tcHGF and active MET in the developing and regenerating stomach,
employing human tcHGF-specific monoclonal antibody and human HGF-knock-in mice.
2. Results
2.1. Specificity of Antibodies
Our previous study indicated that the t5A11 anti-human HGF monoclonal antibody recognizes
both biologically inactive scHGF and active tcHGF, whereas the t8E4 monoclonal antibody exclusively
recognizes tcHGF [26]. We first confirmed the cross-reactivity and specificity of the antibodies usedin this study using western blot analysis and enzyme-linked immunosorbent assay (ELISA). Mouse
HGF and human HGF (composed of scHGF and tcHGF) were subjected to SDS-PAGE and western
blotting under non-reducing conditions (Figure 1 and Figure S1). A commercial polyclonal anti-mouse
HGF antibody was reactive to mouse and human HGF (Figure 1A), while t5A11 and t8E4 recognizedhuman HGF but not mouse HGF (Figure 1A and Figure S2). t5A11 was reactive to scHGF and tcHGF,
whereas t8E4 selectively recognized tcHGF but not scHGF (Figure 1B). Thus, t5A11 and t8E4 antibodies

Int. J. Mol. Sci. 2019, 20, 2955

3 of 14were only reactive to human HGF, t8E4 was specific to tcHGF, and t5A11 was reactive to both scHGF
J. Mol. Sci. 2019, 20, x
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andInt.
tcHGF.

Int. J. Mol. Sci. 2019, 20, x

3 of 15

Figure1. Specificity
of ofanti-human
HGF
antibodies.
Figure1. Specificity
anti-human
HGF
antibodies.(A)(A)Western
Westernblot
blotanalysis
analysisfor
forthethecross-reactivity
cross-reactivityof
anti-mouse
HGF
polyclonal
antibody
andt5A11
anti-human
HGF
monoclonal
antibody
betweenmouseof anti-mouse HGF polyclonal antibody and t5A11 anti-human HGF monoclonal antibody
betweenandmouse
human
HGF.
(B)
Specificity
ofanti-HGF
monoclonal
antibodies(t5A11
and
t8E4)
to
mouse
and human HGF. (B) Specificity of anti-HGF monoclonal antibodies (t5A11 and t8E4)HGF,
toFigure1. Specificity
of anti-human
HGF antibodies.
(A) Western
blottoanalysis
for the cross-reactivity
scHGF,
andtcHGF.
Mouse
and
human
HGF
subjected
SDS-PAGE
non-reducing
mouse HGF,
scHGF,and HGF
tcHGF.
Mouse
HGF
andwerehuman
HGF were
subjected tounder
SDS-PAGE
underof anti-mouse HGF polyclonal antibody and t5A11 anti-human HGF monoclonal antibody between
conditions.
Human
HGF (composedof scHGF
and tcHGF),
and tcHGF
were
and
non-reducing
conditions.
Human HGF
(composedof scHGFscHGF,
and tcHGF),
scHGF,
andexpressed
tcHGF weremouse and human HGF. (B) Specificity of anti-HGF monoclonal antibodies (t5A11 and t8E4) topurified
as describedin theMethods
section.
expressed
and purified
as Materials
described andin theMaterials
and Methods section.

mouse HGF, scHGF, and tcHGF. Mouse HGF and human HGF were subjected to SDS-PAGE under
non-reducing conditions. Human HGF (composed of scHGF and tcHGF), scHGF, and tcHGF were
2.2.2.2.
HGF
andandMET
Receptor
Localizations ininHuman
Knock-in
Mice
HGF
MET
Receptor
Human
Knock-inMicesection.
expressed
andpurifiedLocalizations
as described in the
Materials
and Methods

Based
on on
the thespecificity
of t5A11and andt8E4t8E4
antibodies
to human
HGF,HGF,
we decided
to usetohumanBasedspecificity
of t5A11
antibodies
to humanwe decideduse
2.2. HGF and MET Receptor Localizations in Human Knock-in Mice
tm1.1(HGF)Aveo Prkdcscid /J).
HGF
knock-in(hHGF-ki)
mice
obtainedfromtheJacksonLaboratory(Hgfhuman HGF knock-in (hHGF-ki) mice obtained from the Jackson Laboratory
Basedmice,on thet5A11
t8E4
antibodies
to exons
human
HGF,
weendogenous
decidedto usetm1.1(HGF)Aveo
scidspecificity
In thebothofexonsand
3–6
ofboththeendogenous
murinegene were
replaced(HgfhHGF-ki
Prkdc/J).alleles
In the ofhHGF-kimice,
alleles of
3–6
of HGFthemurinehuman
HGF
knock-in(hHGF-ki)
mice
obtainedfromtheJacksonLaboratory
witha cDNA
sequence
encodingexons
2–18sequenceof the human
HGFexons
gene. 2–18
Human
HGF
was detectablebut
HGF
genetm1.1(HGF)Aveo
were replaced
with
a
cDNA
encoding
of
the
human
HGF
gene.
(Hgf
Prkdcscid/J). In the hHGF-ki mice, both alleles of exons 3–6 of the endogenous murine
mouse
HGF
was
not
detectableintheplasmaofhHGF-kimice[27].
Human HGF was detectable but mouse HGF was not detectable in the plasma of hHGF-ki mice
HGF gene were replaced with a cDNA sequence encoding exons 2–18 of the human HGF gene.
[27].
To confirm
the compatibility
themouse
expression
andnotlocalizationof the
HGF
betweenwild-typeC57BL/6
Human HGF
was detectableinbut
HGF wasdetectable inplasmaof hHGF-ki
mice
To
confirm
thecompatibility
intheexpression
andlocalizationof
HGF
betweenwild-typeand hHGF-ki
mice,
immunohistochemical
andimmunofluorescence
detection
was
performed
using
[27].
C57BL/6
andhHGF-kimice,
immunohistochemical
andimmunofluorescence
detection
wasTo
confirm
thecompatibility
intheexpression
andlocalizationof
HGF
betweenwild-type16.5 days post-coitum mouse embryos from wild-type C57BL/6 and hHGF-ki mice (Figure 2 and
C57BL/6
hHGF-ki
mice, immunohistochemical
anddetection
was mice
performed
16.5
days post-coitum
embryos
fromimmunofluorescencewild-type
and washHGF-kiFigureS3).
In using
theanddeveloping
stomachandmouse
intestineof wild-type
C57BL/6C57BL/6
mice, HGF
distributed
performed
using
16.5
days
post-coitum
mouseembryos
fromwild-typeC57BL/6
andhHGF-kimice(Figureand Figure S3).
In theintestineof wild-type
mice, HGF
mainlyin 2mesenchymal
cells
butdeveloping
faintly in stomach
epithelialandcells.
α-SmoothmuscleC57BL/6
actin (α-SMA)
was(Figure 2 andmainly
Figure S3).
In the developing
stomachand intestineof wild-type
C57BL/6 mice,
HGF actin
wasdistributedinmesenchymal
cells
but
faintlyinepithelial
cells.
α-Smoothmuscleexpressedin smooth muscle cells and myofibroblasts. α-SMA staining indicated that HGF-positive
was distributed mainly in mesenchymal cells but faintly in epithelial cells. α-Smooth muscle actin(α-SMA)was expressedin smoothmusclecells and myofibroblasts.
α-SMA staining indicated
that
cells
were
mainlysmoothmusclecells
and myofibroblasts
in the sub-epithelial
hHGF-ki(α-SMA) was
expressedin smoothmusclecells and myofibroblasts.
α-SMA stainingregion.
indicatedInthat
HGF-positive cells were mainly smooth muscle cells and myofibroblasts in the sub-epithelial
mice, HGF
was mainly
localizedin smoothmusclecells,
it was weakly inpresent
in myofibroblasts
HGF-positive
cells
were mainlysmoothmusclecellswhile
and myofibroblasts
the sub-epithelial
region.
In hHGF-ki
mice,
HGF
was
mainlylocalized
insmoothsmoothmuscle
musclecells,
while
it wasweaklyregion.
In
hHGF-ki
mice,
HGF
was
mainlylocalizedincells,
while
itwasweaklyin the sub-epithelial region and in epithelial cells. These results indicate that smooth muscle
cells were
present in myofibroblasts
in inthethesub-epithelial
regionandininepithelial
epithelial
cells.
These
results
indicatein myofibroblasts
regionandcells.
These
results
indicate
the mainpresent
cellularsource of HGF
andsub-epithelial
that hHGF-ki
mice
were an appropriate
tool
to investigate
thethat smoothmusclecells
weresourceofofHGF
HGFandandthat
hHGF-kithat smoothmusclecells
werethethemainmain cellular
cellular source
that
hHGF-kimicemicewere werean anlocalization of HGF.
appropriate
tooltoolto investigatethe
HGF.
appropriate
to investigate
thelocalization
localization ofof HGF.

Figure 2.Figure
Localizationof HGFin thedeveloping
andintestine
intestineof wild-type
C57BL/6
and2. Localizationof HGFin thedeveloping stomach
stomach andof wild-type
C57BL/6
andhHGF-kihHGF-ki
mice. Immunohistochemistry
wasusinganti-mouseanti-mouse
HGF
polyclonal
antibodies
mice. Immunohistochemistrywasperformed
performed using
HGF
polyclonal
antibodies
2.
Localizationof monoclonal
HGF
the antibodies,developing
stomachandinintestine
ofandwild-typeC57BL/6
and
and
t5A11
anti-human
HGF inmonoclonal
antibodies,
respectively,
in C57BL/6
andhHGF-kimice. Similar
andFiguret5A11
anti-human
HGF
respectively,
C57BL/6
hHGF-kimice.
hHGF-kimice.
Immunohistochemistrywasperformed
using
anti-mouse
HGF
polyclonal
antibodies
Similarandpatterns
localization
patterns
wereinobtained
sections
two different
mice. wereexpression
andexpression
localization
were
obtainedsectionsinfrom
twofrom
different
mice. Tissues
Tissuesanti-humanwere obtainedHGF
from monoclonalday 16.5 embryos.
Scale bars
represent 200inµm.
and t5A11
respectively,
C57BL/6 and hHGF-ki mice.
obtainedfrom day16.5 embryos.
Scale bars antibodies,represent
200µm.
Similar expression and localization patterns were obtained in sections from two different mice.
Tissues were obtained from day 16.5 embryos. Scale bars represent 200 µm.

Int. J. Mol. Sci. 2019, 20, 2955
Int. J. Mol. Sci. 2019, 20, x

4 of 144 of 15

By day 16.5, the embryos of hHGF-ki mice had already developed a variety of tissues/organs.
By day 16.5, the embryos of hHGF-ki mice had already developed a variety of tissues/organs.
HGF-positive cells were mainly smooth muscle cells of several organs including the esophagus,
HGF-positive cells were mainly smooth muscle cells of several organs including the esophagus, trachea,
trachea, lung, stomach, intestine, and urinary bladder (Figure S4). The MET receptor was mainly
lung, stomach, intestine, and urinary bladder (Figure S4). The MET receptor was mainly localized inlocalized in epithelial cells. Previous studies indicated that HGF regulates the growth andepithelial cells. Previous studies indicated that HGF regulates the growth and morphogenesis
morphogenesis of different types of epithelial cells and tissues, mainly as a mesenchymal-derivedof different types of epithelial cells and tissues, mainly as a mesenchymal-derived paracrine
paracrine factor [4,5,9–13]. Thus, these expression patterns of HGF and the MET receptor infactor [4,5,9–13]. Thus, these expression patterns of HGF and the MET receptor in developing
developing tissues suggest that HGF and MET play roles in the development of several organs.
tissues suggest that HGF and MET play roles in the development of several organs.
2.3.
2.3.tcHGF
tcHGFand
andPhosphorylated
PhosphorylatedMETMETReceptor
Receptorininthe
theDeveloping
DevelopingStomach
StomachTo
the potential
potentialinvolvement
involvement
HGF–MET
pathway
the developmentof theToclarify
clarify theof ofthethe
HGF–MET
pathwayin theindevelopmentof the stomach,stomach,thelocalization
of
total
HGF
and
MET
was
analyzed
using
day
16.5
embryos(Figure3
the localization of total HGF and MET was analyzed using day 16.5 embryos (Figure 3 and Figure S5).
andFigureS5). α-SMA
staining
line ofcells
smoothcells inandthe hind-stomach.
fore-stomach andα-SMA
stainingdelineateda linedelineatedof smooth amuscle
in themuscle
fore-stomach
HGF
hind-stomach.
HGF
waslocalizedinsmoothmusclecells(blackarrowsin
Figure3;whiteS5),
arrowsin
was localized in smooth muscle cells (black arrows in Figure 3; white arrows in Figure
whereasFigureS5), whereas
weak
HGF
stainingcells
wasinseenin epithelial(black
cells arrowheads
in the fore-stomach(blackweak HGF
staining wasseen
in epithelial
the fore-stomach
in Figure 3;
white
arrowheads
inFigure3;
whitearrowheads
inFigureS5).
METexpression
was
localizedinepithelial
arrowheads in Figure S5). MET expression was localized in epithelial cells of the fore-stomach (redcellsof the fore-stomach(red Figure
arrowheads
in Figureand FigureS5), whilestrongMET
expression
arrowheads
in Figure 3 andS5), whilestrong3 MET
expression
was seenin thebasal
region of
was
seen
in
the
basal
regionofdeveloping
glandular
structuresinthehind-stomach(reddeveloping glandular structures in the hind-stomach (red arrowheads in Figure 3 and Figure S5).
arrowheads in Figure 3 and Figure S5).

Figure 3.
of HGF
METandreceptors
developing
Immunohistochemical
Figure3. Localizations
Localizationsof andHGF
METin thereceptors
in stomach.
the developing
stomach.
staining
was
performed
using
t5A11
anti-human
HGF
monoclonal
antibody
or anti-METantibody
antibody.
Immunohistochemical staining was performed using t5A11 anti-human HGF monoclonalday 16.5 embryos
weredividedthe anterior/fore-stomach
posterior/hind-stomach,orStomachs
anti-METinantibody.
Stomachsin day16.5into
embryos
were divided into andthe anterior/fore-stomach
distinguishedbydottedlines.
Black
arrowsindicate
HGF
localizationinsmoothcells. Blackand posterior/hind-stomach, distinguished by dotted lines. Black arrows indicate muscle
HGF localization
arrowheads indicate HGF localized in the epithelial cells. Red arrowheads indicate MET expression inin smooth muscle cells. Black arrowheads indicate HGF localized in the epithelial cells. Red
epithelial cells. Similar localization patterns were obtained in sections from two different mice. Tissues
arrowheads indicate MET expression in epithelial cells. Similar localization patterns were obtained
were obtained from day 16.5 embryos of hHGF-ki mice. Scale bars represent 200 µm.
in sections from two different mice. Tissues were obtained from day 16.5 embryos of hHGF-ki mice.
Scale
µm.
We bars
nextrepresent
analyzed200
thelocalization of tcHGF and tyrosine-phosphorylated MET (pMET) in day

16.5 embryos (Figure 4). In the fore-stomach and hind-stomach, HGF (scHGF + tcHGF) was mainly
We next analyzed the localization of tcHGF and tyrosine-phosphorylated MET (pMET) in day
localized in smooth muscle cells, while tcHGF was diffusely localized in sub-epithelial cells (black
16.5 embryos (Figure 4). In the fore-stomach and hind-stomach, HGF (scHGF + tcHGF) was mainly
arrows) and epithelial cells (yellow arrows). In the hind-stomach, strong tcHGF staining was seen
localized in smooth muscle cells, while tcHGF was diffusely localized in sub-epithelial cells (blackin the base region of glandular structures (red arrow). These results suggest that some of the scHGF
arrows) and epithelial cells (yellow arrows). In the hind-stomach, strong tcHGF staining was seenderived from smooth muscle cells was processed into tcHGF in sub-epithelial and epithelial cells. In thein the base region of glandular structures (red arrow). These results suggest that some of the scHGF
derived from smooth muscle cells was processed into tcHGF in sub-epithelial and epithelial cells. In

Int. J. Mol. Sci. 2019, 20, 2955

5 of 14Int. J. Mol. Sci. 2019, 20, x

5 of 15fore-stomach, strong MET staining was mainly seen in epithelial cells (red arrowheads), while strong
the fore-stomach,strongstaining
was mainly
seen in epithelial(red arrowheads),
while
MET
expression wasseenMETin thebase regionof developing
glandularcells
structures(red arrowheads)
strong
MET expression
waswasseenin the inbase
regioncellsof developing
glandular(redinthe hind-stomach.
pMET
localized
epithelialof the fore-stomach
andstructures
hind-stomach
arrowheads)inthehind-stomach.
pMET
waslocalizedinepithelial
cells
of
the
fore-stomachand(yellow arrowheads). Therefore, generation and localization of tcHGF may have played a role in MET
hind-stomach(yellow
arrowheads).
Therefore,
generation
andlocalization
of
tcHGF
may
have
activation in the developing stomach.
played a role in MET activation in the developing stomach.

Figure 4. Localizations of HGF, tcHGF, MET, and phosphorylated MET (pMET) in the developing
Figure 4. Localizations of HGF, tcHGF, MET, and phosphorylated MET (pMET) in the developing
stomach. Immunohistochemical staining was performed using t5A11 (for scHGF and tcHGF), t8E4
stomach. Immunohistochemical staining was performed using t5A11 (for scHGF and tcHGF), t8E4
(for tcHGF), anti-MET, or anti-phospho-MET antibody. The fore-stomach and hind-stomach were(for tcHGF), anti-MET, or anti-phospho-MET antibody. The fore-stomach and hind-stomach wererespectively characterized by multilayered squamous epithelia and developing glandular epithelium.
respectively
characterizedby tcHGF
multilayered
epitheliaand
anddeveloping
glandularBlack
and yellow
arrows indicate
localizedsquamousin the sub-epithelial
epithelial
cells, respectively.
epithelium.
Black
and
yellowarrowsindicate
tcHGF
localizedinthesub-epithelial
andepithelial
The red arrow indicates strong tcHGF localization in developing glandular regions. Red arrowheads
cells,
respectively.
The
red
arrow
indicates
strongtcHGF
localizationindeveloping
glandular
indicate strongly MET-positive cells in the epithelial cells. Yellow arrowheads indicate pMET inregions. Red
indicatestrongly
MET-positive
cells inthe boxed
epithelial
epithelial
cells.arrowheads
The images in
the lower
panel aremagnified imagesof theareascells.
in theYellow
upper
arrowheads
pMET in epithelial
cells. The
imagesin thelower inpanel
are magnified
imagespanel.
Similarindicate
immunohistochemical
localization
patterns
wereobtainedsectionsfrom two differentof theTissues
boxed wereareasobtainedin the upper
panel.
localization
patterns
were
mice.
from day
16.5Similarembryosimmunohistochemicalof hHGF-ki mice. Scalebars represent
200 µm.
obtained in sections from two different mice. Tissues were obtained from day 16.5 embryos of
2.4. tcHGF
andmice.
Phosphorylated
MET Receptor
hHGF-ki
Scale bars represent
200 µm.in the Adult Stomach

To determine the potential involvement of HGF in gastric homeostasis, the localizations of tcHGF
2.4. tcHGF and Phosphorylated MET Receptor in the Adult Stomach
and pMET were analyzed in the stomach of 6–8-week-old hHGF-ki mice. In the adult stomach, theTo determine
potential
involvement
ofcorpus
HGF inandgastric
ofglandularepithelialthe
structureis organized
intoantralhomeostasis,regions. In thethe localizations
corpus, tcHGF
tcHGF
and localized
pMET wereanalyzedin basethe stomachof 6–8-week-old
hHGF-kimice. Intothe
wasmainlyin theglandular
region (Figure
5, red arrow),and extended
the adult
neck
stomach,the glandular
epithelial
structure iscells
organized
into corpus
antralregions.
theregion(yellow
arrows), whereas
HGF-positive
were mainly
seen in andsmoothmusclecells In
(blackcorpus, MET
tcHGF
was mainlyin the glandular
regionstructures(Figure (black
5, red arrowheads).
arrow), andarrow).
expression
was localized
present alongside
glandularbase
epithelial
extended
the neck inregion(yellowregions:arrows),
HGF-positive
cells were
seen
in
pMET
wasto
distributed
two distinct
thewhereas
base region(red arrowhead),wheremainly
chief andstemsmoothmusclearrow).
MET
expression
waspresent
alongside
epithelial
cells
were
locatedcells[25],(blackand theisthmusregionand/or thesurfaceregion(yellowglandulararrowheads).
Thus,
structures
pMET wasindistributedin twodistinct
the baseusing
region(red
tcHGF
and(blackpMETarrowheads).
showed co-localization
the base region,
which
wasregions:also confirmed
doublearrowhead), where chiefand stem
cells
were
located [25],and theisthmus region
and/orimmunofluorescencestaining(FigureS6).
Substantiallysimilarlocalization
patterns
were theseensurface
in theregion (yellow
arrowheads). Thus, tcHGF and pMET showed co-localization in the base region,
antrum(Figure S7).
which was also confirmed using double immunofluorescence staining (Figure S6). Substantiallysimilar localization patterns were seen in the antrum (Figure S7).

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FigureHGF,
tcHGF,
MET,
and pMET
in thein
corpusepitheliumof the adultstomach.
Figure 5.5. Localizations
Localizationsofof
HGF,
tcHGF,
MET,
and pMET
the corpus
epitheliumof theadult
Immunohistochemical
staining
was
performed
using
t5A11
(for
scHGF
and
tcHGF),
t8E4(for
tcHGF),
stomach. Immunohistochemical staining was performed using t5A11 (for scHGF and tcHGF),
t8E4
anti-MET,oranti-phospho-MET
antibody.
Blackarrows,HGF
insmoothmusclecells;
redarrow,
tcHGF(for tcHGF), anti-MET, or anti-phospho-MET antibody. Black arrows, HGF in smooth muscle cells;in the glandular base region; yellow arrows, tcHGF in the neck region; black arrowheads, MET in
red arrow, tcHGF in the glandular base region; yellow arrows, tcHGF in the neck region; black
glandular epithelial structure; red arrowhead, pMET in the glandular base region; yellow arrowhead,arrowheads, MET in glandular epithelial structure; red arrowhead, pMET in the glandular basepMET in the isthmus region and the surface region. The images in the lower panel are magnified
region; yellow arrowhead, pMET in the isthmus region and the surface region. The images in theimages of the boxed areas in the upper panel. Similar immunohistochemical localization patterns werelower panel are magnified images of the boxed areas in the upper panel. Similarobtained in sections from two different mice. Tissues were obtained from hHGF-ki mice. Scale bars
immunohistochemical localization patterns were obtained in sections from two different mice.
represent 200 µm.
Tissues were obtained from hHGF-ki mice. Scale bars represent 200 µm.

Leucine-rich, repeat-containing G-protein coupled receptor 5 (Lgr5) expression is a definitive
Leucine-rich, repeat-containing G-protein coupled receptor 5 (Lgr5) expression is a definitivemarker for gastric epithelial stem cells [25]. Since tcHGF and/or pMET showed close localization
marker for gastric epithelial stem cells [25]. Since tcHGF and/or pMET showed close localization toto gastric stem cells, we performed an immunostaining analysis in Lgr5-reporter mice in which thegastric stem cells, we performed an immunostaining analysis in Lgr5-reporter mice in which thediphtheria toxin receptor-EGFP cassette was knocked into the endogenous Lgr5 locus [25]. Consistent
diphtheria toxin receptor-EGFP cassette was knocked into the endogenous Lgr5 locus [25].
with previous reports [25], GFP expression defined the localization of gastric stem cells at the baseConsistent with previous reports [25], GFP expression defined the localization of gastric stem cells
region of the corpus (Figure 6A,B) and antrum (Figure S8). Similar to the results in human HGF
at the base region of the corpus (Figure 6A,B) and antrum (Figure S8). Similar to the results inknock-in mice, pMET was distributed in two distinct regions: the base region (red arrowheads) andhuman HGF knock-in mice, pMET was distributed in two distinct regions: the base region (redthe isthmus and/or surface regions (yellow arrowheads) (Figure 6A). Double immunofluorescencearrowheads) and the isthmus and/or surface regions (yellow arrowheads) (Figure 6A). Double
staining also indicated the co-localization of pMET and GFP (white arrows) (Figure 6B); the numbers of
immunofluorescence staining also indicated the co-localization of pMET and GFP (white arrows)
GFP- and pMET-positive cells per corpus gland were 18.2 ± 2.9 and 17.7 ± 2.7, respectively (Figure 6C).
(Figure 6B); the numbers of GFP- and pMET-positive cells per corpus gland were 18.2 ± 2.9 and 17.7
Co-localization of GFP and pMET in the base region was also seen in the antrum (Figure S8).
± 2.7, respectively (Figure 6C). Co-localization of GFP and pMET in the base region was also seen in
the antrum (Figure S8).

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Figure 6. Localizations of Lgr5+ stem cells and pMET in the corpus epithelium of the adult stomach.
+ stem cells and pMET in the corpus epithelium of the adult stomach.
Figure6. Localizations of Lgr5
(A)
Immunohistochemical
staining
for GFP and pMET. (B) Double immunofluorescence staining for(A) Immunohistochemical
staining
forLgr5-driven
GFP and pMET.
(B) arrowheads,
Double immunofluorescencestainingfor
GFP
and pMET. In (A), blackarrows,
GFP; redpMET in the glandular
base
GFP
and
pMET.
In
(A),
black
arrows,Lgr5-driven
GFP;
redarrowheads,
pMET
in
the
glandular
region; yellow arrowheads, pMET in the isthmus and/or surface regions. In (B), white arrows indicate
baseco-localization
region; yellow
pMET
in theisthmussurfaceregions.
(B), arewhite
arrows+ stemtheof arrowheads,
pMET and GFP
in Lgr5cells. and/or
The images
in thelowerIn
panel
magnified+stemcells. The
imagesin the lower
panel
are
indicateofthe
GFPTissues
in Lgr5
imagestheco-localization
boxed areas in of
thepMET
upperandpanel.
were
obtainedfromLgr5-DTR-EGFP
mice.
Scale
magnifiedimages
of
the
boxed
areas
in
the
upper
panel.
TissueswereobtainedfromLgr5-DTRbars represent 200 µm. (C) The numbers of GFP- and pMET-positive cells per corpus gland. These
EGFP mice.
Scale barsbyrepresentµm.
(C) TheGFPand for
pMET-positive
cells pervalueswere obtained
counting 200
in 10
glands(n = numbers
10). Dataof
weretestedsignificance usingancorpusgland.
These
valueswereobtainedbycounting
in10
glands
(n
=
10).
Data
were
testedfor
unpaired two-tailed t-test. NS, not significant.
significance using an unpaired two-tailed t-test. NS, not significant.

2.5. Promotion of Gastric Organoids
2.5. Promotion of Gastric Organoids
Because the localization of pMET in Lgr5+ gastric stem cells suggests the involvement of the
HGF–MET
pathwayin the growthand/orinmaintenanceof stemgastriccells
stemsuggests
cells, wethe
employed
an organoid
Becausethe localizationof pMET
Lgr5+ gastricinvolvementof theculturethat allows
three-dimensional
using various
tissuestemcells,
cells,weincludinggastric
HGF–MET
pathwayin the growth reconstruction
and/or maintenanceof gastric
stem
employedancorpusorganoids[25].
Corpusglandular
basal
cells
were
preparedandsubjected
toathree-dimensionalorganoid culture that allows three-dimensional reconstruction using various tissue stem cells,
culture(Figure7).corpus
In the organoids
control culture,organoid
formation
wascells
scarcely(Figureandincludinggastric[25]. Corpus
glandular
basal
were seen
preparedand7A,B),subjected
viablegastric stem cells werenot(Figure
maintained
culture(Figureorganoid
7C). However,in thewaspresenceof
to a three-dimensional
culture7). Induring
the control
culture,formation
scarcely
epidermal
growthfactorstandard
growthfactornot
for gastricorganoid
culture,or HGF,typicalseen (Figure
7A,B),and (EGF),
viable agastric
stem
cells weremaintained
duringculture(Figure7C).
organoid
were seen.
Quantitative
analysisof thesize adistributionof organoids
7B)
However,structuresin the presenceof epidermal
growthfactor(EGF),
standard growth
factor (Figure
for gastricandviablecells(Figure7C)
indicatedthat
HGF
promotedorganoid
formation
andviabilityofgastricorganoid culture, or HGF, typical organoid structures were seen. Quantitative analysis of the size
stem
cells in aof
mannercomparable
thatandof EGF.
Becausestem cells thatare indispensablefor
distribution
organoids(Figureto7B)
viablecells Lgr5+(Figuregastric
7C) indicated
HGF promoted
organoid
plays
a rolestemin thecells
growthmaintenanceof gastriccells.
organoidformation,formationMET
and activation
viability ofgastric
in aand/or
mannercomparable
to thatstem
of EGF.
Because Lgr5+ gastric stem cells are indispensable for organoid formation, MET activation plays arole in the growth and/or maintenance of gastric stem cells.

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FigureFigure7.7.Promotion
Promotionofofgastricgastriccorpus
corpusorganoids
organoidsby
byHGF.
HGF.(A)(A)TheTheappearance
appearanceofoforganoids
organoidscultured
cultured
inintheabsenceorpresence
of
EGF
or
HGF.
Scale
bars
represent
200
µm.
(B)
The
size
the absence or presence of EGF or HGF. Scale bars represent 200 µm. (B) The sizedistribution
distributionofof
organoids.
organoids.(C)(C)TheTheviability
viabilityofoforganoids.
organoids.InIn(A),(A),corpus
corpusgland
glandstem
stemcells
cellsisolated
isolatedfrom
fromaaC57BL/6
C57BL/6
mouse
were
culturedin
Matrigelintheabsenceorpresenceof
EGF(50
ng/mL)
orrecombinant
HGF
mouse were cultured in Matrigel in the absence or presence of EGF (50 ng/mL) or recombinant(100
ng/mL)
for
21
days.
In
(B),
the
size
distribution
wasanalyzedbyusing
Image-J.
In
(C),
the
viability
HGF (100 ng/mL) for 21 days. In (B), the size distribution was analyzed by using Image-J. In (C),® 3D cell viability assay. Values are presented as mean ± S.D.
was
using
the CellTiter-Glo® 3D cell viability assay. Values are presented
themeasured
viability wasmeasuredusing the CellTiter-Glo(n = 6). Data were tested for significance using an unpaired two-tailed t-test. NS, not significant.
as mean ± S.D. (n = 6). Data were tested for significance using an unpaired two-tailed t-test. NS,
Organoid culture was performed twice, from which similar results were obtained.
not significant. Organoid culture was performed twice, from which similar results were obtained.

2.6. Changes in tcHGF and pMET Distributions Post-Injury
2.6. Changes in tcHGF and pMET Distributions Post-Injury
tcHGF and pMET were detected in the area of the stomach where stem cells were present, and HGF
tcHGF
and pMET
wereformation.
detected in
the areaof encouraged
the stomachus
where
stemwhether
cells wereandfacilitatedstomachorganoid
These
results
to assessthepresent,
HGF–MET
HGF
facilitatedstomachorganoid
formation.
These
results
encouragedustoassesswhetherthepathway plays a role in stomach regeneration. For this purpose, we used repetitive tamoxifen treatment
HGF–MET
pathwayplays
a rolechanges
in stomach
regeneration.
Forlocalization
this purpose,used(Figure
repetitiveusing
hHGF-kimice andanalyzedin tcHGF
and pMET
overwe7 days8).
tamoxifen
treatment
using
hHGF-kimice
andanalyzedchanges
in
tcHGF
and
pMET
localization
Tamoxifen treatment damages the corpus epithelium, and is associated with the loss of parietal cells,
over 7 daysof(FigureTamoxifen
treatment
damagesofthe
corpusneck
epithelium,
and is associatedwithproliferation
Lgr5+ 8).
gastric
stem cells,
and expansion
mucouscells [25,28,29].
On day 3 after+
thetamoxifen
loss of parietal
cells, proliferation
Lgr5
gastric
and expansionof mucouscells
thetreatment,
proliferating of
cells
were
seen stemin thecells,
glandularbase region(Figureneck
8, black[25,28,29].
On
day
3
after
the
tamoxifen
treatment,
proliferating
cells
were
seen
in
the
glandulararrow) where strong pMET staining was detected (red arrowheads). pMET was also seen in the neckbase region
black arrowheads)
arrow) wherestrongstaining
was detected
arrowheads).
and/orsurface(Figure
regions 8,(yellowwhere
the pMET
expansionof mucousneck cells(redoccurred[28,29].
pMET
was
also
seen
in
the
neck
and/orsurfaceregions(yellow
arrowheads)
where
the
expansion
tcHGF staining was strong in the base region (red arrows) and diffuse in the neck and/or
surfaceof
mucous(yellow
neck cells
occurred[28,29].
tcHGF staining
strongin thebase region
arrows)andregions
arrows).
On day7, proliferating
cellswaswere
mainlylocalizedin the (redisthmusregiondiffuseinrecovery
the neck of
and/orsurface(yellow
arrows).
Onofday7, proliferating
mainlywheretheparietalcellsregions
occurred.
Strongstaining
pMET
and tcHGF cells
was wereseen inthelocalizedintheisthmusregionwhere
therecoveryofparietalcells
occurred.
Strongstainingbase region (red arrowheads and red arrows, respectively); pMET positivity was still present in theof
pMET andtcHGF
was regions.
seen in thebase
region (redof
arrowheads
andstem
red arrows,
respectively);pMET
isthmus
and/orsurfaceThe
proliferation
Lgr5+ gastriccells hasbeen reported
to+positivity
stillpresent treatmentin the isthmus
and/orsurface
The toproliferation
gastric+ cellsoccur
2 dayswas
aftertamoxifen
and Lgr5
haveregions.
been shownparticipate ofin Lgr5
subsequent+ cells have beenstem cells
has been reported
to occur the
2 daysafter tamoxifen
treatment
andepithelial
regeneration[25]. Collectively,
localizationsof pMETand tcHGF
in Lgr5
glandular
structuresshown
toparticipate
insubsequent
epithelial
regeneration[25].
Collectively,
thelocalizations
and their changes post-injury suggest that the HGF–MET pathway played a role in the homeostasisof
pMET
and tcHGF
andregenerationof intheglandular
stomach. structures and their changes post-injury suggest that the HGF–MET
pathway played a role in the homeostasis and regeneration of the stomach.

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Figure 8. Changes in the localizations of tcHGF and pMET following stomach injury. hHGF-ki mice
Figure 8. Changes in the localizations of tcHGF and pMET following stomach injury. hHGF-ki mice
were subjected to a tamoxifen treatment. The black arrow indicates PCNA-positive proliferatingwere subjected to a tamoxifen treatment. The black arrow indicates PCNA-positive proliferating
cells. Red arrowheads and red arrows indicate pMET and tcHGF localizations, respectively, in thecells. Red arrowheads and red arrows indicate pMET and tcHGF localizations, respectively, in theglandular base region. Yellow arrowheads and yellow arrows indicate pMET and tcHGF localizations,
glandular base region. Yellow arrowheads and yellow arrows indicate pMET and tcHGF
respectively, in the neck and/or surface regions. H&E, hematoxylin and eosin. Similar results werelocalizations, respectively, in the neck and/or surface regions. H&E, hematoxylin and eosin. Similarobtained in sections from two different mice independently subjected to tamoxifen treatment. Scale
results were obtained in sections from two different mice independently subjected to tamoxifen
bars represent 200 µm.
treatment. Scale bars represent 200 µm.

3. Discussion
3. Discussion
A variety of bioactive molecules are secreted or stored as inactive forms and activated in response
A variety of programs,
bioactive molecules
are pathology.
secreted orProteolytic
stored as conversion
inactive forms
and activatedin
to developmental
injury, and/orof inactive
scHGF toresponse
to developmental
injury,ofand/or
pathology.
Proteolytic
conversion
inactive
activetcHGF
has a key role programs,
in the activation
HGF–METsignaling;
however,
because of the
lack
scHGF
to activetool
tcHGF
a key role capturein the activationof spatial
HGF–MET
however, becauseofofa molecularable hasto selectively
tcHGF, theand signaling;
temporal regulationof tcHGF
the lack of ainmolecular
tool able
selectively
tcHGF,unknown.
the spatial In
andtemporalregulationofgeneration
development
and totissuerepair capture
has remained
thisstudy, weobserved
in development
tissuerepairhas remained
unknown.
In regeneration
this study, we
atcHGFchangegenerationin the localizationof tcHGF andduringgastric
development,
homeostasis,
andbyobserveda changein the localization
tcHGF during
gastricmice.
development,
and
using
a humantcHGF-specificmonoclonalofantibody
and hHGF-ki
tcHGF washomeostasis,
localized in areas
regeneration
byfromusingthea humanmonoclonal
antibody andhHGF-kimice. tcHGF
wasthat
differ bothprimarytcHGF-specificsite of synthesis
and the localizationof scHGF.
Conversely,
tcHGF
localized in withareasactivatedthat differ
both from the primary site of synthesis and the localization of scHGF.
co-localized
MET.
Conversely,withactivated
MET.
PrevioustcHGF
studiesco-localized
suggested that
HGF
is a mesenchymal-derived
factor that plays a role in epithelial
Previousmovement,
studies suggested
that HGF
is a mesenchymal-derived
factorthat plays
a roleincell growth,and branchedtube formation.
In the developing
stomach,we foundthat
epithelial
growth,movement,
and branchedtubewhereas
formation.
In thestomach, we
scHGF
wascellmainlylocalizedin smoothmuscle cells,
tcHGF
wasdeveloping
diffusely distributedinfound that scHGF
wasalsomainlylocalizedin smooth
cells, whereas
tcHGF was ofdiffusely
sub-epithelial
cells and
localized
in epithelial
cells.muscle
Our findingthat the localization
tcHGF
distributed
in wassub-epithelial
cellsto andalso
localizedagrees
in epithelial
cells. studies.
Our findingthat also
thebut
not scHGF
closely related
pMET
localization
with previous
Our work
localization
ofthe
tcHGF
but notof
scHGF
to pMET
localizationagreeswith cells
previous
indicatedthatconversion
scHGFwasintoclosely
tcHGFrelated
occurredin sub-epithelial
andepithelialandstudies.
Our
work
also indicated epithelial
that the conversion
ofglycosaminoglycans
scHGF into tcHGFand
occurred
inservesubthattcHGF
acted
on MET-expressing
cells. Because
collagensepithelial and epithelial cells and that tcHGF acted on MET-expressing epithelial cells. Because

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as binding sites for HGF in the extracellular matrix [30,31], scHGF was moderately diffusible, and wasconverted to tcHGF in the pericellular microenvironment.
The proteolytic processing of scHGF to tcHGF is catalyzed by several serine proteases [32].
Matriptase, a membrane-type serine protease, is broadly expressed in a variety of epithelial cells
including human gastric epithelia [33,34]. The ablation of matriptase resulted in the impaired integrityand barrier function of epithelial tissues in the large intestine and epidermis [35,36]. HGF-activator issynthesized mainly in the liver and at a lower level in gastrointestinal tissue [32,37,38]. HGF-activator
present in the plasma is activated by thrombin in injured tissues. Mice deficient in HGF-activator showedno abnormal tissue homeostasis, but the injured mucosa was not sufficiently covered by regenerated
epithelium following colitis-induced injury [39]. A recent study reported that HGF-activator induced
Go-to-GAlert cell-cycle transition in stem cells in various tissues [40]. Taking these findings together,matriptase and HGF-activator are potential serine proteases responsible for tcHGF generation in
the stomach.
Lgr5+ stem cells in gastrointestinal organs play important roles in homeostasis, regeneration, andcancer development [41]. The Lgr5+ stem cell-specific disruption of MET indicated that the MET receptor
is dispensable for the normal homeostasis of intestinal epithelial cells [20]. However, MET deficiency inintestinal stem cells mildly attenuated intestinal regeneration after radiation-induced injury, presumably
by impairing stem cell fitness [20]. The involvement of the HGF–MET pathway in regeneration was also
supported by intestinal crypt organoid cultures, where HGF–MET signaling induced the outgrowthof intestinal stem cells into mini-guts, with an equivalent potency to EGF [20]. In addition, tumor
microenvironment-derived HGF augmented Wnt/β-catenin signaling and clonogenicity in colon cancer
stem cells characterized by high Wnt reporter activity [42]. Our study revealed that pMET and
tcHGF were localized in the base region of the gastric corpus gland, and MET activation occurred in
Lgr5+ gastric stem cells. Moreover, HGF induced organoid formation by gastric stem cells at a levelcomparable to that of EGF. Taken together, these findings show that although HGF is dispensable forthe normal homeostasis of gastric stem cells, potentially because of redundant growth and survival
signals, the HGF-MET pathway plays a supportive role in stem cell fitness, growth, and maintenance,
and in stem cell-derived epithelial tissue expansion.
The HGF–MET pathway participates in homeostasis and regeneration in a variety of tissues.
Studies of conditional MET-deficient mice have revealed that MET activation is important for theregeneration of organs such as the liver and skin [17–19], although the extent of its contribution
depends on the tissue type. Ligand-dependent MET activation is closely associated with the acquisitionof resistance to anticancer drugs in the cancer microenvironment [22]. Elucidation of the spatial
and temporal patterns of tcHGF generation should promote our understanding of the precise
mechanisms involved in tissue regeneration, stem cell activation, and cancer progression. To thisend, molecular tools that selectively capture tcHGF should provide experimental opportunities toinvestigate these mechanisms.
4. Materials and Methods
4.1. Animal Experiments
All mouse experiments were performed in accordance with the Guide for the Care and Useof Laboratory Animals and were approved No. AD-173859 by the ethical committee of the Cancer
Research Institute of Kanazawa University, Kanazawa, Japan. hHGF-ki mice were purchased from theJackson Laboratory Office of Development (stock number: 014553, NSG-hHGFki; Bar Harbor, ME,
USA). C57BL/6 mice were purchased from Sankyo Labo Service (Tokyo, Japan). Male hHGFki and
C57BL/6 mice (8–10 weeks old) were maintained at the Advanced Science Research Center Institute forExperimental Animals, Kanazawa University. The fetuses were isolated from the uterus and dissected
free of embryonic membranes in PBS. Tamoxifen (Merck, Kenilworth, NJ, USA) was dissolved insunflower seed oil and injected intraperitoneally into 6–8-week-old mice at 4 mg/20 g body weight forInt. J. Mol. Sci. 2019, 20, 2955

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3 days. Tissues were prepared at 1, 3, and 7 days after treatment. Lgr5-DTR-EGFP mice were prepared
as described previously [25].
4.2. Recombinant HGF
Full-length human HGF cDNA (NM_001010932.2) was used in all plasmid constructions
throughout this study. Residue numbering was based on the sequence of variant 1, which contained
five additional amino acids in the K1 domain. HGF cDNA with or without point mutations to eliminate
N-linked glycosylation sites was as previously described [43]. Recombinant human HGF was expressedin Chinese hamster ovary cells, and secreted proteins were purified on an AKTA Purifier system (GEHealthcare Life Sciences, Pittsburgh, PA, USA), using a HiTrap Heparin HP Column (GE Healthcare)followed by size-exclusion chromatography on a Superdex 200 10/300 GL column (GE Healthcare)equilibrated in 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. Recombinant HGF was mainly composedof tcHGF and the content of scHGF was less than 1%. Recombinant HGF (Xa), in which the cleavage
site of wild-type HGF (KQLR/V) was mutated to the recognition sequence of Factor Xa (IEGR/V),
was prepared as previously described [26]. Briefly, HGF (Xa) was appended with a hexahistidine tag
at the C-terminus and expressed in Expi293F cells (Thermo Fisher Scientific, Waltham, MA, USA).
Secreted proteins were purified on a Ni-nitrilotriacetic acid (NTA) agarose column (Qiagen, Hilden,
Germany). The C-terminal His-tag was eliminated using overnight incubation with Tobacco Etch
Virus (TEV) protease. To prepare tcHGF (Xa), the TEV-treated samples as described above were further
digested with 6 µg/mL Factor Xa (Novagen, Darmstadt, Germany). Recombinant scHGF (Xa) andtcHGF (Xa) proteins were further purified on a HiTrap Heparin HP Column (GE Healthcare) followedby size-exclusion chromatography on a Superdex 75 10/300 GL column (GE Healthcare) equilibrated in
20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. Murine HGF was purchased from PeproTech, Inc. (Rocky
Hill, NJ, USA).
4.3. Western Blotting
Proteins were subjected to SDS-PAGE in non-reducing conditions and electroblotted onto
polyvinylidene difluoride (PVDF) membranes. The membrane was incubated in Tris-buffered saline
containing 50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, and 3% (w/v) bovine serum albumin for 1 h
at room temperature, and then treated with the following primary antibodies (diluted 1:1000): rabbit
polyclonal mouse HGF antibody (LS-C294476; LifeSpan BioSciences, Inc., Seattle, WA, USA), mousemonoclonal anti-human HGF antibody (t5A11), or mouse monoclonal anti-human HGF antibody(t8E4). Monoclonal antibodies were prepared as described previously [26]. HRP-conjugated anti-rabbit
IgG or anti-mouse IgG (Dako, Carpinteria, CA, USA) was used at 1:1000 dilution. Chemiluminescence
was visualized and quantified using an ImmunoStar LD (Fujifilm Wako Pure Chemical, Osaka, Japan).
4.4. Immunohistochemical Staining
Tissues were fixed for 16–24 h in 4% paraformaldehyde in PBS at 4 ◦ C. Tissues were dehydrated
through a series of ethanol concentrations, immersed in xylene, and embedded in paraffin. Tissue
sections at 5 µm were deparaffinized and rehydrated. Antigen retrieval was carried out by heating slides
in a microwave oven for 10 min in 10 mM Tris-HCl buffer (pH 9.0) containing 1 mM EDTA. Endogenous
peroxidase was inactivated via incubation in 3% hydrogen peroxide in PBS for 5–30 min. Sections
were then blocked with PBS containing 3% bovine serum albumin for 30 min at room temperature.
The following primary antibodies were used: anti-phospho MET (phospho Y1230 + Y1234 + Y1235)
antibody (1:200 dilution, ab5662; Abcam), anti-proliferating cell nuclear antigen (1:4000, M0879;
Dako, Glostrup, Denmark), anti-α-SMA antibody (at 1:200 dilution, ab5694; Abcam, Cambridge,UK), anti-MET (SP260)-R antibody (1:50 dilution, Sc-162-R; Santa Cruz Biotechnology), anti-GFP
antibody biotin-conjugated (at 1:2000 dilution, 600-106-215; Rockland), anti-human HGF monoclonal
antibody (t5A11) (1:250 dilution), and anti-human HGF monoclonal antibody (t8E4) (1:500 dilution).
For immunohistochemical staining, tissue sections were visualized using a horseradish peroxidase

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(HRP)-labeled polymer system (Nichirei Bioscience, Tokyo, Japan) and DAB substrate (SK-4100; Vector
Laboratories, Burlingame, CA, USA). For immunofluorescent staining, tissue sections were visualizedusing Alexa 488/594-labeled goat anti-mouse/rabbit antibodies or Alexa 488-labeled donkey anti-goatantibody (1:200 dilution; Thermo Fisher Scientific). Tissue sections were photographed using an
Olympus BX51 microscope (Olympus, Tokyo, Japan) or Biozero BZ-9000 (Keyence, Osaka, Japan).
The negative controls for immunohistochemical staining were performed without using primary
antibodies. HRP-labeled anti-rabbit IgG or anti-mouse IgG was used as the secondary antibody(Figure S9).
4.5. Organoid Culture and Viable Cell Assay
Isolation of the murine corpus and organoid culture were performed as described previously [25].
Briefly, a corpus isolated from a C57BL/6 mouse was incubated in chelation buffer and glands wereisolated via repeated pipetting, filtration, and centrifugation. Glands were digested to single cells
in TrypLE (Life Technologies, Carlsbad, CA, USA) with DNaseI (0.8 U µL−1 ) (Sigma, Darmstadt,
Germany), and single corpus cells isolated using fluorescence-activated cell sorting were seeded in
Matrigel and cultured in medium supplemented with EGF, gastrin, fibroblast growth factor-10 (FGF10),
50% L-cell line to secrete Wnt3a, R-spondin 3 and Noggin (L-WRN) conditioned medium containing
noggin, Wnt3a, and R-spondin, and Rock-inhibitor (Y27632). Organoid cultures were passaged every
3 days. To test the effect of HGF on the growth of organoids, L-WRN conditioned medium was reduced
to 10%, and EGF and FGF10 were omitted from the culture medium. Cell viability and growth in
the 3D organoid culture were measured using the CellTiter-Glo® 3D cell viability assay kit (Promega,
Madison, WI, USA), in accordance with the manufacturer’s instructions. The luminescence signal wasmeasured at 560 nm.
Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/20/12/
2955/s1.
Author Contributions: Conceptualization, K.S. and K.M. (Kunio Matsumoto); Formal analysis, N.J. and H.S.;
Investigation, N.J., H.S., R.I., Y.T. and K.M. (Kazuhiro Murakami); Methodology, N.J., H.S., R.I., Y.T., K.M.
(Kazuhiro Murakami), N.B., H.O. and M.O.; Resources, J.T., Y.K. and S.Y.; Supervision, K.M. (Kunio Matsumoto);
Writing—original draft, N.J., R.I., K.S. and K.M. (Kunio Matsumoto); Writing—review and editing, N.J. and K.M.
(Kunio Matsumoto).
Funding: This work was supported in part by the Project for Cancer Research and Therapeutic Evolution(P-CREATE) from the Japan Agency for Medical Research and Development (AMED) to K.M., and Platform
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 JP18am0101078. This work was performed
under the Cooperative Research Program of the Institute for Protein Research, Osaka University (CR15-05), andan Extramural Collaborative Research Grant from the Cancer Research Institute (Kanazawa University).
Acknowledgments: We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.

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