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Observation of Molecular Changes of a Necrotic Tissue from a Murine Carcinoma by Fourier-Transform Infrared Microspectroscopy¹

Departments of Dentistry and Oral Surgery [T. Y., T. Ogaw., T. Ogas., Y. K., K. S.] and Pathology [N. M., M. F.], Fukui Medical University, Matsuoka, Fukui 910-1193, and Application Laboratory Spectroscopic Instruments Division, JASCO Co., Ltd., Tokyo 192-8537 [K. A.], Japan

	ABSTRACT

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Purpose: Our purpose is to develop infrared (IR) microspectroscopy as a new optical diagnostic tool to support conventional lightscopic techniques in investigating the viability of carcinoma tissues and to develop its use in the evaluation of the early effects of anticancer therapy by monitoring the IR spectra in the necrotic area.

Experimental Design: We evaluated the tissue which amassed for 4 weeks after the isotransplantation of mouse squamous cell carcinoma into the thigh of mice. The borders of the necrotic area of frozen tissue specimens were investigated by Fourier-transform IR microspectroscopy and conventional histological staining.

Results: A significantly higher accumulation of cholesterol was observed in the necrotic tissue of a carcinoma. The mechanism of this phenomenon is hitherto unrecognized. We proposed that the accumulated cholesterol may lie extracellularly as a result of the ruptured plasma and internal membranes after the swelling of the necrotic cells brought on by hypoxia. The analysis of the secondary structure of protein revealed that the amounts of ß-sheet increased significantly in striking contrast to the decreasing amounts of -helix in a necrotic area of a carcinoma. It is plausible that this structural conversion of protein was because of lipid-autooxidation products, such as cholesterol oxide but not cholesterol itself, which possesses cell toxicity and could be generated in a necrotic area.

Conclusions: We conclude that it will be possible to evaluate the efficacy of the clinical treatment of carcinoma by monitoring subtle biological changes of cholesterol absorbance in the early stage of necrosis because of anticancer treatment.

	INTRODUCTION

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Over the last century, histological examination has become the standard method in pathology for clearly visualizing various structures in a specimen (1) . Most histological examinations require the histochemical staining of sample sections, and identification of various structures often requires the use of several different staining methods. Moreover, the extraction of detailed information using histology sometimes requires the use of more specific and complex immunohistochemical staining (2) . On the other hand, while performing fixation or staining, some specific property in a specimen might disperse or be denatured. This signals the need in pathology for an advanced diagnostic technique that does not require fixation or staining.

However, FT-IRM³ has an advantage in that it enables the visualization of a molecular component that is invisible in a specimen unless it is stained (2 , 3) . In IR spectroscopy, low-energy photons are used to excite the vibrational motion of covalently bonded moieties by direct absorption of the photon (4) . FT-IRM has become a powerful method for the investigation of complex molecular structures in samples (5 , 6) . FT-IRM has been applied to clinical use in the form of Infrared Molecular Pathology (4) . Pressure-tuning FT-IRM has been applied specifically to human malignant colon cell lines and tissues, where differences of the spectra between normal and malignant tissue were demonstrated, these differences also depending on the degree of differentiation of the carcinoma cell lines (3) . FT-IRM spectra are made of a highly complex combination of overlapping components, because of numerous chemical groups that absorb in a given region of a spectrum (7) . After completion of the spectral data collection, specific band intensities that are identified by the Fourier self-deconvolution or curve-fitting are plotted as a function of spatial coordinates to create FT-IRM images of tissue sections. This process is ideal for creating large maps where thousands of sampling points are required. Therefore, an FT-IRM image can be correlated with an image obtained using a conventional microscopic technique, and attention has increasingly been paid to its biological use (2 , 8) , although FT-IRM has also been applied to technological or industrial fields. FT-IRM allows the investigation of tissue samples that have not had artifacts imposed on them by fixation or staining (2 , 3) .

Many FT-IRM studies have focused on viable malignant cells or tissues (3 , 4 , 9 , 10) . Rigas et al. (3) examined the IR spectroscopic features of human colon adenocarcinoma and found that the relative amount of ß-sheet segment with respect to that of the -helix segment is larger in human colon carcinoma tissue than in that of normal human colon tissue. Mountford et al. (10) reported that an increase in membrane lipids was demonstrated in viable neoplasm tissue and could be a feature of the malignant phenotype. However, little attention has been paid to the spectroscopic approach to necrotic cell death. Necrosis is considered a nonspecific mode of cell death induced by different stimuli, including hypoxia, lytic viral infection, attack by complement, and a variety of toxic chemicals (11) . Cell death caused by necrosis is accompanied by ion and water efflux and dispersal of organelles, resulting in a rupture of cytoplasmic and nuclear membranes, the swelling of mitochondria, and the flocculation of chromatin (11) . It is known that the necrotic core in an untreated carcinoma tissue is a result of hypoxic change (11) . These findings on "necrotic cell death" generate interesting questions: (a) What is the biochemical component in a necrotic tissue? (b) Are there any changes with respect to protein conformation in a necrotic tissue? and (c) What is the mechanism whereby necrotic cell death develops? To address these questions in regard to FT-IRM, an experimental model of regional necrosis in mouse squamous cell carcinoma was needed. Our purpose is to develop IR microspectroscopy as a new optical diagnostic tool to support conventional lightscopic techniques in investigating the viability of carcinoma tissues and to develop its use in the evaluation of the early effects of anticancer therapy by monitoring the IR spectra in the necrotic area. Hence, we scrutinized the changes of molecular components in a necrotic area of well-developed carcinoma and deduced how molecular conformation changes in the necrotic area. We describe here the interesting overlap of FT-IRM findings and histological images stained with H&E and Sudan IV by serial sectioning.

	MATERIALS AND METHODS

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Animals.
C3H/HeNCrj mice (8-week-old, male, 23–25 grams; Charles River Japan, Inc., Yokohama, Japan) were used as the experimental tumor model. All mice were kept under normal laboratory conditions and were given free access to water and food (M. F., Oriental Yeast Co., Ltd., Tokyo, Japan). All mice had been acclimatized to the animal housing facility for 1 week before the start of the experiment. The experiment was approved by the Guidelines for Animal Experiments, Fukui Medical University.

Sample Preparation.
NR-S1 murine squamous cell carcinoma (Ref. 12 ; National Institute of Radiological Sciences, Chiba, Japan) was transplanted by injection via an 18-gauge needle into the thigh when the mice were under anesthesia by ether inhalation. By 2 weeks after the transplantation, their tumors were well vascularized, and the spontaneous necroses were minimal. Regional necrosis was established when the tumor developed in size to 20 x 20 x 10 mm³ at 4 weeks after transplantation. The experimental mice were sacrificed by ether inhalation, and their tumor tissues were removed. An incision of the tumor revealed multiple scattered necrotic areas. The fresh samples were embedded immediately in a Tragacanth Gum (#344-09; Nacalai Tesque Inc., Kyoto, Japan) because the ornithine carbamyl transferase-embedding medium normally used in mounting cryostat sections produces interfering IR absorption (2) . After being instantaneously frozen with liquid nitrogen, serial transverse sections were prepared at 7-µm thickness from the frozen blocks through the entire tumor. Each sample section was set onto a 10 mm-diameter and 0.8 mm-thick barium fluoride IR-transparent substrate window and air dried for 1 h before measurement to prevent interference because of moisture. Neighboring sections were mounted onto glass slides, fixed in 10% formalin, stained with H&E and Sudan IV after the standard methodology (13) , and then examined using a light microscope to identify different microstructures.

FT-IRM Measurement.
Lattice mapping spectra in the 4000–400 cm^-1 range were collected by a JASCO FT-IRM-410M spectrometer equipped with an Irtron IRT-30M IR microscope (JASCO Co., Ltd., Tokyo, Japan), a motorized X-Y-Z stage, and a liquid nitrogen-cooled mercury-cadmium-telluride-detector. A screen image recorder camera attached to the microscope enabled the acquisition of a photomicrograph of the investigated area. The object area imaged by an individual aperture size was 30 x 30 µm². Sequential spectra were collected in 3025 points (55 x 55 points) in the specimen. The area of spectral acquisition amounted to a total of 2.286 mm². For each spectrum, 50 interferograms were collected, signal-averaged, and Fourier-transformed to generate spectra with a resolution of 4 cm^-1 in the transmission mode.

Assignments and FT-IRM Image.
The peak positions of the derived spectra in each necrotic area and area of viable carcinoma tissue were confirmed by Fourier self-deconvoluted and the second-derivative spectra with a band width of 35 cm^-1. The wave numbers of characteristic absorption band in each of the selected spectra were assigned identities (2 , 3 , 5 , 9) . To estimate biochemical components, the obtained data were referred to a computerized spectral library data of the FT-IRM-410M software. To evaluate the membrane lipid components, we created a FT-IRM image by using a spectral calculating software for integrating a group of spectra at 2999–2775 cm^-1, which is a result of C-H symmetric and asymmetric stretching vibration modes of CH₂ and CH₃. The obtained FT-IRM image was correlated to the images obtained by light microscopy after staining with H&E and Sudan IV.

Creation of Images on Protein Secondary Structure Analysis.
We investigated the conformational change of protein secondary structure in the necrotic area by a new spectral analytic program (IR-SSE; JASCO Co., Ltd.). This new analytic program applies singular value decomposition techniques to estimate the mathematical relationship between the secondary structures of known proteins revealed by X-ray and the IR spectra of the known proteins in the amide I region (14) . The results of the conformational information are calculated as a relative percentage and plotted as a function of spatial coordinates to create images that are dependent on the classification of secondary structure types. The program classified the protein conformations into four categories: -helix, ß-sheet, ß-turn, and random coil.

	RESULTS

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H&E Staining Image.
Fig. 1A shows the light micrograph of the central necrotic area. The NR-S1 tumor is composed of poorly differentiated squamous cell carcinoma with central necrosis. Vascular formation was poor in the necrotic area. The viable cancer cells diffusely occupied the rim of the section that was measured by an FT-IRM.

View larger version (70K): [in this window] [in a new window]   Fig. 1. The overlap of FT-IRM and histological images and spectra. A, the light micrograph of the investigated area by H&E staining. The viable cancer cells diffusely occupied the rim of the investigated section (V1 and V2). Note the ghostly cell outlines and absence of nuclear staining in the necrotic region (N1 and N2). Vascular formation was poor in the necrotic area (x40; Bar = 200 µm). B, a light micrograph of the serial section by Sudan IV staining. The central necrotic area shown by H&E staining matched the area stained reddish-brown by Sudan IV, whereas the viable carcinoma tissue apparently corresponded to the negative staining area (x40; Bar = 200 µm). C, the superimposed representative spectra of viable and necrotic tissues of the carcinoma. N1 (red trace), a spectrum of a necrotic area, indicated by "N1" in A and B. V1 (blue trace), a spectrum of a viable area, indicated by "V1" in A and B. D, the superimposed spectra of a cholesterol in the library data (black trace) and necrotic tissue of the carcinoma (red trace). E, a FT-IRM image produced by calculating software for integrating a group of spectra between 2999 and 2775 cm-1; methylene and methyl stretching vibration modes that include typical bands of cholesterol can be seen. Each distance between the small dots is 30 µm. Each mark is common to A and B. Note the similarity of this image to that of the necrotic area shown by Sudan IV staining (x40; Bar = 200 µm). F, the distribution of the -helix structure. The area of the -helix closely matches the image of the negative area demonstrated by the Sudan IV staining. The amount of -helix was significantly higher in the viable carcinoma than in the necrotic area. (B). G, the distribution of the ß-sheet structure. Note the similarity of the distribution of the ß-sheet with the accumulation of cholesterol shown in B and E. The distribution of the ß-sheet structure was in striking contrast with that of the -helix. Each mark in F and G corresponds to staining and peak-integrated images (A–C and E).

Biochemical Components in Necrotic and Viable Tissue of the Carcinoma.
Fig. 1C shows the superimposed spectra of viable tissue and necrotic tissue of the NR-S1 carcinoma. The red trace corresponds to the representative spectra of the necrotic tissue (marked "N1," common use in Fig. 1, A–C ). The blue trace corresponds to the representative spectra of the viable carcinoma tissue (marked "V1," used commonly in Fig. 1, A–C ). FT-IRM spectra shows a distinct difference in peak intensity between the necrotic and viable carcinoma tissue (Fig. 1C) . The different absorption bands between the two spectra included the C-H symmetric and asymmetric stretching vibration modes of CH₂ and CH₃ at 3000–2775 cm^-1, the C = O stretching vibration mode of amide I at 1700–1600 cm^-1, and the C-O ester stretching mode and phosphodiester stretching mode at 1200–1000 cm^-1 (Fig. 1C) .

As shown in Fig. 1D , according to the computerized spectral library in the FT-IRM software, the spectra were attributed to those of cholesterol that bear a strong similarity to the wave numbers and absorption profile (Fig. 1D) . In a comparison between the spectrum of the necrotic area and the library data of the spectrum of cholesterol, the characteristic peaks are attributable to asymmetric and symmetric stretching vibrations of CH₂ and CH₃, the C-H bending of lipid, and the phosphodiester stretching (Fig. 1D) . This indicated that the significant accumulation of lipids in the necrotic area was attributable to the cholesterol (Fig. 1, B–D) .

Fig. 1E shows the FT-IRM image after the integration of the methylene (CH₂) and methyl (CH₃) stretching vibration modes that include typical bands of cholesterol. "V1" and "V2" are the coordinates of viable carcinoma tissue, and "N1 " and "N2 " are the coordinates of the necrotic ones depicting the orientation (Fig. 1E) . Each mark was common in Fig. 1, A–C and E , respectively. This integrated image effectively correlated to the Sudan IV image (Fig. 1, B and E) . Hence, we derived evidence that there are significantly higher accumulations of cholesterol in the necrotic area than in the viable carcinoma tissue.

Conformational Changes of Protein in Necrotic and Viable Tissue of the Carcinoma.
The distribution of the -helix structure is shown in Fig. 1F . The amount of -helix was significantly greater in the viable carcinoma than in the necrotic area. The area of -helix closely matched the image of the area negatively stained by Sudan IV (Fig. 1, B and F) .

As shown in Fig. 1G , the distribution of the ß-sheet structure also significantly reflected the Sudan IV-stained image, as it spread out over the necrotic area (Fig. 1, B and G) . The distribution of the ß-sheet structure correlated with the accumulation of cholesterol (Fig. 1E) and was in striking contrast with that of the -helix (Fig. 1, F and G) . It is interesting to note that the -helix and ß-sheet structure showed a close morphological similarity to those of the specimen. Each mark in Fig. 1, F and G corresponded to staining and peak-integrated images (Fig. 1, A–C and E) .

	DISCUSSION

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Cholesterol is a common sterol that is contained in a cell’s membrane, lysosome, endosome, and Golgi body. As for the cell membrane, cholesterol amounts to 30% of the membrane lipids. The cell lipids play several significant roles in the regulation of membrane function (15) . Our results showed significantly higher accumulations of cholesterol in necrotic tissue. The mechanism of this is hitherto unrecognized. Wyllie and Duvall (11) examined the necrotic core of carcinoma tissue and reported that after tumor development, the core of the tumor presents hypoxic and necrotic change because of poor vascularization or imbalance between the progress of a carcinoma and regional blood perfusion. The findings of their report along with our data might suggest that cholesterol accumulates extracellularly in the necrotic area, which may occur after the rupture of the plasma and internal membranes as a result of swelling of the necrotic cell brought on by hypoxia.

The result obtained by our method revealed the novel finding that the amounts of ß-sheet structure significantly increased in striking contrast to the significantly decreasing -helix structure in a necrotic area. It is plausible that the protein’s structural conversion from an -helix to a ß-sheet was because of lipid-autooxidation products, such as cholesterol oxide (16 , 17) but not cholesterol itself, the former possessing cell toxicity and which could be generated in a necrotic area. Furthermore, the lipid-autooxidation products probably deformed the protein’s secondary structure from an -helix to a ß-sheet.

The stained tissue was processed differently (e.g., using formaldehyde, paraffin, ethanols, and xylenes) than the crude tissues that are used in an FT-IRM study. Hence, FT-IRM has the advantage of potentially elucidating lipid components, such as cholesterol, unlike the conventional staining methods. Moreover, FT-IRM has other advantages in that it can scan rapidly and estimate the secondary structure of the protein. In this study, we proposed the spectral characteristics of FT-IRM imaging that reflect the viability of necrotic carcinoma tissue.

As for Sudan IV staining, the minimum area that it could confirm as necrotic was 42 x 42 µm², whereas for FT-IRM, the aperture size is very small; the minimum area it can confirm as necrotic is 30 x 30 µm². FT-IRM has a sensitivity of detection 1.96 (1.4²) times that of a conventional lightscopic image. FT-IRM can be clinically useful in its ability to evaluate subtle and early necrotic changes and constitutes a new optical tool for the support of conventional lightscopic diagnosis. Although this preliminary study used frozen sections in the transmission mode, FT-IRM allows easy sample preparation (e.g., 1 mm³ of scratched bone or biopsy samples) in the reflectance mode.

We conclude that it will be possible to easily and rapidly evaluate the efficacy of treatment of carcinoma tissue by monitoring subtle biological changes of cholesterol absorbance in the early stage of necrosis because of anticancer treatment, which will be of use in the clinical field.

	ACKNOWLEDGMENTS

 
We thank Drs. Koichi Ando and Sachiko Koike in the National Institute of Radiological Sciences, Chiba, for giving a portion of NR-S1 tumor and Hisataka Kato of the Fukui Medical University for his expert technical assistance. T. Y. thanks the advice of Dr. Joe Zeljko Sostaric of the NIH.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by Grants-in-aid for Scientific Research (B)-(2) 11557116 and (C)-(2) 11672293 and the Fukui Industrial Support Center.

² To whom requests for reprints should be addressed, at Department of Dentistry and Oral Surgery, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan. Phone: 81-776-61-3111, extension 2402 or 2409; Fax: 81-776-61-8128; E-mail: sano{at}fmsrsa.fukui-med.ac.jp

³ The abbreviations used are: FT-IRM, Fourier-transform infrared microspectroscopy; IR, infrared.

Received 8/27/01; revised 3/ 4/02; accepted 3/19/02.

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