- Email: [email protected]
Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging
SAA-15862; No of Pages 8 Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging Monika Kopeć, Halina Abramczyk ⁎ Lodz University of Technology, Faculty of Chemistry, Institute of Applied Radiation Chemistry, Laboratory of Laser Molecular Spectroscopy, Wroblewskiego 15, 93-590 Lodz, Poland.
a r t i c l e
i n f o
Article history: Received 20 December 2017 Received in revised form 19 February 2018 Accepted 21 February 2018 Available online xxxx Keywords: Angiogenesis Cancer Raman spectroscopy Raman imaging AFM
a b s t r a c t Combined micro-Raman imaging and AFM imaging are efficient methods for analyzing human tissue due to their high spatial and spectral resolution as well as sensitivity to subtle chemical, structural and topographical changes. The aim of this study was to determine biochemical composition and mechanical topography around blood vessels in the tumor mass of human breast tissue. Significant alterations of the chemical composition and structural architecture around the blood vessel were found compared to the normal breast tissue. A pronounced increase of collagen-fibroblast-glycocalyx network, as well as enhanced lactic acid, and glycogen activity in patients affected by breast cancer were reported. © 2018 Elsevier B.V. All rights reserved.
1. Introduction The development of a variety of cancers is linked to changes in the biochemical properties of living cells. Oncogenically transformed cells are expected to have differences in all cell and tissue layers, but it still remains unclear whether biochemical alterations are a cause or a consequence of cancer development. The study of biochemistry of cancer cells by Raman and AFM imaging is presumably one of the most promising directions of cancer research, because the acquired knowledge will help design new treatments for cancer. It has been reported that the processes occurring in tumor microenvironment composed of noncellular (i.e. vascular and interstitial) and cellular compartments are as important as the processes in epithelial cells where most cancers including the ductal breast cancer develop [1]. These processes must be coupled because the epithelial cells are avascular, and the blood supply from the microenvironment to the tumor mass is one of the most important factors of cancer development. In this paper we will concentrate on biochemical signatures of tumor mass around vascular compartments and interstitial environment. There is a great number of abnormalities in the tumor vessels comparing with normal ones. Among them the most important are: increased vessel tortuosity, deficient pericytes, increased numbers of proliferating endothelial cells and abnormalities in the basement membrane [2,3]. Biochemical changes (upregulation of growth factors, bradykinin, prostaglandins, nitric oxide) are responsible for hyperpermeability what is revealed in high interstitial pressure and ⁎ Corresponding author. E-mail address: [email protected] (H. Abramczyk).
absence of functioning lymphatic network in tumor interstitium. This good vascularization of tumor helps with delivery of drugs, however because of high heterogeneity this effect is not effective in poorly vascularized regions [1,4,5]. The development of new blood vessels is a crucial step in breast cancer growth, progression and metastasis, because like healthy cells, cancer cells cannot live without oxygen and nutrients. The diversity of responsible angiogenic pathways that encourage new blood vessels to grow into the tumor in breast cancer for different tumors has been studied for many years [6–15]. The most important angiogenic factor is VEGF (Vascular Endothelial Growth Factor) [16]. When VEGF is overexpressed, cancer cells encourage the growth of blood vessels to feed a tumor by producing the hormone-like protein, vascular endothelial growth factor. This guarantee a good blood supplies which are needed for growth and in consequences for metastases [17]. Large number of researches suggest that angiogenesis (new blood vessel formation process) promote transformation of mammary hyperplasia to malignancy [10]. Clinical data show that microvessel density (MVD) is highest in aggressive breast tumors, and is associated with increased VEGF expression [13]. It seems appropriate to design drugs, which interrupt and inhibit angiogenesis in breast. The most commonly used drug is bevacizumab, which is a humanized monoclonal antibody directed against the VEGF-A ligand, but there is not yet definitive evidence for the efficacy of agents that specifically target angiogenesis [6]. Understanding mechanisms of angiogenesis as well as biological predictive markers is crucial in development of antiangiogenic therapies in clinical practice. One route to optimize the efficacy of these targeted agents is to better understand biology and biochemistry
https://doi.org/10.1016/j.saa.2018.02.058 1386-1425/© 2018 Elsevier B.V. All rights reserved.
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
2
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
around the vessels in the tumor environment. Highly advisable is therefore seeking new methods breaking the previous limitations and with potential clinical applications in order to satisfy the increasing demand of molecular biology. Reports of world literature in recent years clearly indicate that the particular role in the development of innovative techniques are played by multimodal imaging technologies, among which the dominant role belongs to Raman-AFM-fluorescence imaging, which allow simultaneous monitoring of morphological, biochemical properties with very high spectral and spatial resolution [18]. The Raman imaging is capable of simultaneously recording vibrational spectra from multiple regions and thereby map out the spatial distribution of proteins, lipids, nucleic acids, and metabolites in surrounding of the vessel supplying blood to tumors in contrast to the classical methods LC/ MS, NMR, HPLC, based on the analysis of samples in the mass subjected to homogenization that prevents spatial characteristics of the systems investigated [19–24]. We will show that Raman spectroscopy and imaging are capable of monitoring biochemical composition of tumor vascularity, which is markedly heterogeneous with densely vascularized areas supplying oxygen and nutrients to rapidly growing parts of the tumor. Raman imaging can monitor also areas where there is a decreasing amount of oxygen available to tumor cells which are further away from blood vessels. This information will help understand how new blood vessels are synthesized by tumors in a process known as angiogenesis. The aim of this study is to use micro-Raman and AFM imaging to analyze biochemical composition around blood vessels in cancerous and normal human breast tissue. The biochemical composition of human normal and cancerous breast tissues will be analysed by Raman and AFM imaging. 2. Experimental 2.1. Patients We have employed Raman spectroscopy, Raman imaging, AFM topography imaging to measure tissue biochemical composition around a vessel supplying blood to duct tumor in human breast tissue. These techniques have been applied for ex vivo fresh breast tissue samples without any fixation. The ex vivo samples were obtained during the resection surgery from the tumor mass (cancerous tissue) and the safety margin (normal tissue). All tissue samples were frozen and stored at −80 °C. Before the measurements the frozen samples were cryosectioned at −20 °C with a microtome (Microm HM 550, Sermed) into a few 6 μm-thick slices. Some of them were used for Raman and AFM analysis without typical for histology examination paraffin embedding procedure. The thin sections were placed onto calcium fluoride windows (CaF2, 25 × 1 mm) and examined by Raman and AFM imaging. After the Raman and AFM measurements these sections were stained and histologically examined. The samples were stained in hematoxylin for 3 min, rinsed in water and stained in eosin for 2 min. The adjacent tissue was analysed by histopathologists. All methods were performed in accordance with relevant guidelines and regulations. All tissue procedures were conducted under a protocol approved by the institutional Bioethical Committee at the Medical University of Lodz, Poland (RNN/323/17/KE/17/10/2017). Written informed consent was obtained from patients. 2.2. Chemicals All chemicals have been purchased from Sigma-Aldrich: palmitic acid (P0500; Sigma Aldrich), stearic acid (S4751; Sigma Aldrich), arachidic acid (A3631; Sigma Aldrich), oleic acid (O1008; Sigma Aldrich), linoleic acid (L1376; Sigma Aldrich), γ-linolenic acid (L2378; Sigma Aldrich), arachidonic acid (10,931; Sigma Aldrich), docosapentaenoic acid (D-120; Sigma Aldrich), eicosapentaenoic acid (E2011; Sigma Aldrich), α-linolenic acid (L-039; Sigma Aldrich),
eicosatetraenoic acid (H7768; Sigma Aldrich), tetracosahexaenoic acid (153,745; Sigma Aldrich), docosahexaenoic acid (D2534; Sigma Aldrich). 2.3. Raman Imaging Raman spectra and images were obtained with an alpha 300 RSA+ (WITec, Ulm, Germany), and preprocessed with WITec Control/Project Plus 2.1. Detailed description of equipment and methodology on data pre-processing and multivariate data analysis used in the paper is available elsewhere [19,21,25–27]. The 2D array images of tens of thousands of individual Raman spectra were evaluated by the basis analysis method (BAM). In BAM method, each measured spectrum of the 2D spectral array is compared to basis spectra using a least squares fit. Such basis spectra are created as the average spectra from different areas in the sample. The weight factor at each point is represented as a 2D image of the corresponding color and mixed coloring component. The color code of Raman maps were based on the integrated Raman intensities in specific regions (sum option in the filter manager in the WITec project 2.1). Using a lookup table, bright colors indicate the highest intensities, whereas dark colors indicate the lowest intensities of the chosen region. 2.4. AFM Measurements To obtain the topography of the samples AFM measurements were performed on an alpha 300 RSA+ (WITec, Ulm, Germany) based on an Olympus microscope. The AFM module was equipped with a 25 mm x- and y-range linearized piezoelectric scanner and 980 nm laser. The AFM was operating in the constant force mode, in which the feedback loop controls the z-height of the piezo table to keep the cantilever deflection constant. To generate AFM images we used the pyramidal cantilevers, with the spring constant of 0.2 N/m. 3. Results and Discussion 3.1. Raman Imaging Raman images allow looking inside the biochemical composition of cancerous cells around the lumen of the vessels supplying blood to the tumor mass and the extracellular matrix surrounding the tumor. To understand information that is provided from Raman images and vibrational spectra around the blood vessels in normal and cancerous breast tissues, we need to associate these features with the vessel morphology. Briefly, the walls around the lumen of artery are lined by an exceedingly thin single sheet of endothelial cells, the endothelium, separated from the surrounding outer layers by a basal lamina followed by many layers of smooth muscle cells and a thick, tough wall of connective tissue. Endothelial lining is present regardless amounts of connective tissue and smooth muscle in the vessel wall. Fig. 1 shows a cross section through a small artery supplying blood to a tumor mass in human breast tissue (G3, ductal cancer, P149) obtained by Raman imaging, compared with the H&E-stained histological image, microscopy image, AFM topography image and the characteristic vibrational Raman spectra for different areas of the cancerous tissue. One can see that there is an almost perfect match between the morphological features obtained from the histological, microscopy, AFM and Raman images. Raman imaging is a powerful technique which has many advantages over other imaging techniques, because it offers not only morphological image, but also a very detailed biochemical characterization by probing individual chemical bond vibrations. As a result, Raman spectra and images are information rich, and contain data related to the specific chemical structure and biocomposition of the biological material being analysed. The Raman spectra presented in Fig. 1g show the biochemical composition of the various substructures around a small artery supplying blood to a tumor mass in human breast tissue. The inside wall of the
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
3
Fig. 1. Histological image, Raman images, AFM image and Raman spectra of tumor mass in human breast tissue in the high frequency region. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
artery covered by a thin single sheet of endothelial cells, and the basal lamina cannot be seen at the relatively low resolution chosen for the large area. However, many layers of cells and connective tissue around the artery are clearly visible. One can see from Fig. 1c that the artery is surrounded by an extensive network full of fibers that vary in diameter from 2 to 10 μm that can be easily identified without any staining from the Raman spectra (Fig. 1g). The network constitutes mainly of collagen (red color in Fig. 1c and f) and lipids (fibroblasts –blue color, in Fig. 1c and f). Cancer cells invade in the collagen and fibroblast network [25]. Each collagen fiber is made-up of a variable number of smaller collagen fibrils. These fibrils are composed predominantly of type I collagen, because a characteristic feature of these fibrils is that they exhibit axial periodicity due to the way in which type I collagen assembles to form collagen fibrils, which are synthesized primarily by fibroblasts aligning themselves along collagen fibers.
H&E-stained histological image (a), stitching microscopy image (b), Raman images (360 μm × 660 μm) obtained from basis analysis (c), microscopy image (d), AFM topography image (e), Raman images for specific spectral filters (f), as well as the normalized (model: divided by norm) Raman spectra (g) in the high frequency region of the cancerous breast tissue (P149), integration time 0.3 s, resolution: 0.5 μm, laser excitation power: 10 mW. Objective 40×, 6 μm-thick slices on calcium fluoride window (CaF2, 25 × 1 mm). The line colors of the spectra correspond to the colors of the Raman maps. A clear branching pattern of collagen fibers of smaller diameter in Fig. 1c suggests contribution from reticular fibers consisting of type III collagen fibrils. These structures are usually distinguished by PAS (Periodic acid–Schiff) staining, because unlike collagen I fibers, reticular III type fibers are PAS-positive owing to their greater carbohydrate content. The advantage of the Raman spectroscopy is its capability of
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
4
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
identifying type III collagen fibrils containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans) without any PAS staining. The turquoise color in Fig. 1c, f and g represents an amorphous carbohydrate rich ground substance. More information on the ground substance can be obtained from the Raman fingerprint region. Fig. 2 shows Raman images and Raman spectra the cross section through a small artery supplying blood to a tumor mass in human breast tissue in the fingerprint region (the same as in Fig. 1). The fingerprint region provides additional information about the distribution of main biochemical components of the tumor mass around the artery – proteins (amide I and III), lipids (saturated CH2 bonds), glycogen (840 cm−1), glucose (1125 cm−1) and lactic acid (917 cm−1) [28] partially overlapping with O-P-O backbone of DNA (836 cm−1), collagen (855 cm−1), (874 cm−1) C\\C stretch of 4 hydroxy-proline, (920 cm−1) C\\C stretch
of proline ring, (937 cm−1). The Raman vibrations of these components provide information about distribution of carbohydrate content in intracellular matrix and about altered metabolism in cancer cells. These carbohydrate rich ground substance structures are presented in Fig. 2c, f and g by the turquoise color. The enhanced Raman intensity of carbohydrates is clearly visible in the walls around the lumen of the artery and along the collagen and fibroblast fibers. The carbohydrate content in intracellular matrix originating from an amorphous ground substance consists of glycosaminoglycans synthesized by fibroblasts in loose connective tissue. The vibrations of glycosaminoglycans are partially overlapped with the vibrations of glycogen and lactic acid. Monitoring of the distribution of glycogen and lactic acid by label-free Raman imaging provides very important information about metabolism of cancerous cells. The altered metabolism is one of the important features of cancer and is usually described by Otto Warburg effect with enhanced
Fig. 2. Histological image, Raman images, AFM image and Raman spectra of tumor mass in human breast tissue in the fingerprint spectral region. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
anaerobic glycolysis [29]. The elevated glucose catabolism produces an excess of the glycolytic end-product, pyruvate. Most of the pyruvate is converted to lactate, whereas some of it is converted to acetyl-CoA, which, in turn, is used in de novo fatty-acid synthesis [30]. H&E-stained histological image (a), stitching microscopy image (b), Raman images (360 μm × 660 μm) obtained from basis analysis (c), microscopy image (d), AFM topography image (e), Raman images for specific spectral filters (f) as well as the normalized (model: divided by norm) Raman spectra (g) in the low frequency region of the cancerous breast tissue (P149), integration time 0.5 s, resolution: 0.5 μm, laser excitation power: 10 mW. Objective 40×, 6 μm-thick slices on calcium fluoride window (CaF2, 25 × 1 mm). The line colors of the spectra correspond to the colors of the Raman maps. Comparison between the Raman images obtained from the high frequency (Fig. 1c) and the fingerprint (Fig. 2c) regions demonstrates that the images are coherent and almost identical for the components that have vibrations in both regions (collagen in fibers, lipids in fibroblasts and glycans). Additional information about distribution of carbohydrate content in intracellular matrix originating from an amorphous ground substance, glycogen and lactic acid in cells around the artery supplying
5
blood to the breast tumor is provided by the Raman image for the 800–920 cm−1 filter (turquoise color in Fig. 2c and f). The results presented so far demonstrate the central importance of the network of collagen and fibroblast matrix formed around the blood artery. This matrix seems to be required to protect the balance between rigidity of the environment and deformability of cancer cells to forma stable vascular structure for supplying nutrients and oxygen to the tumor mass. Collagen matrix plays a role of a scaffold that protect the deformable cancer cells. The formation of first primitive vascular plexus is possible through the migration of endothelial cells and angioblasts on a matrix formed from collagen and fibroblasts with the guidance of Vascular Endothelial Growth Factor (VEGF) leading to vascularization of a tumor mass [31]. It is very important to compare the morphology and biochemistry around the blood artery supplying nutrients and oxygen to the tumor mass with the normal structure of the breast tissue. Fig. 3 shows the results for a normal breast tissue. H&E-stained histological image (a), microscopy image (b), Raman images (60 μm × 60 μm) obtained from basis analysis (c), microscopy image (d), AFM topography image (e) Raman images for specific
Fig. 3. Histological image, Raman images, AFM image and Raman spectra of a normal breast tissue from a negative margin of a tumor mass from the same patient (G3, ductal cancer P149) as presented in Figs. 1 and 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
6
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
spectral filters (f) and normalized (model: divided by norm) Raman spectra (g) from the high frequency region for different areas of the normal breast tissue. Integration time 0.3 s, resolution: 1 μm, laser excitation power: 10 mW. Objective 40×, 6 μm-thick slices on calcium fluoride window (CaF2, 25 × 1 mm). The line colors of the spectra correspond to the colors of the Raman maps. Fig. 3 shows Raman images of the normal breast tissue from a negative margin of the tumor mass from the same patient (G3, ductal cancer, P149) as presented in Figs. 1 and 2 compared with the H&E-stained histological image, microscopy image, AFM topography image and the characteristic vibrational spectra for different areas of the normal breast tissue. One can see that in contrast to the cancerous tissue presented in Figs. 1 and 2 collagen is not the most abundant component of the normal breast tissue. In contrast to the tumor mass presented in Figs. 1 and 2 the collagen-fibroblast network is much less pronounced feature in the normal breast tissue from the negative safety margin (lack of cancer cells). The Raman image is dominated by adipose fat (dark blue color) and the normal cells (turquoise color in Fig. 3c) with rich protein structure at 2911 cm−1 which represents CH2 vibrations of nonmethylated and non-acetylated proteins in contrast to the cancerous structures in Fig. 1c that contain dominant component of collagen and methylated/acetylated proteins [25]. Fig. 4 shows Raman images of a normal breast tissue from a negative margin of a tumor mass from the same patient (G3, ductal cancer, P149)
as presented in Figs. 1 and 2 compared with the H&E-stained histological image, microscopy image, AFM topography image and the characteristic vibrational spectra for different areas of the normal breast tissue in the fingerprint region. Comparison of the vector normalized Raman intensities of glycogen (840 cm−1), glucose (1125 cm−1) and lactic acid (917 cm−1) for normal breast tissue in Fig. 4g with the corresponding intensities for tumor tissue in Fig. 2g demonstrates that glycolytic path of metabolism is significantly less efficient (about twice) in the normal tissue. H&E-stained histological image (a), microscopy image (b), Raman images (60 μm × 60 μm) obtained from basis analysis (c), microscopy image (d), AFM topography image (e) Raman images for specific spectral filters (f) and normalized (model: divided by norm) Raman spectra (g) from the fingerprint frequency region for different areas of the normal breast integration time 0.5 s, resolution: 1 μm, laser excitation power: 10 mW. Objective 40×, 6 μm-thick slices on calcium fluoride window (CaF2, 25 × 1 mm). The line colors of the spectra correspond to the colors of the Raman maps. 3.2. Iodine Value Iv Third important feature that reveals from the fingerprint regions in Fig. 2g and Fig. 4g is information about fatty acid composition in tumor environments. To address these important questions on lipid
Fig. 4. Histological image, Raman images, AFM image and Raman spectra of a normal breast tissue from a negative margin of a tumor mass from the same patient (G3, ductal cancer P149) as presented in Figs. 1 and 2.
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
phenotypic alterations we used the iodine value Iv to characterize tissues. The iodine value Iv is the mass of iodine in grams that is consumed by 100 g of a chemical substance. Knowing the molecular weight of each acids and the number of double bonds, we calculated the iodine value according to the formula (1):
Iv ¼
100xNd xMI2 Ma
ð1Þ
where: Nd number of double bonds. MI2 molecular mass of I2. Ma molecular mass of acid. The iodine value Iv is accepted as a good source of information on the amount of unsaturation in fatty acids, where the double bonds react with iodine compounds. The higher iodine value indicates that there is more C_C bonds in the fat [26,32,33]. This formula has been used for all fatty acids to create the calibration curve of the iodine value as a function of the Raman intensity ratio of selected Raman bands. We have applied the iodine value Iv plot vs the Raman intensity ratio 1268/1444 measured at 1268 cm−1 and 1444 cm−1 used as a source of information on the ratio of unsaturated/saturated lipids. Next we recorded the ratio 1268/1444 for the cancerous and noncancerous tissues and from the calibration curve the iodine values of the tissues were determined.
Fig. 5. The iodine value Iv plot vs the Raman intensity ratio 1268/1444 for PUFA and human breast normal (a) and cancer (b) tissues of studied samples. The equation of the curve: y = −34.94x2 + 255.18x − 21.263 R2 = 0,9775 green triangles indicate the iodine value for patient P149 palmitic acid (PA), stearic acid (SA), arachidic acid (ARA), oleic acid (OA), linoleic acid (LA), γ-linolenic acid (GLA), arachidonic acid (AA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosatetraenoic acid (ETA), tetracosahexaenoic acid (THA), docosahexaenoic acid (DHA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
7
Fig. 5 shows the iodine value plot vs the Raman intensity ratio 1268/ 1444 for polyunsaturated fatty acids (PUFA) and breast tissues of the studied samples. The iodine value Iv for lipids in normal and tumor breast tissues are 65 and 136, respectively. The iodine value for the normal tissue is close to the value for oleic acid (Iv = 87), while the abnormal tumor microenvironment shows the numbers between oleic acid (OA) and linoleic acid (LA) (Iv = 181). It indicates that tumor microenvironment in human breast cancer shows altered fatty acid composition in comparison with the normal tissue. 4. Conclusions Understanding mechanisms of angiogenesis as well as biological predictive markers are crucial in development of antiangiogenic therapies in clinical practice. The results presented in the paper demonstrate the central importance of the network of collagen, fibroblasts, and glycocalyx matrix formed around the blood artery supplying blood to tumor mass. The Raman spectra provide information on collagen network, particularly identification of type III collagen fibrils that is not available directly in other visualization techniques. The advantage of the Raman spectroscopy is its capability of identifying type III collagen fibrils containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans) without any PAS staining. The collagen- fibroblast-glycocalyx matrix seems to be required to protect the balance between rigidity of the environment and deformability of cancer cells to form a stable vascular structure for supplying nutrients and oxygen to the tumor mass. The collagen matrix plays a role of a scaffold that protect the deformable cancer cells. The glycocalyx serves as mechanotransducer, because the epithelium cancer cells are exposed to mechanical forces of the rigid collagen environment. However, the molecules responsible for the translation of biomechanical forces into biochemical signals (mechanotransduction) have not been identified yet. The paper on angiogenesis of cancer opened a new window for Raman exploration of processes occurring in non-cellular spaceglycocalyx–a coat on the external surface of plasma membranes of epithelial cells. Because of the functional importance of the epithelial glycocalyx, development of direct visualization techniques is crucial to establish its exact role. We have demonstrated that label-free Raman imaging can easily monitor location and distribution of the epithelial glycocalyx with high spatial and spectral resolution. We have demonstrated that the Raman imaging combined with AFM are capable of simultaneous identification of breast cancer specific biochemical and mechanical alterations in tumor mass around blood vessels in cancerous and normal human breast tissues. This combination of biochemical and topographic signatures has potential to provide predictive Raman cancer biomarkers and treatment targets during cancer development. High-resolution Raman and AFM maps show that matrix stiffening in tumor progression is due to alteration of biochemical composition of the tumor microenvironment composed of noncellular (ie, vascular and interstitial) and cellular compartments. We found significant alterations in chemical composition, and structural architecture around the blood vessel with a pronounced increase of collagen-fibroblast-glycocalyx network. We found that oncogenically transformed cells exhibit enhanced lactic acid, and glycogen activity of cancer tissue that can be easily monitored by Raman-based methods. Author Contributions HA conceived the idea, designed and directed the Raman and AFM experiments, analysed the Raman data, analysed force spectroscopy data wrote the manuscript and edited successive drafts of the paper. MK collected all clinical data, performed Raman imaging and AFM experiments, analysed Raman spectroscopy data, contributed to the discussion.
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
8
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
Competing Interests The authors declare no competing financial interests. Acknowledgements This work was supported by the National Science Center of Poland (grant UMO-2015/19/B/ST4/01878), Dz. St. 2017. References [1] A.S. Thakor, S.S. Gambhir, Nanooncology: the future of cancer diagnosis and therapy, CA Cancer J. Clin. 63 (2013) 395–418. [2] T.M. Allen, P.R. Cullis, Drug delivery systems: entering the mainstream, Science 19 (2004) 1818–1822. [3] I. Brigger, C. Dubernet, P. Couvreur, Nanoparticles in cancer therapy and diagnosis, Adv. Drug Deliv. Rev. 54 (2002) 631–651. [4] H. Koo, M.S. Huh, I.C. Sun, S.H. Yuk, K. Choi, K. Kim, I.C. Kwon, In vivo targeted delivery of nanoparticles for theranosis, Acc. Chem. Res. 44 (2011) 1018–1028. [5] H. Maeda, The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting, Adv. Enzym. Regul. 41 (2001) 189–207. [6] B.P. Schneider, K.D. Miller, Angiogenesis of breast cancer, J. Clin. Oncol. 23 (2005) 1782–1790. [7] E. Fakhrejahani, M. Toi, Antiangiogenesis therapy for breast cancer: an update and perspectives from clinical trials, Jpn. J. Clin. Oncol. 44 (2014) 197–207. [8] J. Folkman, Tumor angiogenesis: therapeutic implications, N. Engl. J. Med. 285 (1971) 1182–1186. [9] S.S. Brem, P.M. Gullino, D. Medina, Angiogenesis: a marker for neoplastic transformation of mammary papillary hyperplasia, Science 195 (1977) 880–882. [10] H.M. Jensen, I. Chen, M.R. Devault, A.E. Lewis, Angiogenesis induced by "normal" human breast tissue: a probable marker for precancer, Science 218 (1982) 293–295. [11] J.M. Guinebretiere, G. Le Monique, A. Gavoille, J. Bahi, G. Contesso, Angiogenesis and risk of breast cancer in women with fibrocystic disease, J. Natl. Cancer Inst. 86 (1994) 635–636. [12] A.J. Guidi, L. Fisher, J.R. Harris, S.J. Schnitt, J. Natl. Cancer Inst. 86 (1994) 614–619. [13] A.J. Guidi, S.J. Schnitt, L. Fischer, K. Tognazzi, J.R. Harris, H.F. Dvorak, L.F. Brown, Microvessel density and distribution in ductal carcinoma in situ of the breast, Cancer 80 (1997) 1945–1953. [14] S.B. Fox, D.G. Generali, A.L. Harris, Breast tumour angiogenesis, Breast Cancer Res. 9 (2007) 1–11. [15] G. Gasparini, Angiogenesis in breast cancer, Breast Cancer 16 (1999) 347–371. [16] M. Relf, S. LeJeune, P.A. Scott, S. Fox, K. Smith, R. Leek, A. Moghaddam, R. Whitehouse, R. Bicknell, A.L. Harris, Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor β-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis, Cancer Res. 57 (1997) 963–969.
[17] R. Kalluri, M. Zeisberg, Fibroblasts in cancer, Nat. Rev. Cancer 6 (2006) 392–401. [18] H. Abramczyk, J. Surmacki, M. Kopeć, A.K. Olejnik, A. Kaufman-Szymczyk, K. Fabianowska-Majewska, Epigenetic changes in cancer by Raman imaging, fluorescence imaging, AFM and scanning near-field optical microscopy (SNOM). Acetylation in normal and human cancer breast cells MCF10A, MCF7 and MDA-MB-231, Analyst 141 (2016) 5646–5658. [19] H. Abramczyk, B. Brozek-Pluska, New look inside human breast ducts with Raman imaging. Raman candidates as diagnostic markers for breast cancer prognosis: mammaglobin, palmitic acid and sphingomyelin, Anal. Chim. Acta 909 (2016) 91–100. [20] J. Surmacki, B. Brozek-Pluska, R. Kordek, H. Abramczyk, The lipid-reactive oxygen species phenotype of breast cancer. Raman spectroscopy and mapping, PCA and PLSDA for invasive ductal carcinoma and invasive lobular carcinoma. Molecular tumorigenic mechanisms beyond Warburg effect, Analyst 140 (2015) 2121–2133. [21] H. Abramczyk, B. Brozek-Pluska, Raman imaging in biochemical and biomedical applications. Diagnosis and treatment of breast cancer, Chem. Rev. 113 (2013) 5766–5781. [22] H. Abramczyk, B. Brozek-Pluska, J. Surmacki, J. Musial, R. Kordek, Oncologic photodynamic diagnosis and therapy: confocal Raman/fluorescence imaging of metal phthalocyanines in human breast cancer tissue in vitro, Analyst 139 (2014) 5547–5559. [23] J. Kneipp, T. Bakker Schut, M. Kliffen, M. Menke-Pluijmers, G. Puppels, Characterization of breast duct epithelia: a Raman spectroscopic study, Vib. Spectrosc. 32 (2003) 67–74. [24] C.H. Liu, Y. Zhou, Y. Sun, J.Y. Li, L.X. Zhou, S. Boydston-White, V. Masilamani, K. Zhu, Y. Pu, R.R. Alfano, Resonance Raman and Raman spectroscopy for breast cancer detection, Technol. Cancer Res. Treat. 12 (2012) 371–382. [25] B. Brozek-Pluska, M. Kopec, H. Abramczyk, Development of a new diagnostic Raman method for monitoring epigenetic modifications in the cancer cells of human breast tissue, Anal. Methods 8 (2016) 8542–8553. [26] A. Imiela, B. Polis, L. Polis, H. Abramczyk, Novel strategies of Raman imaging for brain tumor research, Oncotarget 8 (2017) 85290–85310. [27] H. Abramczyk, A. Imiela, The biochemical, nanomechanical and chemometric signatures of brain cancer, Spectrochim. Acta A 188 (2018) 8–19. [28] D. Qi, A.J. Berger, Chemical concentration measurement in blood serum and urine samples using liquid-core optical fiber Raman spectroscopy, Appl. Opt. 46 (2007) 1726–1734. [29] O. Warburg, On the origin of cancer, Science 123 (1956) 309–314. [30] L.M. Phan, S.C. Yeing, M.H. Lee, Cancer metabolic reprogramming: importance, main features, and potentials for precise targeted anti-cancer therapies, Cancer Biol. Med. 11 (2014) 1–19. [31] L. Lamalice, F. Le Boeuf, J. Huot, Endothelial cell migration during angiogenesis, Circ. Res. 100 (2007) 782–794. [32] A. Thomas, Fats and fatty oils, Encyclopedia of Industrial Chemistry, 2002. [33] S. You, H. Tu, Y. Zhao, Y. Liu, E.J. Chaney, M. Marjanovic, S.A. Boppart, Raman spectroscopic analysis reveals abnormal fatty acid composition in tumor micro- and macroenvironments in human breast and rat mammary cancer, Sci. Rep. 6 (2016) 1–10.
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging Monika Kopeć, Halina Abramczyk ⁎ Lodz University of Technology, Faculty of Chemistry, Institute of Applied Radiation Chemistry, Laboratory of Laser Molecular Spectroscopy, Wroblewskiego 15, 93-590 Lodz, Poland.
a r t i c l e
i n f o
Article history: Received 20 December 2017 Received in revised form 19 February 2018 Accepted 21 February 2018 Available online xxxx Keywords: Angiogenesis Cancer Raman spectroscopy Raman imaging AFM
a b s t r a c t Combined micro-Raman imaging and AFM imaging are efficient methods for analyzing human tissue due to their high spatial and spectral resolution as well as sensitivity to subtle chemical, structural and topographical changes. The aim of this study was to determine biochemical composition and mechanical topography around blood vessels in the tumor mass of human breast tissue. Significant alterations of the chemical composition and structural architecture around the blood vessel were found compared to the normal breast tissue. A pronounced increase of collagen-fibroblast-glycocalyx network, as well as enhanced lactic acid, and glycogen activity in patients affected by breast cancer were reported. © 2018 Elsevier B.V. All rights reserved.
1. Introduction The development of a variety of cancers is linked to changes in the biochemical properties of living cells. Oncogenically transformed cells are expected to have differences in all cell and tissue layers, but it still remains unclear whether biochemical alterations are a cause or a consequence of cancer development. The study of biochemistry of cancer cells by Raman and AFM imaging is presumably one of the most promising directions of cancer research, because the acquired knowledge will help design new treatments for cancer. It has been reported that the processes occurring in tumor microenvironment composed of noncellular (i.e. vascular and interstitial) and cellular compartments are as important as the processes in epithelial cells where most cancers including the ductal breast cancer develop [1]. These processes must be coupled because the epithelial cells are avascular, and the blood supply from the microenvironment to the tumor mass is one of the most important factors of cancer development. In this paper we will concentrate on biochemical signatures of tumor mass around vascular compartments and interstitial environment. There is a great number of abnormalities in the tumor vessels comparing with normal ones. Among them the most important are: increased vessel tortuosity, deficient pericytes, increased numbers of proliferating endothelial cells and abnormalities in the basement membrane [2,3]. Biochemical changes (upregulation of growth factors, bradykinin, prostaglandins, nitric oxide) are responsible for hyperpermeability what is revealed in high interstitial pressure and ⁎ Corresponding author. E-mail address: [email protected] (H. Abramczyk).
absence of functioning lymphatic network in tumor interstitium. This good vascularization of tumor helps with delivery of drugs, however because of high heterogeneity this effect is not effective in poorly vascularized regions [1,4,5]. The development of new blood vessels is a crucial step in breast cancer growth, progression and metastasis, because like healthy cells, cancer cells cannot live without oxygen and nutrients. The diversity of responsible angiogenic pathways that encourage new blood vessels to grow into the tumor in breast cancer for different tumors has been studied for many years [6–15]. The most important angiogenic factor is VEGF (Vascular Endothelial Growth Factor) [16]. When VEGF is overexpressed, cancer cells encourage the growth of blood vessels to feed a tumor by producing the hormone-like protein, vascular endothelial growth factor. This guarantee a good blood supplies which are needed for growth and in consequences for metastases [17]. Large number of researches suggest that angiogenesis (new blood vessel formation process) promote transformation of mammary hyperplasia to malignancy [10]. Clinical data show that microvessel density (MVD) is highest in aggressive breast tumors, and is associated with increased VEGF expression [13]. It seems appropriate to design drugs, which interrupt and inhibit angiogenesis in breast. The most commonly used drug is bevacizumab, which is a humanized monoclonal antibody directed against the VEGF-A ligand, but there is not yet definitive evidence for the efficacy of agents that specifically target angiogenesis [6]. Understanding mechanisms of angiogenesis as well as biological predictive markers is crucial in development of antiangiogenic therapies in clinical practice. One route to optimize the efficacy of these targeted agents is to better understand biology and biochemistry
https://doi.org/10.1016/j.saa.2018.02.058 1386-1425/© 2018 Elsevier B.V. All rights reserved.
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
2
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
around the vessels in the tumor environment. Highly advisable is therefore seeking new methods breaking the previous limitations and with potential clinical applications in order to satisfy the increasing demand of molecular biology. Reports of world literature in recent years clearly indicate that the particular role in the development of innovative techniques are played by multimodal imaging technologies, among which the dominant role belongs to Raman-AFM-fluorescence imaging, which allow simultaneous monitoring of morphological, biochemical properties with very high spectral and spatial resolution [18]. The Raman imaging is capable of simultaneously recording vibrational spectra from multiple regions and thereby map out the spatial distribution of proteins, lipids, nucleic acids, and metabolites in surrounding of the vessel supplying blood to tumors in contrast to the classical methods LC/ MS, NMR, HPLC, based on the analysis of samples in the mass subjected to homogenization that prevents spatial characteristics of the systems investigated [19–24]. We will show that Raman spectroscopy and imaging are capable of monitoring biochemical composition of tumor vascularity, which is markedly heterogeneous with densely vascularized areas supplying oxygen and nutrients to rapidly growing parts of the tumor. Raman imaging can monitor also areas where there is a decreasing amount of oxygen available to tumor cells which are further away from blood vessels. This information will help understand how new blood vessels are synthesized by tumors in a process known as angiogenesis. The aim of this study is to use micro-Raman and AFM imaging to analyze biochemical composition around blood vessels in cancerous and normal human breast tissue. The biochemical composition of human normal and cancerous breast tissues will be analysed by Raman and AFM imaging. 2. Experimental 2.1. Patients We have employed Raman spectroscopy, Raman imaging, AFM topography imaging to measure tissue biochemical composition around a vessel supplying blood to duct tumor in human breast tissue. These techniques have been applied for ex vivo fresh breast tissue samples without any fixation. The ex vivo samples were obtained during the resection surgery from the tumor mass (cancerous tissue) and the safety margin (normal tissue). All tissue samples were frozen and stored at −80 °C. Before the measurements the frozen samples were cryosectioned at −20 °C with a microtome (Microm HM 550, Sermed) into a few 6 μm-thick slices. Some of them were used for Raman and AFM analysis without typical for histology examination paraffin embedding procedure. The thin sections were placed onto calcium fluoride windows (CaF2, 25 × 1 mm) and examined by Raman and AFM imaging. After the Raman and AFM measurements these sections were stained and histologically examined. The samples were stained in hematoxylin for 3 min, rinsed in water and stained in eosin for 2 min. The adjacent tissue was analysed by histopathologists. All methods were performed in accordance with relevant guidelines and regulations. All tissue procedures were conducted under a protocol approved by the institutional Bioethical Committee at the Medical University of Lodz, Poland (RNN/323/17/KE/17/10/2017). Written informed consent was obtained from patients. 2.2. Chemicals All chemicals have been purchased from Sigma-Aldrich: palmitic acid (P0500; Sigma Aldrich), stearic acid (S4751; Sigma Aldrich), arachidic acid (A3631; Sigma Aldrich), oleic acid (O1008; Sigma Aldrich), linoleic acid (L1376; Sigma Aldrich), γ-linolenic acid (L2378; Sigma Aldrich), arachidonic acid (10,931; Sigma Aldrich), docosapentaenoic acid (D-120; Sigma Aldrich), eicosapentaenoic acid (E2011; Sigma Aldrich), α-linolenic acid (L-039; Sigma Aldrich),
eicosatetraenoic acid (H7768; Sigma Aldrich), tetracosahexaenoic acid (153,745; Sigma Aldrich), docosahexaenoic acid (D2534; Sigma Aldrich). 2.3. Raman Imaging Raman spectra and images were obtained with an alpha 300 RSA+ (WITec, Ulm, Germany), and preprocessed with WITec Control/Project Plus 2.1. Detailed description of equipment and methodology on data pre-processing and multivariate data analysis used in the paper is available elsewhere [19,21,25–27]. The 2D array images of tens of thousands of individual Raman spectra were evaluated by the basis analysis method (BAM). In BAM method, each measured spectrum of the 2D spectral array is compared to basis spectra using a least squares fit. Such basis spectra are created as the average spectra from different areas in the sample. The weight factor at each point is represented as a 2D image of the corresponding color and mixed coloring component. The color code of Raman maps were based on the integrated Raman intensities in specific regions (sum option in the filter manager in the WITec project 2.1). Using a lookup table, bright colors indicate the highest intensities, whereas dark colors indicate the lowest intensities of the chosen region. 2.4. AFM Measurements To obtain the topography of the samples AFM measurements were performed on an alpha 300 RSA+ (WITec, Ulm, Germany) based on an Olympus microscope. The AFM module was equipped with a 25 mm x- and y-range linearized piezoelectric scanner and 980 nm laser. The AFM was operating in the constant force mode, in which the feedback loop controls the z-height of the piezo table to keep the cantilever deflection constant. To generate AFM images we used the pyramidal cantilevers, with the spring constant of 0.2 N/m. 3. Results and Discussion 3.1. Raman Imaging Raman images allow looking inside the biochemical composition of cancerous cells around the lumen of the vessels supplying blood to the tumor mass and the extracellular matrix surrounding the tumor. To understand information that is provided from Raman images and vibrational spectra around the blood vessels in normal and cancerous breast tissues, we need to associate these features with the vessel morphology. Briefly, the walls around the lumen of artery are lined by an exceedingly thin single sheet of endothelial cells, the endothelium, separated from the surrounding outer layers by a basal lamina followed by many layers of smooth muscle cells and a thick, tough wall of connective tissue. Endothelial lining is present regardless amounts of connective tissue and smooth muscle in the vessel wall. Fig. 1 shows a cross section through a small artery supplying blood to a tumor mass in human breast tissue (G3, ductal cancer, P149) obtained by Raman imaging, compared with the H&E-stained histological image, microscopy image, AFM topography image and the characteristic vibrational Raman spectra for different areas of the cancerous tissue. One can see that there is an almost perfect match between the morphological features obtained from the histological, microscopy, AFM and Raman images. Raman imaging is a powerful technique which has many advantages over other imaging techniques, because it offers not only morphological image, but also a very detailed biochemical characterization by probing individual chemical bond vibrations. As a result, Raman spectra and images are information rich, and contain data related to the specific chemical structure and biocomposition of the biological material being analysed. The Raman spectra presented in Fig. 1g show the biochemical composition of the various substructures around a small artery supplying blood to a tumor mass in human breast tissue. The inside wall of the
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
3
Fig. 1. Histological image, Raman images, AFM image and Raman spectra of tumor mass in human breast tissue in the high frequency region. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
artery covered by a thin single sheet of endothelial cells, and the basal lamina cannot be seen at the relatively low resolution chosen for the large area. However, many layers of cells and connective tissue around the artery are clearly visible. One can see from Fig. 1c that the artery is surrounded by an extensive network full of fibers that vary in diameter from 2 to 10 μm that can be easily identified without any staining from the Raman spectra (Fig. 1g). The network constitutes mainly of collagen (red color in Fig. 1c and f) and lipids (fibroblasts –blue color, in Fig. 1c and f). Cancer cells invade in the collagen and fibroblast network [25]. Each collagen fiber is made-up of a variable number of smaller collagen fibrils. These fibrils are composed predominantly of type I collagen, because a characteristic feature of these fibrils is that they exhibit axial periodicity due to the way in which type I collagen assembles to form collagen fibrils, which are synthesized primarily by fibroblasts aligning themselves along collagen fibers.
H&E-stained histological image (a), stitching microscopy image (b), Raman images (360 μm × 660 μm) obtained from basis analysis (c), microscopy image (d), AFM topography image (e), Raman images for specific spectral filters (f), as well as the normalized (model: divided by norm) Raman spectra (g) in the high frequency region of the cancerous breast tissue (P149), integration time 0.3 s, resolution: 0.5 μm, laser excitation power: 10 mW. Objective 40×, 6 μm-thick slices on calcium fluoride window (CaF2, 25 × 1 mm). The line colors of the spectra correspond to the colors of the Raman maps. A clear branching pattern of collagen fibers of smaller diameter in Fig. 1c suggests contribution from reticular fibers consisting of type III collagen fibrils. These structures are usually distinguished by PAS (Periodic acid–Schiff) staining, because unlike collagen I fibers, reticular III type fibers are PAS-positive owing to their greater carbohydrate content. The advantage of the Raman spectroscopy is its capability of
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
4
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
identifying type III collagen fibrils containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans) without any PAS staining. The turquoise color in Fig. 1c, f and g represents an amorphous carbohydrate rich ground substance. More information on the ground substance can be obtained from the Raman fingerprint region. Fig. 2 shows Raman images and Raman spectra the cross section through a small artery supplying blood to a tumor mass in human breast tissue in the fingerprint region (the same as in Fig. 1). The fingerprint region provides additional information about the distribution of main biochemical components of the tumor mass around the artery – proteins (amide I and III), lipids (saturated CH2 bonds), glycogen (840 cm−1), glucose (1125 cm−1) and lactic acid (917 cm−1) [28] partially overlapping with O-P-O backbone of DNA (836 cm−1), collagen (855 cm−1), (874 cm−1) C\\C stretch of 4 hydroxy-proline, (920 cm−1) C\\C stretch
of proline ring, (937 cm−1). The Raman vibrations of these components provide information about distribution of carbohydrate content in intracellular matrix and about altered metabolism in cancer cells. These carbohydrate rich ground substance structures are presented in Fig. 2c, f and g by the turquoise color. The enhanced Raman intensity of carbohydrates is clearly visible in the walls around the lumen of the artery and along the collagen and fibroblast fibers. The carbohydrate content in intracellular matrix originating from an amorphous ground substance consists of glycosaminoglycans synthesized by fibroblasts in loose connective tissue. The vibrations of glycosaminoglycans are partially overlapped with the vibrations of glycogen and lactic acid. Monitoring of the distribution of glycogen and lactic acid by label-free Raman imaging provides very important information about metabolism of cancerous cells. The altered metabolism is one of the important features of cancer and is usually described by Otto Warburg effect with enhanced
Fig. 2. Histological image, Raman images, AFM image and Raman spectra of tumor mass in human breast tissue in the fingerprint spectral region. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
anaerobic glycolysis [29]. The elevated glucose catabolism produces an excess of the glycolytic end-product, pyruvate. Most of the pyruvate is converted to lactate, whereas some of it is converted to acetyl-CoA, which, in turn, is used in de novo fatty-acid synthesis [30]. H&E-stained histological image (a), stitching microscopy image (b), Raman images (360 μm × 660 μm) obtained from basis analysis (c), microscopy image (d), AFM topography image (e), Raman images for specific spectral filters (f) as well as the normalized (model: divided by norm) Raman spectra (g) in the low frequency region of the cancerous breast tissue (P149), integration time 0.5 s, resolution: 0.5 μm, laser excitation power: 10 mW. Objective 40×, 6 μm-thick slices on calcium fluoride window (CaF2, 25 × 1 mm). The line colors of the spectra correspond to the colors of the Raman maps. Comparison between the Raman images obtained from the high frequency (Fig. 1c) and the fingerprint (Fig. 2c) regions demonstrates that the images are coherent and almost identical for the components that have vibrations in both regions (collagen in fibers, lipids in fibroblasts and glycans). Additional information about distribution of carbohydrate content in intracellular matrix originating from an amorphous ground substance, glycogen and lactic acid in cells around the artery supplying
5
blood to the breast tumor is provided by the Raman image for the 800–920 cm−1 filter (turquoise color in Fig. 2c and f). The results presented so far demonstrate the central importance of the network of collagen and fibroblast matrix formed around the blood artery. This matrix seems to be required to protect the balance between rigidity of the environment and deformability of cancer cells to forma stable vascular structure for supplying nutrients and oxygen to the tumor mass. Collagen matrix plays a role of a scaffold that protect the deformable cancer cells. The formation of first primitive vascular plexus is possible through the migration of endothelial cells and angioblasts on a matrix formed from collagen and fibroblasts with the guidance of Vascular Endothelial Growth Factor (VEGF) leading to vascularization of a tumor mass [31]. It is very important to compare the morphology and biochemistry around the blood artery supplying nutrients and oxygen to the tumor mass with the normal structure of the breast tissue. Fig. 3 shows the results for a normal breast tissue. H&E-stained histological image (a), microscopy image (b), Raman images (60 μm × 60 μm) obtained from basis analysis (c), microscopy image (d), AFM topography image (e) Raman images for specific
Fig. 3. Histological image, Raman images, AFM image and Raman spectra of a normal breast tissue from a negative margin of a tumor mass from the same patient (G3, ductal cancer P149) as presented in Figs. 1 and 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
6
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
spectral filters (f) and normalized (model: divided by norm) Raman spectra (g) from the high frequency region for different areas of the normal breast tissue. Integration time 0.3 s, resolution: 1 μm, laser excitation power: 10 mW. Objective 40×, 6 μm-thick slices on calcium fluoride window (CaF2, 25 × 1 mm). The line colors of the spectra correspond to the colors of the Raman maps. Fig. 3 shows Raman images of the normal breast tissue from a negative margin of the tumor mass from the same patient (G3, ductal cancer, P149) as presented in Figs. 1 and 2 compared with the H&E-stained histological image, microscopy image, AFM topography image and the characteristic vibrational spectra for different areas of the normal breast tissue. One can see that in contrast to the cancerous tissue presented in Figs. 1 and 2 collagen is not the most abundant component of the normal breast tissue. In contrast to the tumor mass presented in Figs. 1 and 2 the collagen-fibroblast network is much less pronounced feature in the normal breast tissue from the negative safety margin (lack of cancer cells). The Raman image is dominated by adipose fat (dark blue color) and the normal cells (turquoise color in Fig. 3c) with rich protein structure at 2911 cm−1 which represents CH2 vibrations of nonmethylated and non-acetylated proteins in contrast to the cancerous structures in Fig. 1c that contain dominant component of collagen and methylated/acetylated proteins [25]. Fig. 4 shows Raman images of a normal breast tissue from a negative margin of a tumor mass from the same patient (G3, ductal cancer, P149)
as presented in Figs. 1 and 2 compared with the H&E-stained histological image, microscopy image, AFM topography image and the characteristic vibrational spectra for different areas of the normal breast tissue in the fingerprint region. Comparison of the vector normalized Raman intensities of glycogen (840 cm−1), glucose (1125 cm−1) and lactic acid (917 cm−1) for normal breast tissue in Fig. 4g with the corresponding intensities for tumor tissue in Fig. 2g demonstrates that glycolytic path of metabolism is significantly less efficient (about twice) in the normal tissue. H&E-stained histological image (a), microscopy image (b), Raman images (60 μm × 60 μm) obtained from basis analysis (c), microscopy image (d), AFM topography image (e) Raman images for specific spectral filters (f) and normalized (model: divided by norm) Raman spectra (g) from the fingerprint frequency region for different areas of the normal breast integration time 0.5 s, resolution: 1 μm, laser excitation power: 10 mW. Objective 40×, 6 μm-thick slices on calcium fluoride window (CaF2, 25 × 1 mm). The line colors of the spectra correspond to the colors of the Raman maps. 3.2. Iodine Value Iv Third important feature that reveals from the fingerprint regions in Fig. 2g and Fig. 4g is information about fatty acid composition in tumor environments. To address these important questions on lipid
Fig. 4. Histological image, Raman images, AFM image and Raman spectra of a normal breast tissue from a negative margin of a tumor mass from the same patient (G3, ductal cancer P149) as presented in Figs. 1 and 2.
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
phenotypic alterations we used the iodine value Iv to characterize tissues. The iodine value Iv is the mass of iodine in grams that is consumed by 100 g of a chemical substance. Knowing the molecular weight of each acids and the number of double bonds, we calculated the iodine value according to the formula (1):
Iv ¼
100xNd xMI2 Ma
ð1Þ
where: Nd number of double bonds. MI2 molecular mass of I2. Ma molecular mass of acid. The iodine value Iv is accepted as a good source of information on the amount of unsaturation in fatty acids, where the double bonds react with iodine compounds. The higher iodine value indicates that there is more C_C bonds in the fat [26,32,33]. This formula has been used for all fatty acids to create the calibration curve of the iodine value as a function of the Raman intensity ratio of selected Raman bands. We have applied the iodine value Iv plot vs the Raman intensity ratio 1268/1444 measured at 1268 cm−1 and 1444 cm−1 used as a source of information on the ratio of unsaturated/saturated lipids. Next we recorded the ratio 1268/1444 for the cancerous and noncancerous tissues and from the calibration curve the iodine values of the tissues were determined.
Fig. 5. The iodine value Iv plot vs the Raman intensity ratio 1268/1444 for PUFA and human breast normal (a) and cancer (b) tissues of studied samples. The equation of the curve: y = −34.94x2 + 255.18x − 21.263 R2 = 0,9775 green triangles indicate the iodine value for patient P149 palmitic acid (PA), stearic acid (SA), arachidic acid (ARA), oleic acid (OA), linoleic acid (LA), γ-linolenic acid (GLA), arachidonic acid (AA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosatetraenoic acid (ETA), tetracosahexaenoic acid (THA), docosahexaenoic acid (DHA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
7
Fig. 5 shows the iodine value plot vs the Raman intensity ratio 1268/ 1444 for polyunsaturated fatty acids (PUFA) and breast tissues of the studied samples. The iodine value Iv for lipids in normal and tumor breast tissues are 65 and 136, respectively. The iodine value for the normal tissue is close to the value for oleic acid (Iv = 87), while the abnormal tumor microenvironment shows the numbers between oleic acid (OA) and linoleic acid (LA) (Iv = 181). It indicates that tumor microenvironment in human breast cancer shows altered fatty acid composition in comparison with the normal tissue. 4. Conclusions Understanding mechanisms of angiogenesis as well as biological predictive markers are crucial in development of antiangiogenic therapies in clinical practice. The results presented in the paper demonstrate the central importance of the network of collagen, fibroblasts, and glycocalyx matrix formed around the blood artery supplying blood to tumor mass. The Raman spectra provide information on collagen network, particularly identification of type III collagen fibrils that is not available directly in other visualization techniques. The advantage of the Raman spectroscopy is its capability of identifying type III collagen fibrils containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans) without any PAS staining. The collagen- fibroblast-glycocalyx matrix seems to be required to protect the balance between rigidity of the environment and deformability of cancer cells to form a stable vascular structure for supplying nutrients and oxygen to the tumor mass. The collagen matrix plays a role of a scaffold that protect the deformable cancer cells. The glycocalyx serves as mechanotransducer, because the epithelium cancer cells are exposed to mechanical forces of the rigid collagen environment. However, the molecules responsible for the translation of biomechanical forces into biochemical signals (mechanotransduction) have not been identified yet. The paper on angiogenesis of cancer opened a new window for Raman exploration of processes occurring in non-cellular spaceglycocalyx–a coat on the external surface of plasma membranes of epithelial cells. Because of the functional importance of the epithelial glycocalyx, development of direct visualization techniques is crucial to establish its exact role. We have demonstrated that label-free Raman imaging can easily monitor location and distribution of the epithelial glycocalyx with high spatial and spectral resolution. We have demonstrated that the Raman imaging combined with AFM are capable of simultaneous identification of breast cancer specific biochemical and mechanical alterations in tumor mass around blood vessels in cancerous and normal human breast tissues. This combination of biochemical and topographic signatures has potential to provide predictive Raman cancer biomarkers and treatment targets during cancer development. High-resolution Raman and AFM maps show that matrix stiffening in tumor progression is due to alteration of biochemical composition of the tumor microenvironment composed of noncellular (ie, vascular and interstitial) and cellular compartments. We found significant alterations in chemical composition, and structural architecture around the blood vessel with a pronounced increase of collagen-fibroblast-glycocalyx network. We found that oncogenically transformed cells exhibit enhanced lactic acid, and glycogen activity of cancer tissue that can be easily monitored by Raman-based methods. Author Contributions HA conceived the idea, designed and directed the Raman and AFM experiments, analysed the Raman data, analysed force spectroscopy data wrote the manuscript and edited successive drafts of the paper. MK collected all clinical data, performed Raman imaging and AFM experiments, analysed Raman spectroscopy data, contributed to the discussion.
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058
8
M. Kopeć, H. Abramczyk / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2018) xxx–xxx
Competing Interests The authors declare no competing financial interests. Acknowledgements This work was supported by the National Science Center of Poland (grant UMO-2015/19/B/ST4/01878), Dz. St. 2017. References [1] A.S. Thakor, S.S. Gambhir, Nanooncology: the future of cancer diagnosis and therapy, CA Cancer J. Clin. 63 (2013) 395–418. [2] T.M. Allen, P.R. Cullis, Drug delivery systems: entering the mainstream, Science 19 (2004) 1818–1822. [3] I. Brigger, C. Dubernet, P. Couvreur, Nanoparticles in cancer therapy and diagnosis, Adv. Drug Deliv. Rev. 54 (2002) 631–651. [4] H. Koo, M.S. Huh, I.C. Sun, S.H. Yuk, K. Choi, K. Kim, I.C. Kwon, In vivo targeted delivery of nanoparticles for theranosis, Acc. Chem. Res. 44 (2011) 1018–1028. [5] H. Maeda, The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting, Adv. Enzym. Regul. 41 (2001) 189–207. [6] B.P. Schneider, K.D. Miller, Angiogenesis of breast cancer, J. Clin. Oncol. 23 (2005) 1782–1790. [7] E. Fakhrejahani, M. Toi, Antiangiogenesis therapy for breast cancer: an update and perspectives from clinical trials, Jpn. J. Clin. Oncol. 44 (2014) 197–207. [8] J. Folkman, Tumor angiogenesis: therapeutic implications, N. Engl. J. Med. 285 (1971) 1182–1186. [9] S.S. Brem, P.M. Gullino, D. Medina, Angiogenesis: a marker for neoplastic transformation of mammary papillary hyperplasia, Science 195 (1977) 880–882. [10] H.M. Jensen, I. Chen, M.R. Devault, A.E. Lewis, Angiogenesis induced by "normal" human breast tissue: a probable marker for precancer, Science 218 (1982) 293–295. [11] J.M. Guinebretiere, G. Le Monique, A. Gavoille, J. Bahi, G. Contesso, Angiogenesis and risk of breast cancer in women with fibrocystic disease, J. Natl. Cancer Inst. 86 (1994) 635–636. [12] A.J. Guidi, L. Fisher, J.R. Harris, S.J. Schnitt, J. Natl. Cancer Inst. 86 (1994) 614–619. [13] A.J. Guidi, S.J. Schnitt, L. Fischer, K. Tognazzi, J.R. Harris, H.F. Dvorak, L.F. Brown, Microvessel density and distribution in ductal carcinoma in situ of the breast, Cancer 80 (1997) 1945–1953. [14] S.B. Fox, D.G. Generali, A.L. Harris, Breast tumour angiogenesis, Breast Cancer Res. 9 (2007) 1–11. [15] G. Gasparini, Angiogenesis in breast cancer, Breast Cancer 16 (1999) 347–371. [16] M. Relf, S. LeJeune, P.A. Scott, S. Fox, K. Smith, R. Leek, A. Moghaddam, R. Whitehouse, R. Bicknell, A.L. Harris, Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor β-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis, Cancer Res. 57 (1997) 963–969.
[17] R. Kalluri, M. Zeisberg, Fibroblasts in cancer, Nat. Rev. Cancer 6 (2006) 392–401. [18] H. Abramczyk, J. Surmacki, M. Kopeć, A.K. Olejnik, A. Kaufman-Szymczyk, K. Fabianowska-Majewska, Epigenetic changes in cancer by Raman imaging, fluorescence imaging, AFM and scanning near-field optical microscopy (SNOM). Acetylation in normal and human cancer breast cells MCF10A, MCF7 and MDA-MB-231, Analyst 141 (2016) 5646–5658. [19] H. Abramczyk, B. Brozek-Pluska, New look inside human breast ducts with Raman imaging. Raman candidates as diagnostic markers for breast cancer prognosis: mammaglobin, palmitic acid and sphingomyelin, Anal. Chim. Acta 909 (2016) 91–100. [20] J. Surmacki, B. Brozek-Pluska, R. Kordek, H. Abramczyk, The lipid-reactive oxygen species phenotype of breast cancer. Raman spectroscopy and mapping, PCA and PLSDA for invasive ductal carcinoma and invasive lobular carcinoma. Molecular tumorigenic mechanisms beyond Warburg effect, Analyst 140 (2015) 2121–2133. [21] H. Abramczyk, B. Brozek-Pluska, Raman imaging in biochemical and biomedical applications. Diagnosis and treatment of breast cancer, Chem. Rev. 113 (2013) 5766–5781. [22] H. Abramczyk, B. Brozek-Pluska, J. Surmacki, J. Musial, R. Kordek, Oncologic photodynamic diagnosis and therapy: confocal Raman/fluorescence imaging of metal phthalocyanines in human breast cancer tissue in vitro, Analyst 139 (2014) 5547–5559. [23] J. Kneipp, T. Bakker Schut, M. Kliffen, M. Menke-Pluijmers, G. Puppels, Characterization of breast duct epithelia: a Raman spectroscopic study, Vib. Spectrosc. 32 (2003) 67–74. [24] C.H. Liu, Y. Zhou, Y. Sun, J.Y. Li, L.X. Zhou, S. Boydston-White, V. Masilamani, K. Zhu, Y. Pu, R.R. Alfano, Resonance Raman and Raman spectroscopy for breast cancer detection, Technol. Cancer Res. Treat. 12 (2012) 371–382. [25] B. Brozek-Pluska, M. Kopec, H. Abramczyk, Development of a new diagnostic Raman method for monitoring epigenetic modifications in the cancer cells of human breast tissue, Anal. Methods 8 (2016) 8542–8553. [26] A. Imiela, B. Polis, L. Polis, H. Abramczyk, Novel strategies of Raman imaging for brain tumor research, Oncotarget 8 (2017) 85290–85310. [27] H. Abramczyk, A. Imiela, The biochemical, nanomechanical and chemometric signatures of brain cancer, Spectrochim. Acta A 188 (2018) 8–19. [28] D. Qi, A.J. Berger, Chemical concentration measurement in blood serum and urine samples using liquid-core optical fiber Raman spectroscopy, Appl. Opt. 46 (2007) 1726–1734. [29] O. Warburg, On the origin of cancer, Science 123 (1956) 309–314. [30] L.M. Phan, S.C. Yeing, M.H. Lee, Cancer metabolic reprogramming: importance, main features, and potentials for precise targeted anti-cancer therapies, Cancer Biol. Med. 11 (2014) 1–19. [31] L. Lamalice, F. Le Boeuf, J. Huot, Endothelial cell migration during angiogenesis, Circ. Res. 100 (2007) 782–794. [32] A. Thomas, Fats and fatty oils, Encyclopedia of Industrial Chemistry, 2002. [33] S. You, H. Tu, Y. Zhao, Y. Liu, E.J. Chaney, M. Marjanovic, S.A. Boppart, Raman spectroscopic analysis reveals abnormal fatty acid composition in tumor micro- and macroenvironments in human breast and rat mammary cancer, Sci. Rep. 6 (2016) 1–10.
Please cite this article as: M. Kopeć, H. Abramczyk, Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018), https://doi.org/10.1016/j.saa.2018.02.058