Low-in ensi y con inuous
ul asound o inhibi cance cell
mig a ion
I zia González
1
*, Jon Luzu iaga
2
, Alba Valdi ieso
1
, Manuel Candil
1
,
Jesús F u os
3
,
4
, Jaime López
1
, Luis He nández
1
,
Luis Rod íguez-Lo enzo
5
, Vi ginia Yagüe
6
, Jose Luis Blanco
6
,
Albe o Pin o
1
and Julie Ea l
3
,
4
1
G oup o Ul asonic Resona o s RESULT, Ins i u e o Physical Technologies and In o macion, Consejo
Supe io de In es igaciones Cien íficas (CSIC), Mad id, Spain,
2
Signaling Lab, Depa men o Cell Biology and
His ology, Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain,
3
Molecula Epidemiology and P edic i e Tumo Ma ke s G oup, Ramón y Cajal Heal h Resea ch Ins i u e
(IRYCIS), Mad id, Spain,
4
Biomedical Resea ch Ne wo k in Cance (CIBERONC), Mad id, Spain,
5
Ins i u e o
Science and Technology o Polyme s ICTP, CSIC, Mad id, Spain,
6
Uni e sidad Poli écnica de Mad id UPM,
Escuela Técnica Supe io de Ingenie os de Telecomunicación, Mad id, Spain
In ecen yea s, i has been e ified ha collec i e cell mig a ion is a undamen al s ep
in umo sp eading and me as a ic p ocesses. In his pape , we demons a e o he
fi s ime how low-in ensi y ul asound p oduces long- e m inhibi ion o collec i e
mig a ion o epi helial cance cells in wound healing p ocesses. In pa icula , we
show how panc ea ic umo cells, PANC-1, g own as monolaye s in i o espond o
hese wa es a equencies close o 1 MHz and low in ensi ies (<100 mW cm
−2
) o
48–72 h o cul u e a e some minu es o a single ul asound i adia ion. This new
s a egy opens a new line o ac ion o block he sp ead o malignan cells in cance
p ocesses. Despi e ele an spa ial a ia ions o he acous ic p essu e ampli ude
induced in he assay, he cells beha e as a whole, showing a collec i e dynamic
esponse o acous ic pe o mance. Expe imen s ca ied ou wi h samples wi hou
p e ious s a ing showed ema kable e ec s o he LICUs om he fi s hou s o
cul u e, mo e p ominen han hose wi h expe imen s wi h monolaye s subjec ed o
as ing p io o he expe imen s. This new s a egy o con ol cell mig a ion
demons a ing he e ec i eness o LICUS on no s a ed cells opens a new line
o ac ion o s udy e ec s o in i o ul asonic ac ua ion on umo issues wi h
malignan cells. This is a p oo -o -concep s udy o demons a e he physical e ec s
o ul asound s imula ion on umo cell mig a ion. An in-dep h biological s udy o he
e ec s o ul asounds and unde lying biological mechanisms is on-going bu ou o
he scope o his a icle.
KEYWORDS
low-in ensi y ul asounds, cance cells, mig a ion, inhibi ion, umo , long- e m e ec s
1 In oduc ion
Collec i e cell mig a ion plays a c ucial ole in pa hological p ocesses such as cance
in asion and me as asis, phenomena ha depend on he in asi e and mig a o y capaci y o
malignan cells (F iedl and Gilmou , 2009;Yilmaz and Ch is o o i, 2010;Li e al., 2013;Haege
e al., 2015). Du ing collec i e mig a ion, cells mo e as a g oup main aining cell-cell junc ions,
excep o some leade cells a he on , which d i e mig a ion by scanning he en i onmen o
iden i y a pa h, while ollowe cells con ibu e o op imizing collec i e mo emen (Khalil and
F iedl, 2010;Re ay e al., 2011). Mic ofluidic pla o ms ha e made i possible o obse e and
OPEN ACCESS
EDITED BY
Claudia Tanja Mie ke,
Leipzig Uni e si y, Ge many
REVIEWED BY
I ana Dusan Pajic-Lijako ic,
Uni e si y o Belg ade, Se bia
Yilong Zhang,
Uni e si y o Dundee, Uni ed Kingdom
*CORRESPONDENCE
I zia González,
[email p o ec ed]
SPECIALTY SECTION
This a icle was submi ed o Cell
Adhesion and Mig a ion,
a sec ion o he jou nal
F on ie s in Cell and De elopmen al
Biology
RECEIVED 24 Decembe 2021
ACCEPTED 21 Decembe 2022
PUBLISHED 12 Janua y 2023
CITATION
González I, Luzu iaga J, Valdi ieso A,
Candil M, F u os J, López J, He nández L,
Rod íguez-Lo enzo L, Yagüe V, Blanco JL,
Pin o A and Ea l J (2023), Low-in ensi y
con inuous ul asound o inhibi cance
cell mig a ion.
F on . Cell De . Biol. 10:842965.
doi: 10.3389/ cell.2022.842965
COPYRIGHT
© 2023 González, Luzu iaga, Valdi ieso,
Candil, F u os, López, He nández,
Rod íguez-Lo enzo, Yagüe, Blanco, Pin o
and Ea l. This is an open-access a icle
dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License (CC BY).
The use, dis ibu ion o ep oduc ion in
o he o ums is pe mi ed, p o ided he
o iginal au ho (s) and he copy igh
owne (s) a e c edi ed and ha he o iginal
publica ion in his jou nal is ci ed, in
acco dance wi h accep ed academic
p ac ice. No use, dis ibu ion o
ep oduc ion is pe mi ed which does no
comply wi h hese e ms.
F on ie s in Cell and De elopmen al Biology on ie sin.o g01
TYPE B ie Resea ch Repo
PUBLISHED 12 Janua y 2023
DOI 10.3389/ cell.2022.842965
analyze hese p ocesses o cell mig a ion h ough issues. In i o
expe imen s show ha epi helial leade cells a he edge o a wound
unde go longe elonga ions han o he cells on he on edge o he
epi helial monolaye ha p opaga e pe pendicula o he di ec ion o
mig a ion (Wi z e al., 2011;Vedula e al., 2012;B ugues e al., 2014;
Re ay e al., 2014;Yamaguchi e al., 2015;T epa and Sahai, 2018).
The mechanical ole o leade cells du ing collec i e cell mig a ion and
some oles o mechanical o ces and cell coupling in collec i e cell
ea angemen and mig a ion in i o sys ems ha e been ecen ly
analyzed (T epa e al., 2009;B ugues e al., 2014;Yamaguchi e al.,
2015;T epa and Sahai, 2018).
T ac ion o ce measu emen s ha e been used o analyze epi helial
wound closu e in some s udies in he li e a u e13. While a wound
closes, cells exhibi wo ypes o ac ion o ce: o ces poin ing away
om he gap, as classically obse ed du ing cell mig a ion, and o ces
poin ing owa d he gap. In e es ingly, hese o ces can be a ibu ed o
lamellipodial p o usions and ac omyosin cable con ac ion. A la e
s ages o wound closu e, ac ion o ces mos ly poin owa d he
gap. Analyses o hese o ces wi hin he mig a o y cell monolaye s
show accumula ion o in e cellula s ess om he edge o he inside o
he monolaye , which induces a g ea e ension in he cell-cell
junc ions (Se a-Picamal e al., 2012;De o e e al., 2014). Thus,
mig a o y cell monolaye s can be seen as issues unde ension.
O he ecen s udies ha e shown su p ising oscilla o y mo emen s
o epi helial cells in monolaye s (Kocgozlu e al., 2016;No bohm e al.,
2016;Ladoux and Mège, 2017). These oscilla ions define mo emen s
o he cells owa d he ex e io o he epi helial monolaye , which
al e na e wi h mo emen s owa d he in e io in he adial di ec ion.
The unexpec ed a ie y o cell mo emen is ela ed o he complex
mechanical beha io o biological issues (cells can de o m) and hei
ac i e na u e na u e (Pa k e al., 2016) (cells can modi y hei
con ac ili y o adhesi e p ope ies).
On he o he hand, cell densi y is also an impo an egula o o
collec i e cell dynamics (Sada i e al., 2013;Ga cia e al., 2015;Ga g
e al., 2019). The a e age cell eloci y s abilizes when confluence is
eached and dec eases when cell densi y inc eases, leading o a swi ch
om a fluid-like o a mo e solid-like s a e (Sada i e al., 2013). As cell
densi y inc eases, each cell wi hin he popula ion becomes inc easingly
apped by i s neighbo s, leading o educed mo ion and g ea e
co ela ion o mo ion (la ge clus e s o cells ha mig a e oge he ).
This is known as he “cell jamming”e ec (Cios e al., 2021). The
la ge-scale coo dina ion is also educed wi h a ise in cell densi y,
associa ed wi h sho e cell displacemen s (Bazou e al., 2017;Sua ez-
A nedo e al., 2020).
Recen li e a u e has shown ha collec i e cell beha io s can be
al e ed by he applica ion o ex e nal o ces, including g a i y (Pa k
e al., 2016) and chemical and physical s imuli3. The inhibi o y e ec s
o elec omagne ic fields on cell monolaye s ha e been ecen ly
analyzed (Heal h E ec s o Exposu e o Ul asound and
In asound, 2010;Bazou e al., 2017;Sua ez-A nedo e al., 2020).
Howe e , he e ec s gene a ed by oscilla o y mechanical o ces
applied o cance cell monolaye s ha e no ye been analyzed in
he collec i e mig a ion p ocesses o umo cells and a e discussed
in his pape .
He ein, we demons a e how low-in ensi y ul asound p oduces
long- e m inhibi ion o collec i e mig a ion o epi helial cance cells,
in pa icula , in cell monolaye s a equencies close o 1 MHz and low
in ensi ies (<100 mW cm
−2
) o 48–72 h o cul u e a e some minu es
o a single ul asound i adia ion.
2 Me hods and ma e ials
Con en ional issue cul u e assays, ul asound gene a o s,
incuba o s wi h CO
2
supply and imaging equipmen ha e been
used o expe imen s on cell monolaye s ac ua ed by ul asonic
ansduce s. Sc a ched monolaye s o he confluen panc ea ic cell
line Panc-1 we e selec ed as samples o analyze wound healing
p ocesses wi h and wi hou p e ious ul asound i adia ion.
2.1 Expe imen al se up
Con en ional 8-well pla es wi h ec angula geome y and fla
bo om a e ul asonically d i en om a piezoelec ic ce amic PZ26
(Fe ope m) o a ec angula a ea (30 mm × 15 mm x 1.5 mm). A 10 V
peak- o-peak elec ical signal is supplied o he ansduce a a
equency o 1 MHz, gene a ing a con inuous sine wa e. The
ansduce is a ached unde nea h one well con aining he
sc a ched monolaye o he ansmission o ib a ions.
The imaging equipmen consis s o a b igh -field mic oscope
(Sma Cell Image Paula o Leica Mic osys ems) equipped wi h a
digi al came a unning i s specific so wa e o he acquisi ion and
con ol o images/ ideo du ing he en i e cul u e p ocess (Figu e 1A).
Thus, he dynamics o he cells a e moni o ed in eal ime a e hei
exposu e o he LICU e e y 10 min, o long pe iods o up o 72 h o
cul u e, depending on he s age o he wound healing p ocess filmed by
he mic oscope. NIH Science ImageJ eewa e was used o p ocess he
filmed images, allowing econs uc ion o he single-cell ajec o ies
(h ps://image.ne /so wa e/imagej/, Uni e si y o Wisconsin-
Madison).
Figu e 1A shows he expe imen al se up ha includes an incuba o
wi h a mic oscope inside con olled by a ex e nal compu e , an
ul asonic ansduce , a signal gene a o and well pla e including
he cell samples exposed o he ul asounds.
2.2 Expe imen al p ocedu e
(i) Cell cul u e p epa a ion, (ii) sc a ch-making, (iii) wound
healing assays: da a acquisi ion and de e mina ion o wound
healing a es, (i ) applica ion o ul asounds and wound healing
assays, and ( ) da a analysis.
2.2.1 Cell cul u e p epa a ion
The PANC-1 is an epi helioid ca cinoma cell line de i ed om he
human panc eas (Dee e al., 2010) and o ms pa o he ATCC
human cell cul u e collec ion (h ps://www.a cc.o g/). S anda d cell
cul u e p o ocols o PANC-1 cell lines we e applied o cell sample
p epa a ion ollowing he Cell G ow h P o ocol o PANC-1 cell line,
2009. PANC-1 cell lines we e cul u ed in RPMI (Gibco/In i ogen)
supplemen ed wi h 10% e al bo ine se um (FBS; In i ogen) and
50 uni s/mL penicillin/s ep omycin (In i ogen) and kep in an
incuba o a 5% CO
2
and 37°C. App oxima ely 8.8 × 106 PANC-1
cells we e pla ed in 8-well pla es and cul u ed un il confluen
monolaye s we e o med. Then, he cells we e ei he s a ed in
se um- educed medium (1% se um) o 24 h (s a ing condi ion)
o main ained in common comple e medium (10% se um) (con ol).
Du ing he wound-healing expe imen s, he cells we e exposed o
TGF-βs imula ion by he addi ion o 1 µL o ecombinan TGF-β o
F on ie s in Cell and De elopmen al Biology on ie sin.o g02
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p omo e cell mo emen in i o. All me hods desc ibed we e ca ied
ou in acco dance wi h ele an guidelines and egula ions o he hos
ins i u ions. Samples om human subjec s we e no used in his s udy.
2.2.2 Sc a ch-Making
The sc a ches we e made by using 2-µL pipe e ips wi h a
diame e o app oxima ely 400 mic ons. Once he cell monolaye is
sc a ched, he wound p esen s sha p s aigh bounda ies ha become
blu ed as he healing p ocess p og esses. Thus, he healing p ocess
occu s i egula ly. The a e o cell mig a ion can be quan ified using
wo single me ics: he wound wid h as he a e age dis ance be ween
he edges o he sc a ch and/o he wound a ea, which is calcula ed by
he cell- ee a ea in cap u ed images. Du ing his p ocess, nea by
leade and ollowe cells mig a e in di e en di ec ions, no jus in he
di ec ion o he wid h o he wound. The cells dispe se, making he
con ou s o he wound less sha p and less s aigh .
2.2.3 Wound healing assays
Da a acquisi ion and de e mina ion o wound healing a es
P ocedu e o de e mine wound healing a es. Du ing he wound-
healing expe imen s, he cells we e exposed o TGF-βs imula ion by
he addi ion o 1 µL o ecombinan TGF-β o p omo e cell mo emen
in i o. The wounds we e pho og aphed a egula in e als e e y
10 min o long pe iods o 24, 48 o 72 h. The Alama Blue assay was
used o measu e cell iabili y a e each expe imen acco ding o he
manu ac u e ’s ins uc ions.
Wound healing a es we e also compu ed om he wound a eas
measu ed on filmed images. The wound co e age o he o al a ea and
he a e age and s anda d de ia ion o he sc a ch wid h was analyzed
wi h he aid o he ImageJ plugin: Wound_healing_size_ ool (Pajic-
Lijako ic and MIli oje ic, 2020) (WHST). The esul s o assays we e
ob ained om a leas h ee independen expe imen s. Mig a ion a e
can be exp essed as he pe cen age o a ea educ ion o wound closu e
om he filmed images du ing comple e wound healing p ocesses
(Cukja i e al., 2001). We calcula ed he a e o cell ela i e wound
closu e acco ding o Eq. (1).(Moyano e al., 2022):
Wound Closu e %A 0−A Δh
A 0
x100 (1)
whe e A
=0h
is he a ea o he wound measu ed immedia ely a e
sc a ching ( = 0h) and A
=?h
is he a ea o he wound measu ed ?h
hou s a e he sc a h pe o mance. The closu e pe cen age will
inc ease as cells mig a e o e ime.
2.2.4 Applica ion o ul asounds and wound healing
assays
The cell samples we e exposed o ul asounds om a piezoelec ic
ac ua o a ached unde nea h o he well con aining he sample. I was
exposed only once pe expe imen o acous ic in ensi y le els close o
60 mWa /cm
2
, significan ly lowe han a issue inju y h eshold
desc ibed in he li e a u e (Sa aswa hibha la e al., 2020) du ing a
ime o ei he 10 min, 15min o 20 min in di e en expe imen s
immedia ely be o e he samples we e in oduced in o he incuba o o
hei cul u e.
A e applica ion o LICUs on he monolaye s, he samples we e
cul u ed o 2 o 3 days and filmed e e y 2 hou s. LICUs we e applied
ins ead o LIPUS (pulsed wa es) o con ol he exac ime o ac ion o
he monoch oma ic wa e a 1 MHz on he samples, ha is, he exac
numbe o acous ic cycles a said equency. The use o pulsed wa e
en ails a sho ansi o y p ocess un il eaching he equency o he
desi ed wa e. This unce ain y ime is e y sho o each wa e ain,
bu i becomes ele an h oughou he en i e ul asonic ac ua ion o
20 min, wi h millions o pulses in ol ed.
Figu e 1B shows he spa ial p essu e pa e n es ablished a a
equency o 1.003 MHz in a well selec ed o he expe imen s,
loca ed di ec ly abo e he ul asonic ac ua o . I shows b igh a eas
ha co espond o p essu e nodes and da k zones o maximum
p essu e ampli udes o 0.29 MPa measu ed wi h a needle
hyd ophone (P ecision Acous ics LTD., hyd ophone SN 1423).
Polys y ene beads wi h diame e s o 20 µm we e used o obse e
he 2D p essu e pa e n as hey collec ed a he p essu e nodes o he
s anding wa es gene a ed inside he well once d i en by a adia ion
o ce acous ically induced in he chambe . Nodes and an inodes a e
FIGURE 1
(A) Expe imen al se up including he ul asound gene a o , ansduce , mic oscope inside he incuba o and image p ocesso ; (B) Acous ic p essu e
pa e n es ablished a = 1.003 MHz in he well di ec ly loca ed o e he ul asonic ac ua o . Polys y ene beads (20 µm) collec a he p essu e nodes, d awing
his pa e n.
F on ie s in Cell and De elopmen al Biology on ie sin.o g03
González e al. 10.3389/ cell.2022.842965
sepa a ed sho dis ances o less han 1 mm. The wound made in he
cell monolaye also includes mul iple p essu e nodes along i s leng h
in bo h di ec ions, wid h and leng h o he gap.
A e exposu e o di e en ime in e als o 10, 15 o 20 min o
ul asonic ea men , he cul u e samples we e filmed o 48 o 72 h,
depending on he closu e si ua ion achie ed in he wound healing
p ocess a e he di e en doses o ul asonic i adia ion.
2.2.5 Da a analysis
S a is ical Analysis. All esul s a e p esen ed as he mean ± s anda d
e o mean (SEM). The Mann-Whi ney U es was pe o med o analyze
non-pa ame ic esul s. S a is ical es s we e pe o med using SPSS
S a is ics .22 (IBM). S a is ical significance was conside ed o be
*p≤0.05, **p≤0.01. Comple e analyses o cell mo emen s/ eloci ies
we e mapped and analyzed om a pa icle imaging elocime y PIV
s udy unning MATLAB, p o iding eloci y fields o he mul icell
assemblies and indi idual cells du ing wound closu e, including
leade s explo ing he subs a e and ollowe cells.
3 Resul s and discussion
Cells no mally mig a e when exposed o an emp y space. Unde
no mal condi ions, a wound in monolaye s o panc ea ic cance cells
akes a li le o e 24 h o close. These cells ha e li le mobili y, so hey
equi e longe imes han o he cell popula ions.
Cell mo emen s occu a low Reynolds numbe s (S okes
egime), which means ha iscous d ag o hei su ounding
medium domina es ine ial mo emen s, slowing down he
cell mo emen s. In his way, cell dynamics could be assimila ed
o fluid dynamics. Howe e , cell beha io s canno be a ibu ed o
simple lamina flows. In con as , cells o en show
coo dina ed o a ional mo ions ha span o e dozens o cells
nea he edges o he wound in he monolaye , including
swi ling mo emen s o cell g oups and o ices, also obse ed in
hese p ocesses ( o a ion mo ion is also obse able by change o
di ec ion o consecu i e g een a ows in Figu e 2B)(De o e e al.,
2014).
FIGURE 2
(A) PANC-1 cell eloci ies de i ed om filmed cell ajec o ies by PIV-MATLAB code (g een a ows) be ween 4 and 6 h o cul u e and o e imposed on he
mic oscopic image; (B) cells ajec o ies econs uc ed in ImageJ eewa e (Mul i acke plugin) om elapsed ames o a mo ie du ing a ime o 12 h in a
wound closu e p ocess; (C) de i ed healing eloci y ampli udes measu ed in fi e healing p ocesses s a ing om di e en wound wid hs; Scale ba : 100 µm.
F on ie s in Cell and De elopmen al Biology on ie sin.o g04
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3.1 E ec s o LICUs on cell mig a ion
eloci ies in PANC-1 monolaye s
The e ec s o he LICUS on he p og ession o he wound healing
p ocess we e obse able since he fi s hou s in he expe imen s. In
each expe imen wo pa ame e s, wound wid h and wound a ea, we e
espec i ely analysed o di e en objec i es. The fi s one was
equi ed by MATLAB-PIV so wa e o calcula e a mean app oach
eloci y: ime- a ia ions in he posi ions o he indi idual cells
be ween di e en filmed ames o a mo ie, shown in Figu e 2A by
g een a ows. On he o he , wound a eas we e necessa y o de e mine
he wound healing a es o e ime.
The e ec s o he LICUS on he p og ession o speed o he cells
and wound a ea we e obse ed om he beginning o he wound
healing p ocess, wi h a endency o dec ease g ea e han ha o a
p ocess unde no mal condi ions. Figu e 2A shows a e age eloci ies
o dozens o cells close o bo h sides o he wound bounda y in a ime
in e al anging om
1
= 15 h and
2
= 20 h a e 20min o acous ic
ea men in a mo ie. They a e d awn by g een a ows o e -imposed
o a ame o he mo ie. Leade cells a he edge o he wound desc ibe
longe eloci y ec o s ha poin in hei di ec ion o mig a ion
h ough he subs a e o cell gap.
Figu e 2B shows ajec o ies desc ibed by hund eds o Panc-1 cells
a bo h sides o a wound sc a ched in a monolaye 28 h a e he
ul asonic i adia ion a ~ 1 MHz. These ajec o ies we e
econs uc ed in ImageJ eewa e om di e en cells o he
monolaye nea he wound bounda ies a e 20 min i adia ion o
LICUs be o e cul u e. Du ing he wound healing p ocess, leade cells
enla ge hei shapes and acqui e highe eloci y ampli udes han
ollowe cells o scan he subs a e ee o cells, acco ding o he
FIGURE 3
Filmed PANC-1 cell mig a ion du ing wound healing p ocesses: (A) unde no mal condi ions, wi hou ul asound i adia ion; (B) a e exposu e o LICUs
a
US
= 10 min; (C) a e LICU i adia ion a
US
= 20 min; (D)
US
= 30min be o e he cul u e.
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González e al. 10.3389/ cell.2022.842965
li e a u e. Howe e , a e LICU i adia ion, he leade cells acqui e
di e en unexpec ed beha io s, as desc ibed in he ollowing. The
leading cells a he bo de o he mig a ing issue adhe e and mig a e in
amaeboid mo ion on he subs a e h ough he adhesion assembly and
ex ensions o fillopodia ha p opaga e pe pendicula o he di ec ion
o mig a ion, as desc ibed p e iously in he li e a u e (Wi z e al.,
2011;B ugues e al., 2014;Re ay e al., 2014;Yamaguchi e al., 2015;
T epa and Sahai, 2018).
Figu e 2C shows he e ec s o LICUS on quan ified cell eloci ies
in ou assays, whe e he blue g aphic co esponding o an assay wi h
p e ious i adia ion o 20min-LICUS decays o e ime much as e
han he o he cu es, ob ained om assays wi hou p e ious
ul asonic i adia ion.
Long- e m e ec s we e obse ed on he cell mo ion du ing he
whole cul u e ime, as shown in Figu e 2C (blue g aphic) wi h
quan ified cell eloci ies ob ained om ou assays. The cells did
no pe o m any indi idual o collec i e mo emen s du ing sonica ion
o du ing he ollowing 2 hou s. In ou mo e han 20 expe imen s, he
cells did no show any dynamics o immedia e esponse o he
applica ion o mechanical o ces de i ed om he es ablished
acous ic p essu e g adien in he well. This shows ha he
applica ion o LICUS does no p oduce a New onian beha io on
he cells, wi h immedia e ac ion/ eac ion mechanisms. On he
con a y, long- e m cell al e a ions on he cell dynamics we e
ound se e al hou s a e he LICUS i adia ion o a leas 2 days
o cul u e a e LICUS ea men , depending on he acous ic doses.
Figu e 3 shows his long delay e ec o he ul asonic i adia ion
on he wound healing p og ession o PANC-1 cell monolaye s on
h ee cul u e p ocesses p e iously exposed o di e en doses o
ul asonic i adia ion, o 10min, 20min and 30min–LICUS
espec i ely (Figu es 3B–D), compa ed o a no mal closu e p ocess
(Figu e 3A). Inc easing delays in he wound closu e we e obse ed
wi h he longe imes o ul asound i adia ion. This inhibi o y e ec
o LICUS induced on cell mig a ion was epea edly obse ed in all he
20 expe imen s.
Supplemen a y Video S1 shows compa a i ely wo mo ies aken
om wound healing p ocesses expe ienced by PANC-1 cell
monolaye s du ing 48 h: he uppe mo ie co esponds o a sample
p e iously exposed o 20 min o ul asonic i adia ion and lowe
mo ie o a wound healing de eloped unde no mal condi ions
(wi hou ul asound exposu e) espec i ely. A delay o gap closu e
is obse ed a e acous ic ea men , showing inhibi ion o collec i e
cell mig a ion. The ideo includes supe imposed colou s assigned o
each cell quan i ying hei indi idual displacemen s, including
ampli ude and di ec ion o mo ion. Red colou e e s o igh -
di ec ion mo ion, g een e e s o le -di ec ion, blue one e e s o
e ical displacemen s and yellow o s agnan condi ions. Se e al
leade and ollowe cells behind hem expe ience complex
e e sible and o a ional mo ions o e ime, showing di e en
colo s in he images. Thus, in a na u al wound healing p ocess
(wi hou any ex e nal o ce applied), PANC-1 leade and ollowe
cells in he fi s 6-8 ows behind he edge show la ge displacemen s
owa d he wound gap o find cells coming om he aced edge o he
gap. Bo h se s o aced cells confluence in ela i ely sho imes close o
1 day. In con as , he cance cells in monolaye s p e iously exposed o
20min-LICUs pa alyze hei mig a ion in ime, showing e y slow
displacemen s du ing 48 o e en 72 h o cul u e.
In addi ion o his e ec , we ound cance cell monolaye s
beha ing as single bodies a he han linked cells a e he LICUS
i adia ion: a he equency o ou expe imen s (~1 MHz). This
unique beha io occu s despi e complex 2D sound p essu e
pa e ns being es ablished wi hin he ea men well, wi h p essu e
nodes and an inodes sepa a ed app oxima ely 375 mic ons apa in
bo h ho izon al xand ydi ec ions shown in Figu e 1B. Howe e , he
impac o hese spa ial di e ences on he collec i e dynamic beha io
o cells in he monolaye was no obse ed in any o he mo ies.
3.2 Influence o s a a ion on he wound
healing p ocesses a e LICU i adia ion
Fou di e en ypes o expe imen s we e ca ied ou acco ding o
he combina ion o wo a iables o condi ions: LICU i adia ion/non-
i adia ion and se um s a a ion/non-s a ing condi ions o 24 h
be o e he ea men /cul u e, espec i ely o s udy he cell
mechano-beha io . Each o hese 4 condi ions was epea ed a leas
3 imes, so i was necessa y o ca y ou a leas 12 expe imen s. Thus,
six assays we e pe o med unde s a ing condi ions p io o
sc a ching, o p e en cell p oli e a ion du ing he cell cul u e.
Th ee o hem included LICUS ea men s a e he s a a ion o
be compa ed wi h he o he h ee samples wi hou ul asonic
i adia ion and p o ided di e en esul s.
Quan ified esul s o mean wound wid h and closu e eloci y a e
shown in Figu e 4 o e ime. They we e de e mined om mo ies aken
on s a ed and no s a ed samples espec i ely. Ini ial wound wid hs
o 400 µm we e made on he PANC-1 monolaye s o pe o m he
expe imen s wi h iden ical geome ical configu a ions.
In hese figu es, blue and black g aphics co espond o non-
s a ed samples exposed and no exposed o LICUS ea men a
he beginning o he expe imen espec i ely. Red and magen a
colo ed g aphics desc ibe wound healing wid h and eloci ies o e
ime in s a ed samples (wi h and wi hou p e ious LICUS ea men
espec i ely).
All he cu es in his figu e decay o ze o alues: s a ed samples a
he end o he cul u e p ocess and no s a ed samples be o e. This
means ha hey ei he close ully he gap wi hou ul asonic i adia ion
(blue and ed lines) o hey s op hei wound healing p ocess wi hou
culmina ing he gap closu e a e LICUS ac ua ion (black and magen a
lines). A any o bo h acous ic condi ions, s a ed samples acqui e
smalle eloci y ampli udes o e ime. The wound wid h dec eases
o e ime du ing healing p ocesses unde no mal condi ions (wi hou
applica ion o ex e nal o ces) while aced monolaye s a bo h sides o
he wound app oach each o he , showing a dec ease on cell eloci ies
un il he cell confluence. This happened in bo h, s a ed and no
s a ed samples (blue and ed g aphics o Figu es 4A, B). The non-
s a ed samples no p e iously exposed o LICUS (blue g aph) close
he wound in he sho es imes (be o e 24 h), wi h a e y apid
dec ease in he speed o app oach o he wound on s in an equi alen
ime, un il wound closu e.
Howe e , we ound less empo al dec ease in he wound wid h in
hose samples ha had been p e iously LICUS i adia ed, bo h wi h
s a a ion and wi hou s a a ion. (black and magen a colo ed
g aphics o Figu e 3A). In ac , in he expe imen s he cell
confluence o he wo on s o he wound is no eached a leas
du ing a 48-h cul u e. On he con a y, he wound wid h emains
opened o a leas 2 days a e low in ensi y ul asound i adia ion.
This e idences a long- e m inhibi ion o cell mig a ion in he wound
healing p ocess o cance monolaye s.
F on ie s in Cell and De elopmen al Biology on ie sin.o g06
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In summa y, non-s a ed cell monolaye s show quan i a i ely
ema kable e ec s on he cell mig a ion p ocesses om he fi s
hou s o cul u e when i adia ed wi h ul asound. Howe e , he
monolaye s subjec ed o as ing p io o he expe imen s did no
show he e ec s o LICUs on he collec i e cell mo ion so clea ly
du ing he fi s 24 h o cul i a ion, bu a he hey we e mani es ed
la e , on he second day, and hey a e no as p ominen e ec s as hose
ound in he expe imen s wi hou s a ing.
Figu es 5A, B show quan ified esul s o ela i e wound healing
o e ime o he ou es condi ions desc ibed abo e.
3.3 Rela i e wound closu e in no s a ed
samples
Monolaye s p e iously exposed o s a a ion p o ided a alue o
0.5 ela i e o wound closu e a e 24 h o cul u e in no mal
condi ions (black ba s co esponding o con ol samples), as g ey
ba s show in Figu e 5A. The e ec s o LICUS on hese samples a e
negligible du ing he fi s day o cul u e, bu become mo e
ema kable in he nex 24 h, whe e he ela i e wound closu e
a io eached 0.2 poin s lowe in ea ed cells han in con ol cells
(g ey ba s). A e 48 h, hese e ec s became no iceable, eaching a
di e ence o app oxima ely 20% in he ela i e wound closu e on
samples exposed o he acous ic i adia ion. Fu he mo e, s a is ical
analysis confi med he significan di e ence be ween he wo g oups
a 48 h, *p≤0.05. As s a a ion is commonly used o ensu e ha only
mig a ion in wound healing assays is being assessed, his expe imen
confi ms he e ec o LICUS on mig a ion inhibi ion o PANC-1
cance cells.
3.4 Rela i e wound closu e in no s a ed
samples
Wounds o monolaye samples no s a ed closed much mo e
apidly han hose wi h s a a ion in no mal condi ions wi hou
LICUS (g ey ba s), p obably due o a combined e ec o cell
mig a ion and p oli e a ion. An inc ease o app oxima ely
0.9 was ound a e 24 h in con ol cul u es unde non-s a ing
condi ions. Howe e , a e 48 h, he ela i e wound closu e alue
was simila in bo h cases (s a ing and non-s a ing), showing a
long- e m esponse con e gence. In addi ion, a no able influence o
LICUswas oundon heseno s a ed samples, wi h ela i e wound
closu e a 24 h o cul u e, (black ba in Figu e 4B). Su p isingly,
bo h he ela i e wound closu e and he s a is ical analysis e ealed
FIGURE 4
(A) Wound wid hs measu ed o e ime on he filmed monolaye samples a di e en condi ions o he expe imen s; (B) quan ified wound closu e
eloci ies de i ed om he filmed images o he samples a he di e en condi ions o he expe imen s.
F on ie s in Cell and De elopmen al Biology on ie sin.o g07
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he significan impac o LICUS on non- as ing samples o e ime,
om he fi s 24 h o cul u e a e ul asonic i adia ion. The
ela i e wound closu e a io was dec eased by 0.6 poin s in he
fi s 24 h and 0.5 poin s in he pos e io 24 h compa ed o non-
ea ed cells. This was suppo ed by s a is ical analysis, which
showed a **p≤0.01 in bo h cases.
3.5 Analysis o cell iabili y
Analysis o cell iabili y was pe o med 12 h a e he end o he
expe imen s, once finished he cul u e p ocess o 48 h o 72 h
depending on he expe imen s. Alama BLUE assays we e used o
pe o m his s udy in ou eplica e expe imen s (Figu e 5C). This
analysis was made on bo h se um-s a ed samples and on se um-
con aining samples wi h simila iabili y esul s.
Finally, o he expe imen s we e pe o med in he de ice wi h
fib oblas monolaye s o compa e he e ec s o LICUS on he cell
mig a ion in bo h di e en cell lines, which a e displayed in Figu e 6
wi hou LICUS i adia ion (Figu e 6A) and a e a exposu e o 20min-
LICUS espec i ely (Figu e 6B).
Compa ison o esul s o Figu e 6 show di e en influence o he
LICUS i adia ion on he wound healing p ocesses o PANC-1 and
BJ-hTERT espec i ely pe o med a he same acous ic and
geome ical condi ions. Fib oblas s show g ea e mobili y han
panc ea ic cells, as i can be seen in he filmed ames o
Figu e 6A, whe e i can be obse ed how hey each confluence
in 12 h. Howe e , fib oblas s a e also suscep ible o ul asonic
i adia ion, as shown in Figu e 6B.
A e exposing hese cells o i adia ion o LICUS o 20 min,
he monolaye gap emains pa ially opened du ing he fi s 24 h o
cul u e, in con as o hei beha iou unde no mal
condi ions (wi hou acous ic ea men ), whose wound closes in
app oxima ely 12 h. In con as , PANC-1 cells ha e e y low
mobili y, ei he unde no mal wound healing condi ions o a e
being i adia ed wi h LICUS. In no mal condi ions, PANC-1
monolaye s equi e mo e han 24 h o close a wound wi h a
wid h simila o ha o fib oblas monolaye s. Once exposed o
he same LICUS dose i adia ion, hey main ain a wide wound
space han fib oblas s a leas o 48 h cul u e.
O he analyses a e being pe o med in he expe imen s ega ding
he influence o LICUS on some gene exp essions. In pa icula , a
diminu ion o he ascula endo helial g ow h ac o ecep o 3
(VEGFR-3) as well as VEGFR 5-7 and a ise o some in e leukins
ha e been ound a e he LICUS ac ua ion on he samples, which is
o e exp essed in cance cells. An in-dep h s udy o hese and o he
changes in gene exp ession in ol ed in panc ea ic cance p ocesses is
equi ed as he nex s ep in his s udy.
FIGURE 5
Quan ifica ion o ela i e wound closu e o PANC-1 cells in no mal condi ions (g ey ba s) and a e 20 min o LICU ac ua ion (black ba s) a e 24 h and
48h(n=3)in(A) s a ed monolaye s and; (B) in non-s a ing condi ions. S a is ical significance *p≤0.05, **p≤0,01. UMann-Whi ney es ; (C) Analysis o cell
iabili y a e LICUs ea men using Alama BLUE assays pe o med in ou eplica expe imen s.
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4 Discussion
The e ec s o low-in ensi y ul asound on collec i e cell
mig a ion we e in es iga ed in mo e han 20 expe imen s and
a e desc ibed in his pape . In summa y, LICUs hinde
mig a ion o epi helial cance cells in wound healing-induced
p ocesses. In pa icula , we show how panc ea ic PANC-1
cance cells g own in monolaye s in i o wi h a wound, sense
and espond o hese wa es a equencies close o 1 MHz and low
in ensi ies (<100 mW cm
−2
) o 48–72 h a e ul asound
i adia ion. In pa icula , he wound emainsopen o a leas
48ho e en72ha e sho - e macous ic ea men so less han
25 min, depending on he ul asonic dose selec ed. Despi e he
s ong spa ial g adien s o acous ic p essu e es ablished wi hin he
a ea o he well con aining he wound in he monolaye , o ew
hund eds o mic ons, he cells beha ed as a whole, showing a
collec i e dynamic esponse o acous ic pe o mance, a bulk-like
beha io o he cell monolaye s a e hei exposu e. This beha io
was also obse ed in p e ious expe imen s ca ied ou on umo
panc ea ic explan s ea ed wi h LICUs o longe imes, up o
2 hou s o acous ic i adia ion (Pajic-Lijako ic and MIli oje ic,
2020).
In addi ion, he e ec s o LICUs on samples wi hou s a ing we e
mo e p ominen han hose o he monolaye s p e iously exposed o
s a a ion o 24 h, showing quan i a i ely ema kable e ec s om he
fi s hou s o cul u e, unlike s a ed monolaye s, wi hou appa en
LICUS e ec s du ing he fi s 24 h o cul i a ion.
Low-in ensi y ul asound deac i a es cell mig a ion p ocesses a
ce ain acous ic condi ions, in pa icula hose es ed in his esea ch,
as demons a es a pa alysis in he wound healing p ocess o cance
cell monolaye s du ing a leas 48 h. This wo k is he fi s p oo -o -
concep s udy o demons a e he physical e ec s o ul asound
s imula ion on panc eas umo cell mig a ion. An in-dep h
biological s udy o he e ec s o ul asounds is on-going bu ou
o he scope o his a icle. Biochemical and o he e ec s in ol ed in
he acous ic esponse o he cell monolaye s a e no con empla ed as
well as mechanisms associa ed o he ole o iscoelas ici y in
p o oking appa en ine ial e ec s and gene a ing he oscilla o y
mechanical ins abili ies in he o m o s anding wa es and
p opaga i e wa es caused by collec i e cell mig a ion (Heal h
E ec s o Exposu e o Ul asound and In asound, 2010;Pajic-
Lijako ic, 2021). These wa es could be a ec ed by he ul asound
ac ua ion i hey p esen simila equencies and ampli udes o he
same o de o magni ude o he ul asonic wa es, bu would be
negligible in o he condi ions. This la e is he case in ou
expe imen s, since hese wa es co espond o equencies much
lowe han hose o he applied ul asonic field.
The findings om his s udy highligh ele an inhibi o y e ec s o
ul asound on cell mig a ion no epo ed be o e and i seems ha
LICUS could ha e e ec s on cell p oli e a ion om he expe imen s
ca ied ou on samples wi hou p io as ing. These findings open a
doo o he s udy o he e ec s o low in ensi y ul asound in- i o wi h
umo issues ha canno be exposed o as ing.
As ou line o a u he esea ch, we ha e ound ha ce ain genes
di ec ly in ol ed in he cell mig a ion/mobili y show a ia ions in
hei espec i e exp ession in he samples p e iously exposed o
LICUS.
A non-in asi e echnology capable o join ly inhibi ing mig a ion
and p oli e a ion will be especially ele an in non-in asi e slowing
down umo p og ession.
FIGURE 6
Wound wid h p og ession filmed o e ime in PANC-1 and Fib oblas BJ-hTERT monolaye samples espec i ely: (A) wi hou LICUS exposu e be o e he
cul u e; (B) a e 20min LICUS i adia ion.
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