Probing cell proliferation in the human colon using vibrational spectroscopy: a novel use of FTIR-microspectroscopy

Vibrational Spectroscopy 34 (2004) 301–308

Probing cell proliferation in the human colon using vibrational spectroscopy: a novel use of FTIR-microspectroscopy A. Salmana, R.K. Sahua, E. Bernshtainb, U. Zeliga, J. Goldsteinc, S. Walfischd, S. Argovc, S. Mordechaia,* a

Department of Physics, Ben Gurion University, Beer-Sheva 84105, Israel Institute of Pathology, Ben Gurion University, Beer-Sheva 84105, Israel c Department of Pathology, Soroka University Medical Center, Beer-Sheva 84105, Israel d Colorectal Unit, Soroka University Medical Center, Beer-Sheva 84105, Israel b

Received 16 November 2003; received in revised form 16 November 2003; accepted 19 January 2004

Abstract Recently, Fourier transform infrared (FTIR)-spectroscopy has been used to monitor cell growth by several works. Conventionally, the study of cell and tissue dynamics at molecular levels is carried out through various approaches like histochemical methods, application of molecular biology and immunology. Colonic crypts display a pattern in cell growth along their height. Histologically normal sections obtained from formalin fixed biopsies of colon cancer patients were studied in the present work through vibrational spectroscopy. The evolution and development of the normal human colonic crypts manifested in Fourier transform infrared-microspectroscopy (FTIR-MSP) as spectral changes in the levels of nucleic acids, proteins, carbohydrates and lipids. The results indicate that the level of carbohydrates, nucleic acids and lipids increases only till the middle of the crypt up to which the maturation zone is restricted and thereafter decreases till the top where the cells are exfoliated. These observations are in coherence with earlier reports on crypt proliferation. We identify the normal pattern of various biochemicals along the colonic crypt based on data analyzed from FTIR-MSP. This study affords an important example of the application of microscopic vibrational spectroscopy for understanding basic cell processes from formalin fixed tissues where in vivo studies and immunological methods are not feasible. # 2004 Elsevier B.V. All rights reserved. Keywords: Crypt proliferation; Cell dynamics; Fourier transform infrared microspectroscopy; Metabolites; Vibrational spectroscopy

1. Introduction The use of Fourier transform infrared (FTIR) to monitor biochemical changes in living cells has gained considerable importance in recent years [1,2]. The changes in the cells and tissues, which are subtle and often not obvious in the histopathological studies, are shown to be well resolved using Fourier transform infrared-microspectroscopy (FTIRMSP) and FTIR-spectroscopy [3,4]. Moreover, most techniques designed to detect changes in the cells identify the level of one or a few metabolites. In the case of FTIR however it is possible to identify the changes in the levels of various cellular biochemicals simultaneously under in vivo and in vitro conditions as the different metabolites absorb the IR at different characteristic wavenumber [5,6]. FTIR-MSP can be used to study changes in levels of various metabolites lacking other spectroscopic properties such as * Corresponding author. Tel.: þ972-8-6461749; fax: þ972-8-6472903. E-mail address: [email protected] (S. Mordechai).

0924-2031/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2004.01.009

fluorescence and immunoreactions during processes like cell maturation and tissue differentiation [7,8]. It can also help to identify subtle biochemical changes, which are not apparent in the histological studies. The maturation and migration of cells in the colonic crypt hold important clues to the origin of the premalignant and malignant stages of cancer. Abnormal cell proliferation and biological changes have been known to be an indicator of the initiation of malignancy [9]. Though certain markers have been used to identify these abnormal cell proliferation [9–11] the actual maturation of the crypt in terms of total biochemical changes has not been established. The occurrence of abnormality in crypts was also detected using enzymes as markers [12]. FTIR-microspectroscopy has been shown to provide important indications regarding the changes in the biochemical composition of cells and tissues [13]. It has the potential to differentiate between normal and abnormal cells [14] and holds promises for diagnosis of diseases in vivo. In the present study, we for the first time, study the application of FTIR-MSP to analyze the biochemical changes

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occurring along the height of the human colonic crypt, which is a well-established histopathological system. We study the efficiency of the technique to reveal details of crypt biochemistry in comparison to conventional histology studies. Studies using exfoliated cells and tissues from the colon are difficult due to large amounts of debris, which create problems in sampling. We overcome this by taking biopsies from colon cancer patients and studying them in a non-destructive technique so that the histopathological features are well represented. These changes along the crypt were reported earlier using conventional techniques [15,16]. We compare the apex and the base of the crypts under the same conditions for various biochemical components to conclusively draw a relation between the changes and cell growth and death. The maturation of cells from the cervical epithelium and the biochemical changes has been identified by the use of FTIR spectroscopy of the tissues [7,8]. Vibrational spectroscopy has been also used to monitor cell cycle by several workers [2,17–19]. However, no such work has been done on the colonic crypts using FTIR-MSP to elucidate the biochemical changes associated with the migration of the cells. It is known that the venerable histopathological methods are much more sensitive in these types of studies than FTIR as the molecular biological approaches reach the basis of the changes, while FTIR provides a gross evaluation of the biochemical conditions. However, these techniques are not adequate or suitable in instances where molecular changes have already occurred during the tissue processing and fixation. In this study, we show that such type of processing not withstanding FTIR spectroscopy can still reflect the relative changes in the tissues during processes such as crypt proliferation. This is especially true with nucleic acids, which increase during cell growth and division and have absorbance at specific wavenumbers in the mid IR region. The nucleic acids being large molecules are not susceptible to removal during fixation and thus, can be monitored. Therefore, the changes in RNA and/or DNA levels are used as a parameter to monitor cell proliferation. The present study, attempts to understand the normal changes that occur during the development of colonic crypt using FTIR-MSP. Such information can be used in later stages for diagnoses of abnormal crypt development in vitro and possibly for regular monitoring of patients who are prone to colonic cancers due to heredity and other factors. At present, no method can specifically detect lipid variations along the crypt from paraffin embedded tissues. We show that such information on the lipid levels along the crypt can also be deduced from the vibrational spectroscopy data.

2. Materials and methods 2.1. Sample preparation Formalin fixed, paraffin-embedded tissues from 20 colon cancer patients with different Astler Coller classification

grades were retrieved with their consent from the histopathology files of Soroka University Medical Center (SUMC), Beer-Sheva. The tissue samples used in this study were selected at maximum possible distance away from cancer sites and include clear normal crypts. The method of Argov et al. [20], was followed for sample preparation. Two adjacent paraffin sections were cut from each biopsy; one was placed on zinc–selenium slide and the other on glass slide. This procedure was carefully followed to assure that the tissue sections were practically identical. The thickness of the tissue samples was 10 mm. The first slide was deparaffinized using xylol and alcohol and was used for FTIR measurements. The second slide was stained with haematoxylin and eosin for online parallel histology review. 2.2. FTIR-Microspectroscopy An expert pathologist examined the tissue histology to assure accurate determination of the measured microscopic site and confirms the absence of any premalignant condition, which can be observed through the histological staining. The areas of measurements were taken along the entire height of the crypt keeping in view the gradual migration of cells from base to the apex as shown in Fig. 1. Measurements on biopsies were performed using the FTIR microscope, IRscope II, with a liquid nitrogen cooled mercury– cadmium–telluride (MCT) detector, coupled to the FTIRspectrometer (Bruker Equinox model 55, OPUS software) following the method of Salman et al. [21]. During each measurement, the area of the measured sites was about 40 mm  40 mm, which included few cells. In our study, 60 crypts from 20 different patients were measured. For each crypt, 11 sites along the entire height were measured to monitor gradual changes. To achieve high signal-to-noise ratio (SNR) 128 co-added scans were collected in the wavenumber region 600–4000 cm1 in each measurement. The spectra were baseline corrected using rubber band method and normalized to amide I for the whole range using OPUS software. For subsequent calculations and analyses, each wavenumber region under study was cut and baseline corrected again using the same method to remove any contribution from artifacts. For example, while calculating the change in absorbance due to carbohydrates, phosphates etc in the region 900–1185 cm1 the spectral region was cut and baseline corrected such that the spectral baseline was offset at zero level. We note that alternate normalization methods such as vector normalization or normalization to amide II did not significantly alter the results. To calculate the 2852 cm1 band intensity (corresponding to CH2 symmetric stretching vibrations from membrane lipids) the region 2700–3000 cm1 of the spectra were cut. The cut spectra were normalized to the 2920 cm1 band (corresponding to CH2 stretching vibrations from lipids and proteins which is the maximum peak in this region).

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Fig. 1. (a) Histological longitudinal section of the human colon stained with haematoxylin-eosin showing the crypts and the measurements sites in FTIRMSP. The symbols represent: (S) stroma, (B) base of the crypt, (T) top of the crypt, (L) lumen, (M1, M2, M3 . . . Mn) measurement sites along the crypt taken consecutively where M1 is the measurement at the base and Mn is the measurement at site number n. The arrow indicates the direction of the FTIR-MSP measurements. The magnification is 100. (b) Schematic diagram of the crypt indicating the measurements sites and cell migration.

3. Results and analysis A typical longitudinal histological section of a crypt is shown in Fig. 1a. The measurements were taken along the outer surface of the crypt, as indicated in Fig. 1a and the schematic diagram (Fig. 1b), to prevent any spurious absorbance due to proximity to the lumen of the crypt.

Representative spectra from the different regions of the crypt are shown in Fig. 2. The numerical labels refer to the measurement site along the crypt as shown in Fig. 1. All spectra were normalized with respect to amide I band. The arrow indicates the direction of the measurements where 6 represents a site near the middle of the crypt. The spectra show an increase in the absorbance intensity at most of the

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(a)

6 4 3 2 1

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Wavenumber (cm-1) Fig. 2. (a) Mid-IR spectra of various sites along the colonic crypt in the region 800–1800 cm1. The numbers (traces 1–6) indicate the position starting from the base of the crypt as described in (b). (b) Same as (a) but showing the decrease in absorbance from the mid region to the apex of the crypt (traces 6–11). The inset is the expanded 1700–1750 cm1 region.

wavenumbers as we progress form the base to the middle of the crypt (Fig. 2a), and then again a decrease from the middle to the top (Fig. 2b). This feature is common to the regions between 2800–3000 cm1, 1200–1500 cm1 and 1000–1200 cm1 where the components like lipids, phospholipids, carbohydrates and phosphate are known to absorb in the Mid-IR [6]. Thus, the changes in the spectra at these regions signify the relative changes in the cells with respect to these components. It shows that the metabolic activity of the cells increases in the lower half of the crypt. Similarly in Fig. 2b it is seen that the sites towards the top have decreasing absorbance relative to the middle in the above wavenumbers. The changes in the metabolites are studied in various wavenumber regions of the spectra where they have characteristic absorptions. The deduced absorbance values are estimated after normalization to the amide I band. Since we study the changes along the height of the crypt relative to the composition of the cells at the base of the crypt, the choice of the normalization method does not alter the study processes

under consideration. This also alleviates the complete deconvolution of all metabolites and their individual contribution when there is an overlap in the spectra. The absorption in the region between 800 and 1185 cm1 occurs predominantly due to the stretching vibration of C–C and C–O and deformation of C–O–H and C–O–C bonds present in carbohydrates. The changes in the glycogen (carbohydrate) level at different distances from the base of the crypt are indicated by the absorbance spectra in the region 900–1185 cm1 (Fig. 3a). The figure shows the results for a single representative crypt with normal variation in metabolites as an example. The results were similar for other normal crypts from this and other patients where the levels of metabolites varied in a manner reported in literature. It is seen that there is an increase in carbohydrate level up to the middle of the crypt showing increasing storage of the carbohydrate (traces 1, 3 and 6). Thereafter, the carbohydrate content again decreases up to the apex of the crypt (traces 6, 10 and 11). This distribution is similar along both sides of the crypt showing that the cell carbohydrate

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Wavenamber (cm ) Fig. 3. (a) Expansion of the spectra presented in (a) in the wavenumber region 800–1300 cm1, presenting the built up of carbohydrates (900–1200 cm1) and phosphate (1000–1100 and 1200–1300 cm1) from base to the apex of the crypt. The labels are the same as in Fig. 1. (b) Expansion of the spectra in the wavenumber region 1300–1475 cm1 displaying the build up of phospholipids (1350–1475 cm1) from the base to the apex of the crypt. The labels are same as in Fig. 1. The spectra were baseline corrected in this region using OPUS software.

(glycogen) content increases from the base to the middle and then decreases to the top. As colonic crypts secret glycoproteins to the lumen this could also be due to secretion of mucin and carbohydrate containing glycoproteins from the cells. We note that absorbance in this region can also be due to several other components like ATP, phosphates and other energy metabolites along with nucleic acids. The increased or decreased absorbance signifies the energy metabolism as well as synthesis related to proliferation. The absorption due to the symmetric and antisymmetric bending vibrations of CH3 and CH2 groups, which are present in the lipids, triglycerides, proteins, phospholipids and other molecules occurs in the region 1350–1475 cm1. Thus, the variations in the content of these molecules in the cell which are an essential part of the membranes of cells and cell organelles is seen in the changes occurring in this region (Fig 3b). Since phospholipids are necessary for formation of

cell membrane and organelles during cell division, this may reflect active cell division phase of the cells and also formation of organelles. It is also known that nucleic acids can contribute to absorbance in this region. The relative quantitative variations of the metabolites calculated from the area under the peaks (integrated absorbance) are shown in Fig. 4a-c for specific regions, where the various biochemicals have characteristic absorbance as reported in the literature [5,6]. All the presented data are normalized consistently with respect to the base of crypt (location number 1). The figure shows the data for crypts from three different candidates. Fig. 4a shows the glycogen (carbohydrate) variation calculated from absorbance in the region 900–1175 cm1 as a ratio relative to the base of the crypt. The phosphate levels measured by integrating the antisymmetric phosphate region (1175–1300 cm1) are shown in Fig. 4b. Fig. 4c shows integrated absorbance

Absorbance (n) / Absorbance (1)

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Location along the crypt Fig. 5. Changes in the levels of various metabolites as calculated from the spectra of a single crypt at various sites. The symbols represent the predominant metabolite: carbohydrate (glycogen) ( ), phosphate (mainly from antisymmetric stretching vibrations) (!) and phospholipids (&). The ratios were calculated with respect to the integrated absorbance of the basal measurement (site number 1).

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Location along the crypt Fig. 4. (a) Relative changes in the carbohydrate and phosphates content along the crypt compared to the base as quantified from analysis of the integrated absorbance in the region 900–1175 cm1 for three different candidates. The dashed line indicates the trend when no rubber band correction is undertaken and the area is calculated from the normalized spectra. (b) Relative changes in the phosphate content along the crypt compared to the base as quantified from analysis of the integrated absorbance in the region 1175–1300 cm1 due to antisymmetric phosphate vibrations. (c) Relative changes in the phospholipid and proteins content along the crypt compared to the base quantified from the analysis of the integrated absorbance in the region 1350–1475 cm1.

between 1350 and 1475 cm1. It is seen that there is an increase in the content of the metabolites up to the middle of the crypt and thereafter it begins to decline. We assume that there is a constant proportionality between the symmetric and antisymmetric regions, and thus, the changes in the

symmetric phosphate absorbance vibrations (which are obscured due to predominance of carbohydrates and phosphate compounds in the region 800–1200 cm1) have also a similar behavior. The growth pattern of the crypts has been previously studied [22,23]. While earlier studies related to crypt proliferation [24] have associated only changes in the nucleus as an indicator of cell growth, we show that other components also change in parallel during the growth and maturation, which is a better indicator of normal or abnormal metabolism of the tissues. The comparative changes in the levels of different metabolites for a representative crypt from an additional patient are presented in Fig. 5. It is seen that the carbohydrate (glycogen) content increases up to the middle of the crypt and then decreases (solid circles). The increase and decrease is very sharp compared to the phosphates (inverted solid triangles). This is obvious because the glycogen is used for respiration while the cell migrates towards the apex and exfoliates. On the other hand the phospholipid levels (which are mainly found in the membranes) is decreased gradually very much like the phosphate level (open squares) possibly due to onset of apoptosis [25]. The variation in the phosphate levels denoting the antisymmetric phosphate vibrations, are similar to the variation in the phospholipid levels as shown in Fig. 4a and b. It is not known if this reflects that all the phosphate vibrations in the tissue are the contribution from only phospholipids since it is difficult to clearly calculate the contribution of the symmetric phosphate vibrations due to interference from the carbohydrates. The symmetric stretching of PO2 has absorbance around the wavenumber 1080 cm1 that greatly overlaps with the C–O vibrations from the carbohydrates, and thus, it is difficult to quantify precisely.

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RNA / Lipids

2.0 1.8 1.6 1.4 1.2 1.0 1

2

3

4

5

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7

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Location along the crypt Fig. 6. Changes in the RNA/Lipid ratio along representative crypts from two different patients as calculated from the changes in the absorbance at 1121 and 2848 cm1 where these molecules have characteristic absorption. The dashed lines indicate the variation of the RNA/lipid ratio when 996 cm1 absorbance is taken instead of 1121 cm1 for the above two crypts, respectively. The open and closed symbols represent the same patient.

To further examine such changes in the metabolism, especially protein synthesis, the transcription (RNA) levels, the RNA/lipids ratio (A(1121)/A(2849)) was studied as a parameter [18,26]. This was verified using the absorbance at 996 cm1 values for RNA as well. The calculated trends were similar using absorbance from both RNA wavenumbers (996 and 1121 cm1). This was done keeping in view the reports that it is not always possible to determine exact content of the DNA from spectroscopic data [18,27], due to various physical processes and interference from other biochemicals. However, RNA (during protein synthesis) and membranes/lipids are present in the cytoplasm where the problem due to high density effect is diminished [27]. The membrane lipids absorb at around 2852 cm1 [28,29]. This parameter shows that the level of RNA/lipids increases till the middle of the crypts and then tapers off (Fig. 6). Both crypts have maxima around the middle of the crypts but at slightly different positions where maximum metabolism is taking place. This behavior indicates the increasing level of RNA relative to lipids at the middle, which is due to increased levels of transcription and migration of these RNA molecules to the cytoplasm for translation. Since the crypts produce mucin, it is likely that the synthesis and transfer of RNA molecules is involved with synthesis of mucin and other glycoproteins. It is reasonable to assume that the lipid quantity is fairly constant as the crypts mostly are involved with glycoproteins synthesis and the phospholipid quantity is an indicator of membranes, which is fairly constant after cell division is over. This was further confirmed by examining the changes at 996 cm1 where uracil (which present only in RNA) has its characteristics absorption [30]. The variation in levels of RNA/Lipids showed similar trend using RNA absorbance at either 996 or 1121 cm1 (data not shown).

4. Discussions Under normal conditions of growth, differentiation and proliferation of crypts occur at the lower two third portion [25,31,32]. Most of the earlier works to understand the pathogenesis of colon cancer focused on the study of crypts for abnormal development [33–35]. These studies developed in vitro models or used molecular biology techniques to show the abnormal changes in the metabolism, and correlate the changes in content of DNA, RNA or other markers with cell growth and differentiation. There was however, no study in vivo or ex vivo where several biological molecules were monitored simultaneously. In the present work for the first time we try to monitor several biomolecules (carbohydrate, nucleic acids, lipids) using formalin fixed biopsies, which represent the changes occurring in the colonic tissue. Recently it has been shown that changes occur in the IR spectra of the cells during cell growth and death [2]. The studies included the spectra of single cells using synchrotron radiation. In the present work, we utilize a more simple method of microspectroscopy and show that their observations can be obtained for cells in biopsied tissues and thus afford the elucidation of cell physiology using more convenient means. We elucidate the changes occurring in the crypts so that the abnormal FTIR-MSP spectra of crypts can be identified as a tool to distinguish normal and abnormal crypts. Other biomarkers have been reported earlier for crypts which may not always be possible for utilization in biopsies [36] and in such case FTIR-MSP could be a useful method. The accumulation of glycogen has been shown to occur during the maturation of squamous epithelium of the cervix [7,8]. Since the colonic crypts are also subjected to exfoliation it is likely that in these cases the accumulation of the glycogen is required for the cells to function as they migrate from the base towards the apex where exfoliation takes place. This

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accumulation may be needed so that as the cells reach the apex, their storage glycogen is used up and their dependence on the basal tissues for energy is minimized. The decrease in carbohydrates from the middle of the crypt to the apex shows the gradual utilization of the stored carbohydrates by the cells as they move to the top before they are exfoliated. This also could represent the accumulation and secretion of glycoproteins from the cell, which corresponds to the variation in the absorbance along the crypt with regards to the carbohydrates. This is in accordance with our observation (Figs. 4 and 5). The final level of the metabolites in cells at the bottom is often higher than that at the top but always less than the middle. The protein synthesis (transcription and translation) is slowly decreased while the cells reach the apex. This further supports the observations in Figs. 5 and 6 regarding the RNA metabolic pattern during crypt formation. The RNA/lipid ratio again indicates changes in the levels of transcription, as the membrane lipids are nearly constant in the cell after formation of the organelles during cell division. The variations in the RNA/Lipids (Fig. 6) show that the metabolism of nucleic acids (both RNA and DNA) follows the same trend as that of the phosphates and carbohydrates indicating the complexity of the biological process where the different components are metabolized simultaneously and the synthesis of one is related with the other. The slight shift of the RNA/Lipids parameter towards the top of the crypt possibly occurs due to decreasing levels of lipids just after the middle of the crypt. Several genetic alterations in the crypts have been detected using immunohistochemistry and molecular biology techniques to identify specific proteins, mRNA associated with crypt proliferation [37,38]. The role of cell processes and their relevance to malignancy has been reviewed in [25]. In summary, we show that FTIR-MSP is an effective tool to study formation and degradation of biochemicals in cells and tissues, which has a potential diagnostic value for tissue abnormality. The results are encouraging in the context of crypt dynamics and their monitoring by relevant experimental designing for detection of abnormality during colon carcinogenesis. Acknowledgements This research work was supported by the Israel Science Foundation (ISF grant no: 788/01), and the Israel Cancer Association (ICA). Many thanks are due to Dr. V. Erukhimovitch for data collection. References [1] M. Diem, L. Chiriboga, P. Lasch, A. Pacifico, Biopolymers 67 (2002) 349. [2] H. Holman, M. Martin, E. Blakely, B.K., M. WR, Biopolymers 57 (2000) 329. [3] M. Cohenford, T. Godwin, F.C.F, P. Bhandare, T.C. TA, B. Rigas, Gynecol. Oncol. 66 (1997) 59.

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