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Fluorescence spectroscopy and imaging to improve diagnosis of normal and tumoral cytological pancreatic cells
Pathology – Research and Practice 209 (2013) 1–5
Contents lists available at SciVerse ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original article
Fluorescence spectroscopy and imaging to improve diagnosis of normal and tumoral cytological pancreatic cells Malek Atyaoui a,b,c,∗ , Wissem Dimassi b , Nouemen Tounsi c , Nejm Eddine Jaidan a , Hatem Ezzaouia b a b c
Laboratoire de Spectroscopie Atomique, Moléculaire et Applications, Université Tunis El Manar, Tunis, Tunisia Laboratoire de Photovoltaïque, Centre de recherches et des technologies de l’énergie, technopole de Borj-Cédria, PB: 95, Hammam Lif 2050, Tunis, Tunisia Ecole supérieure des sciences et techniques de Tunis, Université de Tunis, Tunisia
a r t i c l e
i n f o
Article history: Received 3 May 2012 Received in revised form 11 July 2012 Accepted 28 August 2012 Keywords: Fluorescence Cytological cells Tumoral cells Normal cells Pancreas
a b s t r a c t We investigated a simple and effective diagnostic method for normal and malignant pancreatic cells using fluorescence emission of endogenous and exogenous molecules in cytological cells. Fluorescence imaging was performed to assess the spectral properties and the spatial distribution of the fluorescence emitted by pancreatic cells. The results revealed quite different fluorescence distributions between tumor cells, characterized by perimembrane fluorescence localization, and normal cells exhibiting an intracellular fluorescence. This was not caused by differences in the fluorescence emission of the endogenous fluorophores NAD(P)H or porphyrins but by various localizations of the exogenous molecules (the EA 50 Papanicolaou stain). © 2012 Elsevier GmbH. All rights reserved.
Introduction
Materials and methods
Pancreatic cancer leads to death in industrialized countries. Its incidence is high [1,2], and is caused by early metastasis and destructive growth along the endothelium basement membrane and neurons [3]. Most patients with pancreatic cancer have a poor outcome due to the absence of early diagnosis and its highly invasive and metastatic features, which leaves more than 85% of the patients inoperable at the time of diagnosis. Therefore, the application of new approaches and new methods is necessary to identify and detect cancer signatures. Fluorescence has proven to be a versatile tool for studying molecular interactions in analytical chemistry, biochemistry, cell biology, photochemistry, and environmental science [4–6]. It has been recently used for cancer diagnosis [7–15]. The fluorescence emitted by the endogenous and exogenous molecules of cytological pancreatic cells is worth studying in order to improve prognosis and to discriminate between normal and tumoral pancreatic cells. In this work, discrimination between normal and tumoral pancreatic cells was possible by the activation of EA 50 stain. The fluorescence imaging of normal and tumor cells is presented and discussed.
Preparation of cytological slides
∗ Corresponding author at: Laboratoire de Spectroscopie Atomique, Moléculaire et Applications, Université Tunis El Manar, Tunis, Tunisia. Tel.: +216 94 088 223. E-mail address: [email protected] (M. Atyaoui). 0344-0338/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2012.08.007
Pancreatic cytological slides were transferred to the anatomopathological department for cytospinning and staining using the classical Papanicolaou technique [16]. The use of this protocol is necessary to identify the different cell elements (nucleus, cytoplasm, cellular membrane) and to activate their fluorescent molecule. Using this method, three stain mixtures were applied for cell staining with different locations: Harris hematoxylin, specific of cell nucleus staining, orange G6 (OG 6), which interacts with cytoplasmic keratin, and EA 50, a trichromatic stain containing light green, yellow eosin and Bismarck brown with non-specific localization. More details concerning the photophysical properties of the Papanicolaou stains are given elsewhere [10]. Wavelength-resolved confocal laser scanning microscopy Confocal fluorescence images were acquired with a commercial device (Leica TCS SP2 or SP5 AOBS, Mannheim, Germany). A Leica objective (63×/1.4–0.60 NA, oil immersion) was used for acquiring images in direct slow scanning mode. It was checked that the immersion oil (Cargille, Cedar Grove) did not fluoresce over all the spectral range 400–800 nm irrespective of the fluorescence excitation wavelength used. Fluorescence signatures from pancreatic cells, in terms of intensity, spectra and images were obtained under excitation either with the 364-nm line of argon
2
M. Atyaoui et al. / Pathology – Research and Practice 209 (2013) 1–5
Table 1 Transmission and fluorescence intensity images of papanicolaou-stained pancreatic normal cells. Transmission
Fluorescence
Normal cells
laser and 633-nm line of a helium-neon laser (average laser power ∼90 W) or with the 488-nm line of argon lasers (average laser power ∼3 W) to characterize the fluorescence of endogenous fluorophores, NAD(P)H and porphyrins, and Papanicolaou stains, respectively. For spectral measurements, the epifluorescence was imaged on a photomultiplier with a 10-nm spectral slit which was moved by 2-nm steps.
pancreatic cells on the basis of their fluorescence localization: in normal cells, fluorescence intensity was found inside the cell with a higher intensity measured in the cytoplasm than in the nucleus (Table 1), whereas in tumor cells, the fluorescence was localized mainly on the perimembrane surface (Table 2). On the other hand, a fluorescence spectroscopy was performed in order to identify the fluorescent molecule responsible for the above mentioned difference in fluorescence distribution. Fig. 1a and b shows the fluorescence spectra of tumor cells and normal pancreatic cells in different locations (cytoplasm, nucleus and perimembrane surface) recorded in the wavelength range (525–609 nm) and under excitation with the 488 nm Ar laser line of the confocal device. The fluorescence spectra of the normal cells and tumor cells showed identical emission peak positions centered at around 550 nm. The highest fluorescence intensity of the normal cell was obtained in the cytoplasm (Fig. 1a), but was recorded on the perimembrane surface for the malignant cell (Fig. 1b). Steenkeste and Lécart [10] reported that the red emission band centered at around 550 nm in the fluorescence spectrum of urothelial cells was attributed to the presence of EA 50 stain. Therefore, on the basis of the above fluorescence spectra and images, we conclude that the signature of the EA 50 exogenous molecule may be just a clue or phenomenon regarding the separation between normal and tumor cells. There was no cause nor did this affect the relationship. On the other hand, many authors [17–24] reported that for pathological cells or for cells under various stimulations, such as pro-inflammatory, pro-apoptosis, or pro-coagulating signals,
(a) 200
Nucleus Cytoplasm Perimembrane surface
Results and discussion In order to observe the possible change in the fluorescence distribution or fluorescence localization of the obtained samples (tumoral and normal cells), we performed fluorescence imaging (Tables 1 and 2) in the wavelength range of (525–609 nm) with an excitation wavelength of 488 nm. As shown in the two tables, it is obvious that the tumor cells can be distinguished from the normal
Fluorescence intensity (u.a).
180 160 140 120 100
Table 2 Transmission and fluorescence intensity images of papanicolaou-stained pancreatic tumoral cells. Transmission
80 60 40 20 0
520
540
Fluorescence
560
580
Wavelength (nm)
600
620
(b) 55
Nucleus Cytoplasm Perimembrane surface
Tumoral cells
Fluorescence intensity (u.a).
50 45 40 35 30 25 20 15 10 5 520
540
560
580
600
620
Wavelength (nm) Fig. 1. Fluorescence spectrum of cytological pancreatic cells obtained under 488 nm excitation: (a) normal cells and (b) tumor cells.
M. Atyaoui et al. / Pathology – Research and Practice 209 (2013) 1–5 140
0
2
4
6
8
0
10 10
3
2
4
6
8
10 10
80 120
70
100 80
6
Perimembrane intensity of Tumoral Cells 60 4
Perimembrane intensity of Normal Cells
40 20
2
Fluorescence intensity (u.a).
Fluorescence Intensity (u.a)
8
8
60 6
50 40
4 30 20
2
10 0 1
2
3
4
5
6
7
8
9
0
Number of serial Fluorescence tests on 18 pancreatic cells Fig. 2. Serial fluorescence blind tests on 18 pancreatic cells under 488 nm excitation. 9 were classified as normal and 9 as cancerous.
the membrane phospholipid bilayer is restructured: a loss of the membrane asymmetry is observed and correlated to the externalization of phosphatidylserines [25–27]. Therefore, the fluorescence on the perimembrane surface of tumor cells can be attributed to the restructuration in the membrane phospholipid bilayer. The reproducibility of the two above remarkable fluorescence patterns of normal and tumor cells was investigated by repeating the fluorescence intensity measurements on 18 Papanicolaoustained pancreatic cells: 9 were classified as normal cells and 9 as tumoral pancreatic cells. As shown in Fig. 2, there was no change in the fluorescence behavior of the two types of cells (normal and tumoral): all the tumor cells had higher perimembrane fluorescence intensity compared to normal cells. The fluorescence intensity measured on the perimembrane surface of tumor cells was almost 1572 ± 166% times higher than that in normal cells. The error limit of the measurement was determined by repeating the analysis three times per sample. Autofluorescence of cells is produced by endogenous molecules that naturally occur in cells after excitation with a suitable wavelength. The presence of disease changes the concentration of the
0
640
660
680
700
720
740
760
Wavelength (nm) Fig. 3. Fluorescence emission of porphyrins under 633 nm excitation.
endogenous molecules, as well as the light scattering and absorption properties of the tissue or the cells, due to changes in a.o. blood concentration, nuclear size distribution, collagen content and epithelial thickness [28–31,9,32]. Therefore, the activation of the endogenous molecules (NAD(P)H, porphyrins) is necessary to improve the discrimination between normal and pancreatic tumor cells. Fluorescence properties of the endoporphyrins of cytological pancreatic cells have been detailed in the wavelength range 643–750 nm. In this spectral range, there is no overlap with the emission of EA50. The endoporphyrin fluorescence images revealed for each cell type (tumoral and normal) a similar trend: an intracellular distribution of the fluorescence with a higher intensity in the cytoplasm than in the nucleus as shown in Table 3. The corresponding spectra obtained under 633-nm excitation are shown in Fig. 3. Irrespective of the cell type, normal or tumoral, the emission was characterized by two main bands centered at about 675 and 700 nm with a shoulder around 740 nm in agreement with the fluorescence properties of porphyrins [33,34]. The fluorescence spectrum shown in Fig. 4 was obtained after excitation of the NAD(P)H with the 364-nm line of argon laser.
Table 3 Transmission and fluorescence intensity images of papanicolaou-stained pancreatic cells. (a) Tumoral cells and (b) normal cells. Fluorescence intensity images in the 525–609 nm spectral range obtained after activation of EA 50 and in the 643–750 nm spectral range obtained after excitation of porphyrins. Transmission
Tumoral cells (A)
Normal cells (B)
Fluorescence 525–609 nm range
Fluorescence 643–750 nm range
4
M. Atyaoui et al. / Pathology – Research and Practice 209 (2013) 1–5 2
14
4
6
8
10
12
Fluorescence intensity (u.a).
8 10 6
8
6
4
4 2 2
0
400
420
440
460
480
500
520
0
Wavelength (nm) Fig. 4. Fluorescence emission of NAD(P)H under 364 nm excitation.
The NAD(P)H emission coincides well with the in vitro spectrum of NADH [35–37] with a maximum at 460 nm. No changes were found in the emission spectral shape and intensity between normal and tumor cells. Unfortunately, the fluorescence signal of NAD(P)H is low, so that fluorescence images could not be recorded. As observed in the fluorescence spectrum and images (Table 3 and Figs. 3 and 4), it was not possible to discriminate between normal and tumoral cytological pancreatic cells from the autofluorescence emission. Conclusion The aim of this study was to excite endogenous and exogenous molecules in cytological pancreatic cells and, consequently, to use their fluorescence properties in order to improve the diagnosis of normal cells and tumor cells. Fluorescence spectroscopy and imaging showed that the tumor cells and normal cells have different spatial fluorescence distributions. After the EA 50 activation, the fluorescence images showed two fluorescence locations in the cells, one intracellular location for the normal cells, and another perimembrane surface location for the tumor cells. On the other hand, it is not efficient to excite endogenous molecules like NAD(P)H and porphyrins of normal cells and tumor cells. There is a similar trend: the autofluorescence images showed for each cell type (tumoral and normal) an intracellular distribution of the fluorescence. This detection method is now in clinical progress, and the discussion on the diagnostic value of the EA 50 activation in other types of cancer and other pancreatic diseases, such as pancreatitis, should be the topic of future studies. Acknowledgements The authors thank the Centre de Photonique Biomédicale, Centre Laser de l’Université Paris-Sud, Orsay, for supporting the analysis of fluorescence spectroscopy and imaging. The authors are also grateful to Service d’Anatomie Cytologie Pathologiques, Hopital de Bicetre, Le Kremlin-Bicetre, France, for preparing the samples and for their help. References [1] A.B. Lowenfels, P. Maisonneuve, Epidemiology and risk factors for pancreatic cancer, Best Pract. Res. Clin. Gastroenterol. 20 (2006) 197. [2] M.T. Carriaga, D.E. Henson, Liver, gallbladder, extrahepatic bile ducts, and pancreas, Cancer 75 (1995) 171–190.
[3] S.A. Eccles, D.R. Welch, Metastasis: recent discoveries and novel treatment strategies, Lancet 369 (2007) 1742. [4] V. Masilamani, K. Al-Zhrani, M. Al-Salhi, A. Al-Diabb, M. Al-Ageilyc, Cancer diagnosis by autofluorescence of blood components, J. Lumin. 109 (2004) 143–154. [5] M.S. Khot, N.K. Desai, G.B. Kolekar, S.R. Patil, Fluorescence enhancement effect for the determination of adenosine 5 -monophosphate with 9-anthracene carboxylic acid–cetyl trimethyl ammonium bromide system, J. Fluorescence 21 (2011) 1997–2003. [6] X. Gao, L. Niu, X. Su, Detection of DNA via the fluorescence quenching of Mn-doped ZnSe D-dots/doxorubicin/DNA ternary complexes system, J. Fluorescence 22 (2012) 103–109. [7] A.M. Kluftinger, N.L. Davis, N.F. Quenvillei, S. Lam, J. Hung, B. Palcic, Detection of squamous cell cancer and pre-cancerous lesions by imaging of tissue autofluorescence in the hamster cheek pouch model, Surg. Oncol. 1 (1992) 183–188. [8] M. Sato, A. Sakurada, M. Sagawa, M. Minowa, H. Takahashi, T. Oyaizu, Y. Okada, Y. Matsumura, T. Tanita, T. Kondo, Diagnostic results before and after introduction of autofluorescence bronchoscopy in patients suspected of having lung cancer detected by sputum cytology in lung cancer mass screening, Lung Cancer 32 (2001) 247–253. [9] K. Onizawa, N. Okamura, H. Saginoya, H. Yoshida, Characterization of autofluorescence in oral squamous cell carcinoma, Oral Oncol. 39 (2003) 150–156. [10] K. Steenkeste, S. Lécart, A. Deniset, P. Pernot, P. Eschwège, S. Ferlicot, S. LévequeFort, R. Briandet, M.-P. Fontaine-Aupart, Ex vivo fluorescence imaging of normal and malignant urothelial cells to enhance early diagnosis, Photochem. Photobiol. 83 (2007) 1–10. [11] K. Nakanishi, Y. Ohsaki, M. Kurihara, S. Nakao, Y. Fujita, K. Takeyama, S. Osanai, N. Miyokawa, S. Nakajima, Color auto-fluorescence from cancer lesions: improved detection of central type lung cancer, Lung Cancer 58 (2007) 214–219. [12] I. Jose, K.D. Deodhar, U.B. Desai, S. Bhattacharjee, Early detection of breast cancer: synthesis and characterization of novel target specific NIR-fluorescent estrogen conjugate for molecular optical imaging, J. Fluorescence 21 (2011) 1171–1177. [13] T.-D. Way, C.-J. Chang, C.-W. Lin, Bioconjugated fluorescent polymeric nanoparticles for imaging and targeted therapy of HER2-overexpressing cancer cells, J. Fluorescence 21 (2011) 1669–1676. [14] H.S. Tran Cao, S. Kaushal, R. Menen, C.A. Metildi, C. Lee, C.S. Snyder, S. Horgan, M.A. Talamini, R.M. Hoffman, M. Bouvet, Metastatic pancreatic cancer staged by fluorescence laparoscopy in a fluorescent carcinomatosis mouse model, J. Surg. Res. 165 (2011) 223–228. [15] M. Vibin, R. Vinayakan, A. John, V. Raji, C.S. Rejiya, A. Abraham, Fluorescence imaging of stem cells, cancer cells and semi-thin sections of tissues using silicacoated CdSe quantum, J. Fluorescence 21 (2011) 1365–1370. [16] G. Zhu, H. Ju, H. Zheng, Fluorescence spectroscopic determination of dipyridamole binding on pancreas-1 tumor cell membrane, Clin. Chim. Acta 348 (2004) 101–106. [17] F. Leblond, S.C. Davis, P.A. Valdés, B.W. Pogue, Pre-clinical whole-body fluorescence imaging: review of instruments, methods and applications, J. Photochem. Photobiol. B 98 (2010) 77–94. [18] G.N. Papanicolaou, A new procedure for staining vaginal smears, Science 5 (1942) 432–433. [19] O. Robert, Biological Systems: Structures, Dynamics and Functions. In Regards Croises, Institut Curie, Paris, 2003. [20] C. Diaz, A.J. Schroit, Role of translocases in the generation of phosphatidylserine asymmetry, J. Membr. Biol. 151 (1996) 1–9. [21] R.F.A. Zwaal, P. Comfurius, L.L.M. Van Deenen, Membrane asymmetry and blood coagulation, Nature 268 (1997) 358–360. [22] R.F.A. Zwaal, A.J. Schroit, Pathophysiologic implications of membrane phospholipid asymmetry in blood cells, Blood 89 (1997) 1121–1132. [23] V.A. Fadok, D.R. Voelker, P.A. Campbell, J.J. Cohen, D.L. Bratton, P.M. Henson, Exposure of phosphatidyl serine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages, J. Immunol. 148 (1992) 2207–2216. [24] W.-H. Deng, C. Chen, W.-X. Wang, J. Yu, J.-Y. Li, L. Liu, Effects of ORP 150 on appearance and function of pancreatic beta cells following acute necrotizing pancreatitis, Pathol. Res. Pract. 207 (2011) 370–376. [25] M. Van Engeland, L.J.W. Nieland, F.C.S. Ramaekers, B. Schutte, C.P.M. Reutelingsperger, Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure, Cytometry 31 (1998) 1–9. [26] M.C. Martinez, C. Kunzelmann, J.M. Freyssinet, Plasma membrane remodelling and cell stimulation, Med. Sci. 20 (2004) 189–195. [27] T. Utsugi, A.J. Schroit, J. Connor, C.D. Bucana, Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes, Cancer Res. 51 (1991) 3062–3066. [28] H. Woehlecke, A. Pohl, N. Alder-Baerens, H. Lage, A. Herrmann, Enhanced exposure of phosphatidylserine in human gastric carcinoma cells overexpressing the half-size ABC transporter BCRP (ABCG2), Biochem. J. 376 (2003) 489–495. [29] P.F. Devaux, Static and dynamic lipid asymmetry in cell membranes, Biochemistry 30 (1991) 1163–1173. [30] K.T. Moesta, B. Ebert, T. Handke, D. Nolte, C. Nowak, W.E. Haensch, et al., Protoporphyrin IX occurs naturally in colorectal cancers and their metastases, Cancer Res. 61 (2001) 991–999. [31] A.M. Kluftinger, N.L. Davis, N.F. Quenville, S. Lam, J. Hung, B. Palcic, Detection of squamous cell cancer and pre-cancerous lesions by imaging of tissue
M. Atyaoui et al. / Pathology – Research and Practice 209 (2013) 1–5 autofluorescence in the hamster cheek pouch model, Surg. Oncol. 1 (1992) 183–188. [32] J.M. Salmon, E. Kohen, P. Viallet, J.G. Hirscheberg, A.W. Wouters, C. Kohen, B. Thorell, Microspectrofluorometric approach to the study of free/bound NAD(P)H ratio as metabolic indicator in various cell types, Photochem. Photobiol. 36 (1982) 585–593. [33] N. Ramanujam, M.F. Mitchell, A. Mahadevan, S. Thomsen, A. Malpica, T. Wright, N. Atkinson, R. Richards Kortum, Spectroscopic diagnosis of cervical intraepithelial neoplasia (CIN) in vivo using laser-induced fluorescence spectra at multiple excitation wavelengths, Lasers Surg. Med. 19 (1996) 63–74.
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[34] M. Anidjar, O. Cussenot, J. Blais, O. Bourdon, S. Avrillier, D. Ettori, J.-M. Villette, J. Fiet, P. Teillac, A. Le Duc, Argon laser induced fluorescence may distinguish between normal and tumor human urothelial cells: a microspectrofluorometric study, J. Urol. 155 (1996) 1771–1774. [35] M. Gouterman, Optical spectra and electronic structure of porphyrins and related rings, in: D. Dolphin (Ed.), The Porphyrins, vol. III, Academic Press, New York, 1978. [36] F. Adar, Electronic absorption spectra of hemes and hemoproteins, in: D. Dolphin (Ed.), The Porphyrins, vol. III, Academic Press, New York, 1978. [37] S. Huang, A.A. Heikal, W.W. Webb, Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein, Biophys. J. 82 (2002) 2811–2825.
Contents lists available at SciVerse ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original article
Fluorescence spectroscopy and imaging to improve diagnosis of normal and tumoral cytological pancreatic cells Malek Atyaoui a,b,c,∗ , Wissem Dimassi b , Nouemen Tounsi c , Nejm Eddine Jaidan a , Hatem Ezzaouia b a b c
Laboratoire de Spectroscopie Atomique, Moléculaire et Applications, Université Tunis El Manar, Tunis, Tunisia Laboratoire de Photovoltaïque, Centre de recherches et des technologies de l’énergie, technopole de Borj-Cédria, PB: 95, Hammam Lif 2050, Tunis, Tunisia Ecole supérieure des sciences et techniques de Tunis, Université de Tunis, Tunisia
a r t i c l e
i n f o
Article history: Received 3 May 2012 Received in revised form 11 July 2012 Accepted 28 August 2012 Keywords: Fluorescence Cytological cells Tumoral cells Normal cells Pancreas
a b s t r a c t We investigated a simple and effective diagnostic method for normal and malignant pancreatic cells using fluorescence emission of endogenous and exogenous molecules in cytological cells. Fluorescence imaging was performed to assess the spectral properties and the spatial distribution of the fluorescence emitted by pancreatic cells. The results revealed quite different fluorescence distributions between tumor cells, characterized by perimembrane fluorescence localization, and normal cells exhibiting an intracellular fluorescence. This was not caused by differences in the fluorescence emission of the endogenous fluorophores NAD(P)H or porphyrins but by various localizations of the exogenous molecules (the EA 50 Papanicolaou stain). © 2012 Elsevier GmbH. All rights reserved.
Introduction
Materials and methods
Pancreatic cancer leads to death in industrialized countries. Its incidence is high [1,2], and is caused by early metastasis and destructive growth along the endothelium basement membrane and neurons [3]. Most patients with pancreatic cancer have a poor outcome due to the absence of early diagnosis and its highly invasive and metastatic features, which leaves more than 85% of the patients inoperable at the time of diagnosis. Therefore, the application of new approaches and new methods is necessary to identify and detect cancer signatures. Fluorescence has proven to be a versatile tool for studying molecular interactions in analytical chemistry, biochemistry, cell biology, photochemistry, and environmental science [4–6]. It has been recently used for cancer diagnosis [7–15]. The fluorescence emitted by the endogenous and exogenous molecules of cytological pancreatic cells is worth studying in order to improve prognosis and to discriminate between normal and tumoral pancreatic cells. In this work, discrimination between normal and tumoral pancreatic cells was possible by the activation of EA 50 stain. The fluorescence imaging of normal and tumor cells is presented and discussed.
Preparation of cytological slides
∗ Corresponding author at: Laboratoire de Spectroscopie Atomique, Moléculaire et Applications, Université Tunis El Manar, Tunis, Tunisia. Tel.: +216 94 088 223. E-mail address: [email protected] (M. Atyaoui). 0344-0338/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2012.08.007
Pancreatic cytological slides were transferred to the anatomopathological department for cytospinning and staining using the classical Papanicolaou technique [16]. The use of this protocol is necessary to identify the different cell elements (nucleus, cytoplasm, cellular membrane) and to activate their fluorescent molecule. Using this method, three stain mixtures were applied for cell staining with different locations: Harris hematoxylin, specific of cell nucleus staining, orange G6 (OG 6), which interacts with cytoplasmic keratin, and EA 50, a trichromatic stain containing light green, yellow eosin and Bismarck brown with non-specific localization. More details concerning the photophysical properties of the Papanicolaou stains are given elsewhere [10]. Wavelength-resolved confocal laser scanning microscopy Confocal fluorescence images were acquired with a commercial device (Leica TCS SP2 or SP5 AOBS, Mannheim, Germany). A Leica objective (63×/1.4–0.60 NA, oil immersion) was used for acquiring images in direct slow scanning mode. It was checked that the immersion oil (Cargille, Cedar Grove) did not fluoresce over all the spectral range 400–800 nm irrespective of the fluorescence excitation wavelength used. Fluorescence signatures from pancreatic cells, in terms of intensity, spectra and images were obtained under excitation either with the 364-nm line of argon
2
M. Atyaoui et al. / Pathology – Research and Practice 209 (2013) 1–5
Table 1 Transmission and fluorescence intensity images of papanicolaou-stained pancreatic normal cells. Transmission
Fluorescence
Normal cells
laser and 633-nm line of a helium-neon laser (average laser power ∼90 W) or with the 488-nm line of argon lasers (average laser power ∼3 W) to characterize the fluorescence of endogenous fluorophores, NAD(P)H and porphyrins, and Papanicolaou stains, respectively. For spectral measurements, the epifluorescence was imaged on a photomultiplier with a 10-nm spectral slit which was moved by 2-nm steps.
pancreatic cells on the basis of their fluorescence localization: in normal cells, fluorescence intensity was found inside the cell with a higher intensity measured in the cytoplasm than in the nucleus (Table 1), whereas in tumor cells, the fluorescence was localized mainly on the perimembrane surface (Table 2). On the other hand, a fluorescence spectroscopy was performed in order to identify the fluorescent molecule responsible for the above mentioned difference in fluorescence distribution. Fig. 1a and b shows the fluorescence spectra of tumor cells and normal pancreatic cells in different locations (cytoplasm, nucleus and perimembrane surface) recorded in the wavelength range (525–609 nm) and under excitation with the 488 nm Ar laser line of the confocal device. The fluorescence spectra of the normal cells and tumor cells showed identical emission peak positions centered at around 550 nm. The highest fluorescence intensity of the normal cell was obtained in the cytoplasm (Fig. 1a), but was recorded on the perimembrane surface for the malignant cell (Fig. 1b). Steenkeste and Lécart [10] reported that the red emission band centered at around 550 nm in the fluorescence spectrum of urothelial cells was attributed to the presence of EA 50 stain. Therefore, on the basis of the above fluorescence spectra and images, we conclude that the signature of the EA 50 exogenous molecule may be just a clue or phenomenon regarding the separation between normal and tumor cells. There was no cause nor did this affect the relationship. On the other hand, many authors [17–24] reported that for pathological cells or for cells under various stimulations, such as pro-inflammatory, pro-apoptosis, or pro-coagulating signals,
(a) 200
Nucleus Cytoplasm Perimembrane surface
Results and discussion In order to observe the possible change in the fluorescence distribution or fluorescence localization of the obtained samples (tumoral and normal cells), we performed fluorescence imaging (Tables 1 and 2) in the wavelength range of (525–609 nm) with an excitation wavelength of 488 nm. As shown in the two tables, it is obvious that the tumor cells can be distinguished from the normal
Fluorescence intensity (u.a).
180 160 140 120 100
Table 2 Transmission and fluorescence intensity images of papanicolaou-stained pancreatic tumoral cells. Transmission
80 60 40 20 0
520
540
Fluorescence
560
580
Wavelength (nm)
600
620
(b) 55
Nucleus Cytoplasm Perimembrane surface
Tumoral cells
Fluorescence intensity (u.a).
50 45 40 35 30 25 20 15 10 5 520
540
560
580
600
620
Wavelength (nm) Fig. 1. Fluorescence spectrum of cytological pancreatic cells obtained under 488 nm excitation: (a) normal cells and (b) tumor cells.
M. Atyaoui et al. / Pathology – Research and Practice 209 (2013) 1–5 140
0
2
4
6
8
0
10 10
3
2
4
6
8
10 10
80 120
70
100 80
6
Perimembrane intensity of Tumoral Cells 60 4
Perimembrane intensity of Normal Cells
40 20
2
Fluorescence intensity (u.a).
Fluorescence Intensity (u.a)
8
8
60 6
50 40
4 30 20
2
10 0 1
2
3
4
5
6
7
8
9
0
Number of serial Fluorescence tests on 18 pancreatic cells Fig. 2. Serial fluorescence blind tests on 18 pancreatic cells under 488 nm excitation. 9 were classified as normal and 9 as cancerous.
the membrane phospholipid bilayer is restructured: a loss of the membrane asymmetry is observed and correlated to the externalization of phosphatidylserines [25–27]. Therefore, the fluorescence on the perimembrane surface of tumor cells can be attributed to the restructuration in the membrane phospholipid bilayer. The reproducibility of the two above remarkable fluorescence patterns of normal and tumor cells was investigated by repeating the fluorescence intensity measurements on 18 Papanicolaoustained pancreatic cells: 9 were classified as normal cells and 9 as tumoral pancreatic cells. As shown in Fig. 2, there was no change in the fluorescence behavior of the two types of cells (normal and tumoral): all the tumor cells had higher perimembrane fluorescence intensity compared to normal cells. The fluorescence intensity measured on the perimembrane surface of tumor cells was almost 1572 ± 166% times higher than that in normal cells. The error limit of the measurement was determined by repeating the analysis three times per sample. Autofluorescence of cells is produced by endogenous molecules that naturally occur in cells after excitation with a suitable wavelength. The presence of disease changes the concentration of the
0
640
660
680
700
720
740
760
Wavelength (nm) Fig. 3. Fluorescence emission of porphyrins under 633 nm excitation.
endogenous molecules, as well as the light scattering and absorption properties of the tissue or the cells, due to changes in a.o. blood concentration, nuclear size distribution, collagen content and epithelial thickness [28–31,9,32]. Therefore, the activation of the endogenous molecules (NAD(P)H, porphyrins) is necessary to improve the discrimination between normal and pancreatic tumor cells. Fluorescence properties of the endoporphyrins of cytological pancreatic cells have been detailed in the wavelength range 643–750 nm. In this spectral range, there is no overlap with the emission of EA50. The endoporphyrin fluorescence images revealed for each cell type (tumoral and normal) a similar trend: an intracellular distribution of the fluorescence with a higher intensity in the cytoplasm than in the nucleus as shown in Table 3. The corresponding spectra obtained under 633-nm excitation are shown in Fig. 3. Irrespective of the cell type, normal or tumoral, the emission was characterized by two main bands centered at about 675 and 700 nm with a shoulder around 740 nm in agreement with the fluorescence properties of porphyrins [33,34]. The fluorescence spectrum shown in Fig. 4 was obtained after excitation of the NAD(P)H with the 364-nm line of argon laser.
Table 3 Transmission and fluorescence intensity images of papanicolaou-stained pancreatic cells. (a) Tumoral cells and (b) normal cells. Fluorescence intensity images in the 525–609 nm spectral range obtained after activation of EA 50 and in the 643–750 nm spectral range obtained after excitation of porphyrins. Transmission
Tumoral cells (A)
Normal cells (B)
Fluorescence 525–609 nm range
Fluorescence 643–750 nm range
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Fluorescence intensity (u.a).
8 10 6
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Wavelength (nm) Fig. 4. Fluorescence emission of NAD(P)H under 364 nm excitation.
The NAD(P)H emission coincides well with the in vitro spectrum of NADH [35–37] with a maximum at 460 nm. No changes were found in the emission spectral shape and intensity between normal and tumor cells. Unfortunately, the fluorescence signal of NAD(P)H is low, so that fluorescence images could not be recorded. As observed in the fluorescence spectrum and images (Table 3 and Figs. 3 and 4), it was not possible to discriminate between normal and tumoral cytological pancreatic cells from the autofluorescence emission. Conclusion The aim of this study was to excite endogenous and exogenous molecules in cytological pancreatic cells and, consequently, to use their fluorescence properties in order to improve the diagnosis of normal cells and tumor cells. Fluorescence spectroscopy and imaging showed that the tumor cells and normal cells have different spatial fluorescence distributions. After the EA 50 activation, the fluorescence images showed two fluorescence locations in the cells, one intracellular location for the normal cells, and another perimembrane surface location for the tumor cells. On the other hand, it is not efficient to excite endogenous molecules like NAD(P)H and porphyrins of normal cells and tumor cells. There is a similar trend: the autofluorescence images showed for each cell type (tumoral and normal) an intracellular distribution of the fluorescence. This detection method is now in clinical progress, and the discussion on the diagnostic value of the EA 50 activation in other types of cancer and other pancreatic diseases, such as pancreatitis, should be the topic of future studies. Acknowledgements The authors thank the Centre de Photonique Biomédicale, Centre Laser de l’Université Paris-Sud, Orsay, for supporting the analysis of fluorescence spectroscopy and imaging. The authors are also grateful to Service d’Anatomie Cytologie Pathologiques, Hopital de Bicetre, Le Kremlin-Bicetre, France, for preparing the samples and for their help. References [1] A.B. Lowenfels, P. Maisonneuve, Epidemiology and risk factors for pancreatic cancer, Best Pract. Res. Clin. Gastroenterol. 20 (2006) 197. [2] M.T. Carriaga, D.E. Henson, Liver, gallbladder, extrahepatic bile ducts, and pancreas, Cancer 75 (1995) 171–190.
[3] S.A. Eccles, D.R. Welch, Metastasis: recent discoveries and novel treatment strategies, Lancet 369 (2007) 1742. [4] V. Masilamani, K. Al-Zhrani, M. Al-Salhi, A. Al-Diabb, M. Al-Ageilyc, Cancer diagnosis by autofluorescence of blood components, J. Lumin. 109 (2004) 143–154. [5] M.S. Khot, N.K. Desai, G.B. Kolekar, S.R. Patil, Fluorescence enhancement effect for the determination of adenosine 5 -monophosphate with 9-anthracene carboxylic acid–cetyl trimethyl ammonium bromide system, J. Fluorescence 21 (2011) 1997–2003. [6] X. Gao, L. Niu, X. Su, Detection of DNA via the fluorescence quenching of Mn-doped ZnSe D-dots/doxorubicin/DNA ternary complexes system, J. Fluorescence 22 (2012) 103–109. [7] A.M. Kluftinger, N.L. Davis, N.F. Quenvillei, S. Lam, J. Hung, B. Palcic, Detection of squamous cell cancer and pre-cancerous lesions by imaging of tissue autofluorescence in the hamster cheek pouch model, Surg. Oncol. 1 (1992) 183–188. [8] M. Sato, A. Sakurada, M. Sagawa, M. Minowa, H. Takahashi, T. Oyaizu, Y. Okada, Y. Matsumura, T. Tanita, T. Kondo, Diagnostic results before and after introduction of autofluorescence bronchoscopy in patients suspected of having lung cancer detected by sputum cytology in lung cancer mass screening, Lung Cancer 32 (2001) 247–253. [9] K. Onizawa, N. Okamura, H. Saginoya, H. Yoshida, Characterization of autofluorescence in oral squamous cell carcinoma, Oral Oncol. 39 (2003) 150–156. [10] K. Steenkeste, S. Lécart, A. Deniset, P. Pernot, P. Eschwège, S. Ferlicot, S. LévequeFort, R. Briandet, M.-P. Fontaine-Aupart, Ex vivo fluorescence imaging of normal and malignant urothelial cells to enhance early diagnosis, Photochem. Photobiol. 83 (2007) 1–10. [11] K. Nakanishi, Y. Ohsaki, M. Kurihara, S. Nakao, Y. Fujita, K. Takeyama, S. Osanai, N. Miyokawa, S. Nakajima, Color auto-fluorescence from cancer lesions: improved detection of central type lung cancer, Lung Cancer 58 (2007) 214–219. [12] I. Jose, K.D. Deodhar, U.B. Desai, S. Bhattacharjee, Early detection of breast cancer: synthesis and characterization of novel target specific NIR-fluorescent estrogen conjugate for molecular optical imaging, J. Fluorescence 21 (2011) 1171–1177. [13] T.-D. Way, C.-J. Chang, C.-W. Lin, Bioconjugated fluorescent polymeric nanoparticles for imaging and targeted therapy of HER2-overexpressing cancer cells, J. Fluorescence 21 (2011) 1669–1676. [14] H.S. Tran Cao, S. Kaushal, R. Menen, C.A. Metildi, C. Lee, C.S. Snyder, S. Horgan, M.A. Talamini, R.M. Hoffman, M. Bouvet, Metastatic pancreatic cancer staged by fluorescence laparoscopy in a fluorescent carcinomatosis mouse model, J. Surg. Res. 165 (2011) 223–228. [15] M. Vibin, R. Vinayakan, A. John, V. Raji, C.S. Rejiya, A. Abraham, Fluorescence imaging of stem cells, cancer cells and semi-thin sections of tissues using silicacoated CdSe quantum, J. Fluorescence 21 (2011) 1365–1370. [16] G. Zhu, H. Ju, H. Zheng, Fluorescence spectroscopic determination of dipyridamole binding on pancreas-1 tumor cell membrane, Clin. Chim. Acta 348 (2004) 101–106. [17] F. Leblond, S.C. Davis, P.A. Valdés, B.W. Pogue, Pre-clinical whole-body fluorescence imaging: review of instruments, methods and applications, J. Photochem. Photobiol. B 98 (2010) 77–94. [18] G.N. Papanicolaou, A new procedure for staining vaginal smears, Science 5 (1942) 432–433. [19] O. Robert, Biological Systems: Structures, Dynamics and Functions. In Regards Croises, Institut Curie, Paris, 2003. [20] C. Diaz, A.J. Schroit, Role of translocases in the generation of phosphatidylserine asymmetry, J. Membr. Biol. 151 (1996) 1–9. [21] R.F.A. Zwaal, P. Comfurius, L.L.M. Van Deenen, Membrane asymmetry and blood coagulation, Nature 268 (1997) 358–360. [22] R.F.A. Zwaal, A.J. Schroit, Pathophysiologic implications of membrane phospholipid asymmetry in blood cells, Blood 89 (1997) 1121–1132. [23] V.A. Fadok, D.R. Voelker, P.A. Campbell, J.J. Cohen, D.L. Bratton, P.M. Henson, Exposure of phosphatidyl serine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages, J. Immunol. 148 (1992) 2207–2216. [24] W.-H. Deng, C. Chen, W.-X. Wang, J. Yu, J.-Y. Li, L. Liu, Effects of ORP 150 on appearance and function of pancreatic beta cells following acute necrotizing pancreatitis, Pathol. Res. Pract. 207 (2011) 370–376. [25] M. Van Engeland, L.J.W. Nieland, F.C.S. Ramaekers, B. Schutte, C.P.M. Reutelingsperger, Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure, Cytometry 31 (1998) 1–9. [26] M.C. Martinez, C. Kunzelmann, J.M. Freyssinet, Plasma membrane remodelling and cell stimulation, Med. Sci. 20 (2004) 189–195. [27] T. Utsugi, A.J. Schroit, J. Connor, C.D. Bucana, Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes, Cancer Res. 51 (1991) 3062–3066. [28] H. Woehlecke, A. Pohl, N. Alder-Baerens, H. Lage, A. Herrmann, Enhanced exposure of phosphatidylserine in human gastric carcinoma cells overexpressing the half-size ABC transporter BCRP (ABCG2), Biochem. J. 376 (2003) 489–495. [29] P.F. Devaux, Static and dynamic lipid asymmetry in cell membranes, Biochemistry 30 (1991) 1163–1173. [30] K.T. Moesta, B. Ebert, T. Handke, D. Nolte, C. Nowak, W.E. Haensch, et al., Protoporphyrin IX occurs naturally in colorectal cancers and their metastases, Cancer Res. 61 (2001) 991–999. [31] A.M. Kluftinger, N.L. Davis, N.F. Quenville, S. Lam, J. Hung, B. Palcic, Detection of squamous cell cancer and pre-cancerous lesions by imaging of tissue
M. Atyaoui et al. / Pathology – Research and Practice 209 (2013) 1–5 autofluorescence in the hamster cheek pouch model, Surg. Oncol. 1 (1992) 183–188. [32] J.M. Salmon, E. Kohen, P. Viallet, J.G. Hirscheberg, A.W. Wouters, C. Kohen, B. Thorell, Microspectrofluorometric approach to the study of free/bound NAD(P)H ratio as metabolic indicator in various cell types, Photochem. Photobiol. 36 (1982) 585–593. [33] N. Ramanujam, M.F. Mitchell, A. Mahadevan, S. Thomsen, A. Malpica, T. Wright, N. Atkinson, R. Richards Kortum, Spectroscopic diagnosis of cervical intraepithelial neoplasia (CIN) in vivo using laser-induced fluorescence spectra at multiple excitation wavelengths, Lasers Surg. Med. 19 (1996) 63–74.
5
[34] M. Anidjar, O. Cussenot, J. Blais, O. Bourdon, S. Avrillier, D. Ettori, J.-M. Villette, J. Fiet, P. Teillac, A. Le Duc, Argon laser induced fluorescence may distinguish between normal and tumor human urothelial cells: a microspectrofluorometric study, J. Urol. 155 (1996) 1771–1774. [35] M. Gouterman, Optical spectra and electronic structure of porphyrins and related rings, in: D. Dolphin (Ed.), The Porphyrins, vol. III, Academic Press, New York, 1978. [36] F. Adar, Electronic absorption spectra of hemes and hemoproteins, in: D. Dolphin (Ed.), The Porphyrins, vol. III, Academic Press, New York, 1978. [37] S. Huang, A.A. Heikal, W.W. Webb, Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein, Biophys. J. 82 (2002) 2811–2825.