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 Musiał2

• Joanna Jabłońska-Gajewicz

• Radzislaw Kordek2

• Eric Freysz3

• Katherine Lau4

• Agnieszka Sozańska5 • Krystyna Fabianowska-Majewska6

• 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.

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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

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1,0

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1,4

1,6

1,8

2,0

abso

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ce

wavelength (nm)

P8

P1

P7 P6 P5P4

P3P2

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 1014 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+carcinomalobulareinfiltranscarcinoma 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.

safety margin

tumor mass

Tumor mass

Negative safety margin No cancer cells are found by histology

adjacent sections of 5 mm

Tumor mass

Positive safety margin cancer cells extend out of the tumor mass, cancer cells are found by histology

Distribution of chemotherapy drugs in

MCF7 cell cultures (cancerous breast

cells) by Raman imaging.

Clofarabine is a purine nucleoside antimetabolite marketed in the U.S. and Canada as Clolar. In Europe and Australia the product is marketed under the name Evoltra. It is FDA-approved for treating relapsed or refractory acute lymphoblastic leukaemia (ALL) in children after at least two other types of treatment have failed. It is not known if it extends life expectancy. Some investigations of effectiveness in cases of acute myeloid leukaemia (AML) and juvenile myelomonocytic leukaemia (JMML) have been carried out. Ongoing trials are assessing its efficacy, if any, for managing other cancers.

CLOFARABINE

MCF7 cell cultures MORPHOLOGY, BIOCHEMISTRY AND

PHOTOCHEMISTRY

Fatty acids 2830-2900

Proteins 2900-3010

Photosensitizer 3600-4600 cm-1

proteins 1600 - 1700

Raman imaging of a single cell of MCF7

2830 -2950 cm-1

2900 -3010 cm-1

Fatty acids Proteins

CONCLUSIONS The results presented here demonstrate that Raman spectra and images are sensitive

indicators of distribution of different compounds in a normal and cancerous breast tissue, single cells of MCF7

• MORPHOLOGY, BIOCHEMISTRY AND PHOTOCHEMISTRY • OF HUMAN TISSUE, AND SINGLE CELLS

• Gaining a Practical Understanding of Roads to Cell Death:

Apoptosis, Necrosis, and Autophagy

• RAMAN BIOPSY AND VISUALIZATION TUMOR MARGINS

• RAPID BIODIAGNOSTICS OF EARLY STAGES OF CANCER

• THE METHOD IS READY TO BE TRANSLATED INTO A NOVEL CLINICAL DIAGNOSTIC TOOL

• 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.

• Current imaging methods are often limited by inadequate sensitivity, specificity, spatial and spectral resolutions. MRI- limited spatial resolution FLUORESCENCE- limited spectral resolution