HOPE AND INNOVATIVE CANCER DIAGNOSTICS BY RAMAN
SPECTROSCOPY AND RAMAN IMAGING
Fulbright Poland 55th Anniversary Conference
15 May, 2014 Panel 2: Innovation and Technology: Key Drivers for Industrial and Economic Development
Halina Abramczyk
Lodz University of Technology, Laboratory of
Laser Molecular Spectroscopy, Lodz, Poland
• http://mitr.p.lodz.pl/raman
Lodz University of Technology, Faculty of Chemistry , Laboratory of Laser Molecular Spectroscopy, Lodz, Poland.
Lodz
Acknowledgements
• Beata Brozek-Pluska
• Jakub Surmacki
• Jacek Musiał
2• Joanna Jabłońska-Gajewicz
• Radzislaw Kordek
2• Eric Freysz
3• Katherine Lau
4• Agnieszka Sozańska
5• Krystyna Fabianowska-Majewska
6• 1Technical University of Lodz, Institute of Applied Radiation Chemistry, Laboratory of Laser Molecular Spectroscopy, Lodz, Poland.
• 2Medical University of Lodz, Department of Pathology, Chair of Oncology, Paderewskiego 4, 93-509 Lodz, Poland.
• 3 Laboratoire Ondes et Matière d'Aquitaine (LOMA), UMR 5798 Université Bordeaux 1, France
• 4 Spectroscopy Product Division Renishaw plc, Old Town, Wotton-under-Edge, GL12 7DW UK
• 5 Spectroscopy Product Division Renishaw Sp z o.o., Szyszkowa 34, 02-85 Warsaw, Poland
• 6 Medical University of Lodz, Department of Biomedical Chemistry 93-509 Lodz, Poland.
H. Abramczyk, B. Brozek – Pluska
• Raman Imaging in Biochemical and
Biomedical Applications. Diagnosis and
Treatment of Breast Cancer, Chemical Review, 2013, Impact factor 41. 3
The field of cancer diagnostics has become so huge that it is impossible to touch the whole field in a single
lecture. Therefore, I have selected only a few topics , giving preference only to those which were directly related
to our personal contribution: The views expressed in this lecture are highly personal, in the sense that they are
based either on my own laboratory work recently , or on the work I am familiar with
1. H. Abramczyk, B. Brozek – Pluska, Raman Imaging in Biochemical and
Biomedical Applications. Diagnosis and Treatment of Breast Cancer. Chemical Reviews, 2013, DOI: 10.1021/cr300147r, IF:41,3.
2. J. Surmacki, P. Wroński, M., Szadkowska-Nicze, H. Abramczyk, Raman
spectroscopy of visible-light photocatalyst - Nitrogen-doped titanium dioxide generated by irradiation with electron beam, Chemical Physics Letters,
566(2013), 54-59, IF: 2,145.
3. H. Abramczyk, B. Brozek-Pluska, M. Tondusson, E. Freysz, Ultrafast Dynamics of Metal Complexes of Tetrasulfonated Phthalocyanines at Biological Interfaces:
Comparison between Photochemistry in Solutions, Films, and Noncancerous and Cancerous Human Breast Tissues. J. Phys. Chem C, 117 (10), 2013, 4999–
5013, IF:4,814.
4. J. Surmacki, J. Musiał, R. Kordek, H. Abramczyk, Raman imaging at biological interfaces: applications in breast cancer diagnosis. Mol. Cancer, 12, 2013, 48, doi:10.1186/1476-4598-12-48, IF:5,99.
The field of cancer diagnostics has become so huge that it is impossible to touch the whole field in a
single lecture. Therefore, I have selected only a few topics , giving preference only to those which were
directly related to our personal contribution: The views expressed in this lecture are highly personal, in
the sense that they are based either on my own laboratory work recently , or on the work I am familiar with
•
• 5. B. Brozek-Pluska, J. Musial , R.Kordek , E. Bailo , T. Dieing, H. Abramczyk, Raman spectroscopy and imaging: applications in human breast cancer diagnosis. Analyst, 2012,137, 3773.
•
• 6. B. Brozek-Pluska, A. Jarota; J Jablonska-Gajewicz, R. Kordek, W. Czajkowski, H.Abramczyk, Distribution of phthalocyanines and Raman reporters in human cancerous and noncancerous breast tissue as studied by Raman imaging. Technol. Cancer Res. Treat. 2012, 11, 317.
•
• 7. A. Jarota, M. Tondusson, G. Galle, E. Freysz, H. Abramczyk, Ultrafast Dynamics of Metal Complexes of Tetrasulphonated Phthalocyanines. J Phys Chem A. 2012, 116(16), 4000.
• 8. . H.Abramczyk, Mechanisms of energy dissipation and ultrafast primary events in photostable systems: H- bond, excess electron, biological photoreceptors. Vibrational Spectroscopy, 2012 , 58, 1.
•
• 9. H. Abramczyk, B. Brozek-Pluska, J. Surmacki, J. Jablonska-Gajewicz, R. Kordek, Raman ‘optical biopsy’ of human breast cancer. Progress in Biophysics and Molecular Biology, 2012, 108 (1-2) 74
•
• 10. H. Abramczyk, B. Brozek-Pluska, J. Surmacki, J. Jablonska-Gajewicz, R. Kordek, Hydrogen bonds of interfacial water in human breast cancer tissue compared to lipid and DNA interfaces. Journal of Biophysical Chemistry, 2011, 2, 158-169.
• 11. B. Brozek-Pluska, J. Jablonska-Gajewicz, R. Kordek, H. Abramczyk Phase transitions in oleic acid and in human breast tissue as studied by Raman spectroscopy and Raman imaging. J. Med. Chem. 2011, 54, 3386- 3392
• 12 . H. Abramczyk, B. Brozek-Pluska, J. Surmacki, J. Jablonska, R. Kordek The label-free Raman imaging of human breast cancer. J. Mol. Liq. 2011, Vol. 164, 123-13.
• The field of cancer diagnostics has become so huge that it is impossible to touch the whole field in a
single lecture. Therefore, I have selected only a few topics , giving preference only to those which were
directly related to our personal contribution: The views expressed in this lecture are highly personal, in
the sense that they are based either on my own laboratory work recently , or on the work I am familiar
with
Goal
• We will demonstrate that IR and Raman imaging combined
with ultrafast, femtosecond spectroscopy give new hope for
cancer diagnosis. This combination offers unsurpassed
spatio-temporal resolution, sensitivity and multiplexing
capabilities. Researchers have already begun to translate
Raman imaging into a novel clinical diagnostic tool using
various endoscopic strategies.
Biomedical applications
• High spatial resolution (far below the diffraction limit, TERS) RAMAN IMAGING
• High temporal resolution (FEMTOSECOND PUMP-PROBE SPECTROSCOPY)
• Strong signal enhancement enabling monitoring the genetic and immunological responses in biological systems (SERS COMBINED WITH NANOPARTICLES)
• Specificity of interactions (BIOCONJUGATES)
Biomedical applications
• High spacial resolution (far below the diffraction
limit, TERS) RAMAN IMAGING
CONFOCAL RAMAN MICROSCOPY
R>>l
wide-field microscopy
Scanning near-field microscopy (SNOM)
Near-field imaging occurs when a sub-micron optical probe is positioned at a very short distance from the sample and light is transmitted through a small aperture at the tip of this probe.
The near-field is defined as the region above a surface with dimensions less than a single wavelength of the light incident on the surface. Within the near-field region light is not diffraction limited and nanometer spatial resolution is possible. This phenomenon enables non-diffraction limited imaging of a sample that is simply not possible with conventional optical imaging techniques.
d<<l
The next step to material analysis on a smaller scale has been the combination of Raman spectroscopic analysis with near field optics and an Atomic force microscope (AFM). Such systems allow tip enhanced Raman scattering to be explored, making true NanoRaman achievable, with spatial resolution <100nm.
spatial resolution <100nm.
Tip-enhanced Raman spectroscopy (TERS) (cantilever based SNOM)
In a typical TERS experiment a Au- or Ag-coated AFM tip is used as a nanostructure to produce Raman signal
enhancement on a sample surface once the excitation laser is focused on the apex of the tip with the tip brought into
close proximity with the surface. The tip radius, which defines the lateral resolution of an AFM measurement, is
typically in the range of 10-20 nm. In the TERS experiment the lateral resolution depends on the size of the hot-spot
therefore one can expect resolution in the range of 20-50 nm for Raman spectroscopy and imaging measurements. The
TERS tip-apex must be illuminated with the excitation laser from either above, below, or the side.
Biomedical applications
• High spatial resolution (far below the diffraction limit, TERS) RAMAN IMAGING
• High temporal resolution (FEMTOSECOND PUMP-
PROBE SPECTROSCOPY)
Ultrafast nonlinear spectroscopy
pump-probe femtosecond transient absorption
Recently developed techniques of ultrafast nonlinear vibrational spectroscopy allow a
much more effective attack on this problem.
Biomedical applications
• High spacial resolution (far below the diffraction limit, TERS) RAMAN IMAGING
• High temporal resolution (FEMTOSECOND PUMP-PROBE SPECTROSCOPY)
• Strong signal enhancement enabling monitoring the genetic and immunological responses in biological systems (SERS COMBINED WITH NANOPARTICLES)
• Specificity of interactions (BIOCONJUGATES)
SERS methods
• Despite the high specificity (vibrational fingerprint) , traditional Raman spectroscopy was considered limited because of the very poor efficiency of the inelastic scattering processes and thus the relatively weak signal.
• The SERS technique is based on the fact that if a molecule is brought into close proximity with a metal (Au, Ag) nanostructure or nanoparticle that results in significant increase in the intensity of the Raman spectra.
plasmon
the enhancement mechanism for SERS comes from intense localized fields arising from surface plasmon resonance in metallic (e.g. Au, Ag, Cu)
nanostructures with sizes of the order of tens of nanometers, a diameter much smaller than the wavelength of the excitation light.
SERS combined with nanoparticles
300 400 500 600 700 800
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0
absorbance
wavelength (nm) P8
P1
P7 P6 P5 P4
P3
P2
Next step is to enhance the SERS signal with help of nanotechnology.
Nanoparticles produced in our lab
Biomedical applications
• High spacial resolution (far below the diffraction limit, TERS) RAMAN IMAGING
• High temporal resolution (FEMTOSECOND PUMP-PROBE SPECTROSCOPY)
• Strong signal enhancement enabling monitoring the genetic and immunological responses in biological systems (SERS COMBINED WITH NANOPARTICLES)
• Specificity of interactions (BIOCONJUGATES)
How to reach selective interaction?
binding specificity
Answer: antibody-antigen interactions
Fortunately, nature provides a solution.
The antibody has an unique ability to bind with high specificity to the antigen. Each antibody binds to a specific antigen; an interaction is similar to a lock and key.
antibody antigen
Each antibody binds to a specific antigen;
interaction is similar to a lock and key.
antigen antibody
An antigen is a protein molecule that
triggers antibody generation
Biocojugates
When conjugated with biomolecular targeting ligands such as monoclonal antibodies, peptides or small molecules, these nanoparticles can be used to target malignant tumors with high specificity and affinity.
There are a number of formats used to provide Raman signal.
Currently, a promising way to catch cancer lesions early is to use Raman reporters coupled with nanoparticles and
antibodies that recognise and
bind to cancer cells.
Antibody-antigen interaction protein-protein interaction
Mutations affecting EGFR expression or activity could result in cancer
Antigen- EGFR-HER2 EGFR-epidermal
growth factor receptor
Antibody- C225 HAuNs -
hollow gold nanoparticles
C225- human-mouse chimerized
monoclonal antibody directed against the epidermal growth factor receptor (EGFR
.
In a few papers HGNs have been used as sensitive imaging agents for detection in cancer cells. As an optical imaging
target, MCF7 cancer cells (MCF7 ) expressing human epidermal growth factor (HER2) markers on their surface membrane were used. HER2 is a clinically significant molecular marker of breast cancer (Ueda et al., 2004).
SERS detection
By immobilising a coloured molecule (Raman reporter) onto a suitably
roughened metal surface of the nanoparticle, extremely
strong SERRS signals can be obtained with an overall enhancement factor of
up to 10 14 enabling monitoring the genetic and immunological responses
in biological systems.
What?
Human normal and cancerous human breast tissue, neck and head tissues
Cancerous and normal breast cell cultures MCF7 and MCF10A
Drugs and Photosensitizers in cancer
therapy
carcinoma ductale infiltrans carcinoma lobulareinfiltrans
carcinoma ductale infiltrans+carcinoma lobulareinfiltrans
carcinoma multifocale infiltrans papillare intracysticum nonivasium carcinoma mucinosum sinistri carcinoma intraductale fibroadenoma
carcinoma metaplasticum dysplasia benigna
hyperplasia ductalo-lobularis adenosis
Patients Statistics
230 patients
The pathology reports indicated that 70% of the cancer samples were ductal carcinomas;
the remaining samples were lobular or
untyped mammary carcinomas, metastases
were found in 60% of patients
Breast morphology (P94)
One can see that the cross section through the normal organization of ducts and lobules in the human breast
demonstrates luminal epithelial cells aligned in a polar manner so their apical side faces and surrounds the
lumen. These cells are surrounded the basement membrane. Fibroblasts align the basement membrane and
this entire structure is surrounded by the stroma, which is predominantly, but not exclusively, composed of
type I collagen and adipose tissue. During ductal carcinoma in situ (DCIS), the normal polar organization of
the luminal epithelial cells is lost, as these cells proliferate. The cross-section shows the epithelial cells
completely filling the lumen. In invasive, or infiltrating, carcinoma, the epithelial cells migrate and invade
through the basement membrane and into the surrounding stroma.
In order to evaluate the diagnostic value of the Raman biomarkers for monitoring cancer pathology we have applied the principal component analysis. Here we can see a score PCA plot. Without going into the PCA details it is easy to see that the samples in this figure belong to one of two groups. Namely, the samples in the left and the right areas separated along PC1. In the left area there are almost exclusively the tumor tissues. In the right area there are almost exclusively the normal tissues .
Breast cancer biodiagnostics.
Raman biomarkers
Breast cancer progression
2
45
45 23
IDC_G1
IDC_G2
IDC_G3
IDC_GX
Patients statistics for ductal carcinoma (IDC)
The modified Bloom–Richardson–Elston grading system (called also Nottingham Prognostic Index)
We have also studied if Raman spectroscopy is capable of displaying the
difference in the degree of agressiveness.
Microscopy and Raman images of cancerous human breast tissue
Tkanka Pacjent 105 miejsce 2
Video Image_004 40x_Nikon_532 (Top)_Miejsce2Scan_004_3200 0.6 sek 10 mWSpec.Data 2_F: Sum -> Sum [2900 a.u. -> 3010 a.u.]
When cells become cancerous they signal to the
surrounding tissues to increase production of a protein called collagen, which forms a ‘scaffold’ around the tumour that supports the growth and development of the cells.
Cancer Cell, , 2 2011
Professor Michael Olson, who led the study, (Cancer Cell 20111 , said: “Collagen is a protein most people probably associate with cosmetic surgery to create fuller, firmer lips.
But it’s a major component of our connective tissues and also important in tumour growth.
Microscopy and Raman images of normal human breast tissue
stitching image 001 P102
The non-cancerous breast tissue is dominated by adipose tissue .
Area Scan_003_Spec.Data 2 Sum [2800 -2900]
Microscopy Raman images
Fatty acids and triglycerides proteins
Large Area Scan_003_Spec.Data 2 Sum [2900-3000 a.u.]
The breast tissue from the margin of the tumor mass: H&E- stained histological image (a), microscopy image (1000 x 1000
um, 2000 x 2000 pixels, spatial resolution 0.5 x 0.5 um)
composed of 121 single video images (b), Raman image
The breast tissue from the tumor mass: H&E-stained histological image (a), microscopy image (2000 x 2000 um, spatial resolution 0.66 x 0.66 um), Raman image © 80x80 um,
1.3x1.3 um
Patient P104, the breast tissue from the tumor mass single spectra corresponding to different areas of Raman image (colors of the spectra corresponding to colors of the Raman
image)
Fig. 2 Patient P104, the breast tissue from the tumor mass: H&E-stained histological image (a), microscopy image (2000 x 2000 mm, 300 x 300 pixels, spatial resolution 0.66 x 0.66 mm) composed of 400 single video images (b), Raman image (80 x 80 mm, 60 x 60 points per line/lines per image, resolution 1.3 x 1.3 mm) (c), microscopy image (2000 x 2000 mm, 300 x 300 pixels, spatial resolution 0.66 x 0.66 mm), images for
the filters for spectral regions: 1490 – 1580 cm-1, 2850 – 2950 cm-1, and 2900 – 3010 cm-1 (d), average spectra used for the basis analysis method and single spectra corresponding to different areas of Raman image (colors of the spectra corresponding to colors of the Raman image presented in part (c)) (e), microscopy image (2000 x 2000 mm, 300 x 300 pixels, spatial resolution 0.66 x 0.66 mm) and single spectra of
various sites of the sample, colors of the spectra correspond to the colors of the crosses in the microscopy image; integration times 10 sec, 2 accumulations (f).
\
Raman images of the noncancerous (a) and cancerous (b) breast tissue, for the carotenoids (1518 cm
-1), monounsaturated fatty acids (2854 cm
-1), proteins (2930 cm
-1) and autofluorescence (1800
cm
-1) filters
A detailed inspection into figure demonstrates that the noncancerous areas in the safety margin contain a
markedly higher concentration of carotenoids than the cancerous tissue from the tumor mass. Moreover, they are
accumulated in the adipose tissue as the images for the both filters are almost identical.
Raman optical biopsy of human breast cancer tissue
The completeness of the surgical resection is a key factor in the progress of patients with breast tumors. The Raman image indicates that for the patient PX the margin is positive (green colour), which means that not all cancer cells have been removed in the surgery.
Patients with a positive margin often require more surgery to make sure that all the cancer is removed. The advantage of the
‘Raman biopsy’ is that it provides direct biochemical information (vibrational fingerprint) in real time, it is not prone to subjective interpretations, and it monitors biological tissue without any external agents, in contrast to histopathological assessment.
