patlisci2, nano-tera 2016
TRANSCRIPT
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PATLiSci II – Probe Array Technology for Life Sciences /Rapid Sensing of Cancer
cells
Indentation-typeAFM
targetadsorption(sensing)
orce spectroscopy mappingllows to identify breast cancerells among healthy cells inisse by their characteristicistogram of elasticity modleales"
Label free# ampli$cation free and tisse sample basedetection of biomar%ers sing the cantile!ers insensing mode in the same FL&' AF( head com)plements information abot stats of a tmor"Case stdies* melanoma# breast cancer"
Implementation of cantile!er arrays for parallel ac+isitionof force !s" distance cr!es in a commercial FL&' AF(head ,-A-.SRF A0 Liestal1 increases throghpt of thetechni+e"
Partners* ni!ersity of 2asel# ni!ersity 3ospital 2asel#&PFL# CS&(
-A-.SRF A0PATLiSci
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The clinical !ale of newdiagnostic tools
Based on the current diagnostics,
patients are undertreated or more oftenovertreated
Overtreatment includes chemotherapy (and all its side eects) ormore etensive surgery (and its additional ris!s and more di"cult
recovery)#
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TLiSci 4 – Probe Array Technologiese Sciences – Rapid Sensing of Canc
Partners*ni!ersity of 2asel# ni!ersity3ospital 2asel# &PFL# CS&(#
$!in and %reast cancer are among the most comcancers of the &orld
'he faster cancer can %e detected, the %etter e need rapid diagnostic tools#
It has %een found that cancerous %reast cells havdierent elasticity than healthy ones#
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Atomic Force (icroscopy ,AF(1
Palpation Indentation
(acrometer scale -anometer scale
'he tip touches andfeels the
moleclarstrctres
'he e5aminer touches andfeels the
patient6s body
3ow can we measre this7
Cell and Cell ) &C( interactions occr at the nano s
How physical properties of tissues
contribute
to cancer development and
progression?
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Partners*ni!ersity of 2asel# ni!ersity3ospital 2asel# &PFL# CS&(#
*antilevers can pro%e the elasticity of surface loca%y force spectroscopy#
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le!er arrays ha!e been microfabric&()&PFL
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e (apping Spectroscopy on 2iopsincer cell elasticity is pro%ed %y force spectroscopyrce-distance curves) in a grid on the %iopsy surfac
ousands of force +distance curves are evaluateding a stiness histogram#
e shape of the histogram clearly allo&s to distingualthy tissue from cancerous cells
e results from classical histology, uorescent in-sit%ridiation and force spectroscopy maps are comp
omar!er detection %y cantilever sensing#
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2reast Tisse Composition
www.proteinatlas.org
histology
cell sie. /-0 1m
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mary tmor pro$le and the ad8acent tisse re!eal a cphenotype detected in the lymph node metastases
Field eect evaluation
2rognostic32redictive Mar!er
405 specimensmeasured &ithforcespectroscopy.
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Actin367AlamininActin367A
*ancer cells BM Fi%ro%lasts 8*M
*ollagen I
Co)cltre of cancer cells and$broblasts on nati!e 2(
cancer cells
BM
9%ro%lasts
:;
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Cancer cells Re;ection CAFs
2(
Cancer cells
=*'44/ cells > *AFs, 4? FB$, start after : days co-culture, imaging time /5h
CAFs
Cancer cells prepared on basalmembrane for calibration prposes
@; 1m
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*om%ination of force and opticalmicroscopy
for live cell and tissue imaging
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e Spectroscopy 3istogram Analysi
resence of cancer cells in a %iopsy is detectedn the histogram
isadvantage. It ta!es several hours
mprovement &ithin 2A'i$ci .sing a cantilever array speeds up diagnosisaster data acDuisition applied using dedicated
ard&are of a 7ano$urf FleAFM head
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• Cse cantilever array
• Cse E*$8 (vertical-cavity
surface emitting laser) array
&ith 4 E*$8 aligned to
each cantilever
• $&itch E*$8s on (and o)
fast
• $ynchronise readout of
position sensitive detector tomeasure the deection of
each cantilever
• Increase speed of data
acDuisition VCSEL array
Photodetector
Cantilever array
*opyright ;4/ *$8M Mi 2age 4@
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2roof of principle.• E*$8 array. @;µm pitch, λ G
0@;nm
• pac!aged
• dedicated driver electronics
created
• Hay-tracing simulations to optimise
optical design
• Optimisation of readout of position
sensitive detector
• Alignment. many degrees of
freedom *opyright ;4/ *$8M Mi 2age 4/
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• Bench-top set up
2roof of principle.
2$6
*antilever array
Beamsplitters
nd lens
E*$8array > 4stlens
$ample
*opyright ;4/ *$8M Mi 2age 45
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Integration in a commercialAFM.
• Modi9ed FleAFM• Modi9ed optics + tilted
• Addition of alignment
system for E*$8 array
(,y,,θ)
$tandard 7anosurf FleAFM
Modi9ed FleAFM &ith tilted optics and E*$8 alignment
E*$8 alignment
'ilted optics
*opyright ;4/ *$8M Mi 2age 40
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Optics.•
Optics is tilted#• $tandard FleAFM optics not
&ide enough for cantilever
arrays &ithout modi9cation#
• Hay tracing predicts the
eect of a%erration on the
dierent cantilever spots
*opyright ;4/ *$8M Mi 2age 4
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E*$8s.•
7e& E*$8 electronics.• Faster s&itching speed (J;;!=)
for %etter precision &hen
determining the spot position on
the 2$6
• Modulation of the driver signal at
J::M= allo&s us to avoid
oscillations in the E*$8 output
• *an %e controlled from FleAFM
soft&are
*opyright ;4/ *$8M Mi 2age ;
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Alignment.•
7eed to reduce the manydegrees of freedom in the
%enchtop system to %e
compati%le &ith the FleAFM
• J degrees of freedom retained
in the E*$8 positioning. ,y,
and Θ
• eight restrictions on
alignment system*A6 dra&ings of alignment
system for E*$8s
*opyright ;4/ *$8M Mi 2age 4
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o support force spectroscopy 9ndings cantilere also operated in sensing mode to detectiomar!ers for cancer#
peci9c %inding of molecules to pro%es on thantilevers leads to %ending due to surface
tress change
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lanoma tmor progression
PATLiSci
Radial growthphase :erticalgrowth phase
M# Eol!enandt. Maligne Melanome. In.Dermatologie und Venerologie.
O# Braun-Falco u# a# (8d#), Eerlag$pringer, ;;@, $# 4:4:+4:J
Melanoma on a patientLs s!in(source . 7ational *ancerInstitute)#
6ierent gro&th phases.Hadial, vertical, circulating tumor cells
Metastatic malignant melanoma at the heart
i!iMedia *ommons
1960 20060.000
0.002
0.004
0.006
0.008
0.010
r a t e
o f i n c i d e n c e
year
development of lifetime risk for
melanoma in the last 50 years
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e
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rocedre
totalRNA
evice !et"p
functionalization
injectionisolation
c# Hybridization
a)
b)
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psies al&ays contain healthy ,wildtype1 BHAF#
&e &ill al&ays see a BHAF &ildtype signal (green cr
elanoma tmor cells are present, the cantileverctionalied &ith 2RAF :=>>& mar!er &ill detect it#
en &e &ill see a BHAF E/;;8 signal (red cr!e
PATLiSci
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o% for the red cr!e?
ple and rapid diagnostics of melanoma (BHAF E/;;
(elanoma tmor present -o s%in cancer
PATLiSci
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s on melanoma %iopsies. all samples correctly ide
positi!es -o false
PATLiSci
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gram representation* all biopsies correctly id
e positi!es -o false negati!esPATLiSci
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imating the detection limit
s of cell line H7A etracts containing BHAF E/;;8d type to assess the detection limit. %etter than
@ ? mutant in &ild type#PATLiSci
Institut fr 2athologie, Basel;4:N;:N40
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:J
-o labelling# no PCR ampli$cation#
no se+encing
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In 4; ? of melanoma patients a dierent mutatioBHAF E/;; instead of BHAF E/;;8 + is responsi%lfor s!in cancer
By choosing appropriate 67A pro%es on thecantilevers, &e can detect %oth BHAF E/;;8 and
BHAF E/;;
cting di@erent mtations sing cantile! sensors
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9etect 2RAF :=>>& 9etect 2RAF :=>>
PATLiSci
@;? of melanomapatients
4;? of melanomapatients
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se 4* 2reast Cancer
ast cancer is detected %y evaluation of =8H leveman epidermal gro&th factor )
ay, certain aggressive mammary carcinomas can
ated &ith therapeutic anti%odies (trastuuma%)
tection of =8H overepression allo&s to identifyients &ho &ill respond to such a therapyPATLiSci
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perimental procedre
ollecting
%iopsy
'issuesample
3 i s t o
l o g y
& 5 t r a c t R - A
Cantile!ersensing
Florescentin sit hybridi)
Bation ,3&R41
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3&R4Positi!e nmsignal
3&R4 -egati!e)D nm signal
PATLiSci
C il 4 C il
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-mbe
rType of Tmor
R-A
yield"
,Eg1
R-A
conc"
,ng/El1
Cantile!
er
response
s
3&R4
stats
Cantile!
er
sensing
reslt
<
Invasive ductal
carcinoma4;# ::
-@ nm
negati
!e
negati!
e
4Invasive ductal
carcinoma;#@@0 40#/
-; nm
negati
!e
negati!
e
6uctal carcinoma
in situ4#/ /@#J
>4; nm
positi!
e
positi!e
G
=ighly
dierentiated
lo%ular carcinoma
9&0RA9&9 R-A
SA(PL&
4# /J#4J
>@ nm
negati
!e
positi!e
DInvasive ductal
carcinoma#04 :#@4
-@ nm
negati
!e
negati!
e
=
Invasive ductal
carcinoma
9&0RA9&9 R-A
SA(PL&
4#/4 @:#0
>J; nm
negati
!e
positi!e
=8H
Bdetec
t
ion
results
correctly identi$ed" 4 false positi!es de to degraded biopsy R
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-anoSrf Fle5AF( head
.ptics andpositioning stage:CS&L board ,CS&(1
gration of Cantile!er Array Sensinhnology into the Fle5AF( head
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LEDs represent
the eight VCSELs
SDA SCL GND
$ontrol electronics for %$!&'s via 4()it *2$(+"s
PATLiSci
l t ti f i h
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6-levels valve device
Kent Scientific Sringe
!"#p$nverted #icr%sc%pe&le'A&(
Valves indicat%r
!C f%r #%nit%ring the cantilever arra
and the ill"#inati%n %f the VCSELs
lementation of peripheryted microscope# !al!e# syringe pmp
PATLiSci
t d l t ,C>>> A li ti
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>>"Applicatio
PATLiSci
eado t test
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eadot test
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l i
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onclsions
setup for detection of %reast cancer andelanoma %ased on a 7ano$urf FleAFM hea
as %een developed#
reast cancer diagnosis is %ased on %oth forcpectroscopy and cantilever array sensingiomar!er detection
PATLiSci
t t
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emonstrators
Mem%rane $urface$tress$ensors for an8lectronic 7ose
PATLiSci
7ano$urfFleAFMfor Force$pectros-copy using a
canti-lever array
word abot biopsy sample +ality*
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word abot biopsy sample +ality*-A +ality control ,electrophoresis
tal R-A ,
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parison of measrements with total Ri@erent +ality ,sable !s" degraded1
0ood sample*