PATLiSci II Probe Array Technology for Life Sciences /

Rapid Sensing of Cancer

Force spectroscopy mapping

to identify breast cancer

Implementation of cantilever
arrays to increase throughput
Indentation-type
AFM

cells
Label free, amplification free detection of
target
adsorption biomarkers to complement information
(sensing)

Partners: University of Basel, University Hospital Basel,

EPFL, CSEM
PATLiSci
NANOSURF AG
 The clinical value of new
diagnostic tools
Based on the current diagnostics,

patients are undertreated or more often

overtreated

Overtreatment includes chemotherapy (and all its side effects) or

more extensive surgery (and its additional risks and more difficult
recovery).
 How physical properties of tissues
contribute
to cancer development and
- Cell and Cell - ECM interactions occur at the nano sc
progression?
How can we measure this?
Atomic Force Microscopy (AFM)
Palpation Indentation

The examiner The tip

touches and touches and
feels the feels the
patient`s body molecular
structures

Macrometer scale Nanometer scale

Cantilevers can probe the elasticity of surface loca
by force spectroscopy.

Partners:
University of Basel, University
PATLiSci Hospital Basel, EPFL, CSEM,
ilever arrays have been microfabrica
SEM-EPFL

PATLiSci
Breast Tissue Composition

histology

www.proteinatlas.org
cell size: 6-8 m

M. Plodinec and R. Lim: Mammary Stem Cells: Methods and Protocols, Springer
 Prognostic/Predictive Marker
mary tumor profile and the adjacent tissue reveal a c
phenotype detected in the lymph node metastases
Field effect evaluation

End of 2016:
292 breast core
biopsy
specimens
measured with
force
spectroscopy:
M. Plodinec and R. Lim: Mammary Stem Cells: Methods and Protocols, Springer
 Cultivation of cancer cells on native basal
membrane for calibration purposes

Cancer cells

BM

CAFs

Cancer cells Reflection CAFs

50 m

HCT116 cells + CAFs, 1% FBS, start after 3 days co-culture, imaging time 67h
Combination of force and optical
microscopy
for live cell and tissue imaging
 Use 8-cantilever array to
reduce acquisition time
Use VCSEL (vertical-cavity
surface emitting laser) array
with 1 VCSEL aligned to
each cantilever
Switch VCSELs on (and off)
fast
Synchronise readout of
position sensitive detector to
Photodetector
measure the deflection of
each cantilever
Increase speed of data VCSEL array
acquisition
Cantilever array

Copyright 2016 CSEM | | MLi | Page 10

 Integration in a commercial
AFM:
Modified FlexAFM
Modified optics tilted
Integrated micromechanics
for optical alignent of
VCSEL array (x,y,z,z
Parallel AFM instrument
installed on inverted VCSEL alignment
microscope

Tilted optics

Standard Nanosurf
FlexAFM Modified FlexAFM with tilted optics and VCSEL alignment

Copyright 2016 CSEM | | MLi | Page 11

mechanical Sensing of Biomarkers for Ca
a)
isolation
total injection
RNA

c) Hybridization

b) functionalization

Device Setup
PATLiSci F. Huber et al., Swiss Medical Weekly 146,
Case 1: Melanoma
Skin Cancer)
Melanoma on a patient's skin
(source : National Cancer
Institute).

Today, life expectancy of skin cancer patients can be

extended by 1 year, if treated with special drugs

These drugs require the presence of a point mutation

BRAF V600E in the genes, which is present in
50-60 % of all melanoma patients

PATLiSci
A 38-year-old man with BRAF-mutant melanoma and subcutaneous
metastatic deposits
Before After 15 weeks of Relapse after 23
therapy therapy weeks

Therapeutic
Effect of
Zelboraf

Nikhil Wagle et al. JCO 2011;29:3085-3096

2011 by American Society of Clinical Oncology
PATLiSci
PATLiSci F. Huber et al., Swiss Medical Weekly 146,
opsies always contain healthy (wildtype) BRAF.

we will always see a BRAF wildtype signal (green curv

melanoma tumor cells are present, the cantilever

nctionalized with BRAF V600E marker will detect it.

hen we will see a BRAF V600E signal (red curve)

PATLiSci
ook for the red curve!
mple and rapid diagnostics of melanoma (BRAF V600E

Melanoma tumor present No skin cancer

PATLiSci F. Huber et al, Nano Lett. 16, 5373-5377

s on melanoma biopsies: all samples correctly iden

positives No false n
Sensitivity: < 5 % V600E positive tumor
content in biopsy
PATLiSci Better than COBAS
F. Huber test from
et al, Nano Roche
Lett. 16, 5373-5377
ase 2: Breast Cancer

east cancer is detected by evaluation of HER 2 level

uman epidermal growth factor receptor 2)

day, certain aggressive mammary carcinomas can b

eated with therapeutic antibodies (trastuzumab)

etection of HER 2 overexpression allows to identify

tients who will respond to such a therapy
PATLiSci
xperimental procedure
ogy
tol
His

Fluorescent
in situ hybridi-
zation (HER2)

Collecting Tissue Extrac

t RN
a biopsy sample A

Cantilever
sensing
PATLiSci
 HER2 HER2 Negative
Positive -5 nm signal
+10 nm
signal

PATLiSci
 ER2 detection results (updated)
PATLiSci
s II
correctly identified. 3 false positives due to degraded biopsy RNA samples
Conclusions
A setup for detection of breast cancer and
melanoma based on a NanoSurf FlexAFM head has
been developed.
Breast cancer diagnosis is based on both force
spectroscopy and cantilever array sensing
biomarker detection

Collaborations with end users (hospitals)

Scientific Impact: 5 peer-reviewed papers
including 1 book chapter, 1 PhD thesis, 1 Master
thesis
Industry partner: NanoSurf (Nuomedis) AG
3 Patents:
- Plodinec, Lim and Loparic. US Patent 8,756,711 B2
- WO2013087726-A1 EP2791688-A1 US2014338073-A1
CN104067133-A JP2015500990-W, Patent Assignee: UNIV BASEL,
PATLiSci
Inventor(s): PLODINEC M; LOPARIC M; LIM R Y
 PATLiSci Probe Array Technology for Life Science Applications

Project Partners
H. Heinzelmann
CSEM Probe array
E. Meyer (PI) technologies
Ch. Gerber R. Lim
Uni Basel M. Loparic
Cantilever sensors Uni Basel
E-Mapping

N. de Rooij, S. Gautsch R. Zanetti

EPFL-IMT, MEMS design & Hospital Basel
fab Oncology

K. Glatz P. B. Henrich
Hospital Basel Hospital Basel
Pathology Ophthamolmology
A word about biopsy sample quality:
RNA quality control (electrophoresis
otal RNA (18S and 28S bands)

4000

-28S rRNA
2000

1000
-18S rRNA
500

200

25

RNA Quality
Control
PATLiSci
mparison of measurements with total RN
different quality (usable vs. degraded)

Good sample: 18S andBad sample: 18S and

28S bands present 28S bands NOT present
Cantilever sensing: Cantilever sensing: detected a
correctly
PATLiSci identified as false positive (actually HER 2 n
ce Spectroscopy Histogram Analysis

Presence of cancer cells in a biopsy is detected

n the histogram

Disadvantage: It takes several hours

mprovement within PATLiSci 2:

Using a cantilever array speeds up diagnosis
Faster data acquisition applied using dedicated
hardware of a NanoSurf FlexAFM head

PATLiSci
ATLiSci 2 Probe Array Technologies
fe Sciences Rapid Sensing of Cance

Skin and breast cancer are among the most comm

cancers of the world
The faster cancer can be detected, the better !
We need rapid diagnostic tools.

It has been found that cancerous breast cells have

different elasticity than healthy ones.

Partners:
University of Basel, University
PATLiSci Hospital Basel, EPFL, CSEM,
ce Mapping Spectroscopy on Biopsie
ancer cell elasticity is probed by force spectroscopy
orce-distance curves) in a grid on the biopsy surface

undreds of force distance curves are evaluated

sing a stiffness histogram.

he shape of the histogram clearly allows to distingui

ealthy tissue from cancerous cells

he results from classical histology, fluorescent in-situ

ybridization and force spectroscopy maps are compa

iomarker detection by cantilever sensing.

PATLiSci
ecting different mutations using cantileve
y sensors

In 10 % of melanoma patients a different mutation

BRAF V600K instead of BRAF V600E is responsibl
for skin cancer

By choosing appropriate DNA probes on the

cantilevers, we can detect both BRAF V600E and
BRAF V600K

PATLiSci
PATLiSci
 Detect BRAF V600E Detect BRAF V600K

50% of melanoma 10% of melanoma

patients patients
PATLiSci F. Huber et al, Nano Lett. 16, 5373-5377
PATLiSci II Probe Array Technology for
Life Science
(Rapid Sensing of Cancer)
AFM force spectroscopy
Cancer "aggressiveness"
peak

Indentation-type
AFM

cells
target
Plodinec et al., Nature Nanotechnology adsorption
(sensing)
(2012)
Complementary biomarker tests
PATLiSci 2
for decision on therapy
To support force spectroscopy findings cantilev
are also operated in sensing mode to detect
biomarkers for cancer.

Specific binding of molecules to probes on the

cantilevers leads to bending due to surface
stress change

PATLiSci
 Co-culture of cancer cells and
fibroblasts on native BM
Cancer cells BM Fibroblasts ECM

Actin/DNA laminin Actin/DNA Collagen I

cancer cells

30m

BM

fibroblasts
M. Plodinec and R. Lim: Mammary Stem Cells: Methods and Protocols, Springer