Natural fluorescence of normal and neoplastic human colon

Lasers in Surgery and Medicine 1648-60 (1995)

Natural Fluorescence of Normal and Neoplastic Human Colon: A

Comprehensive “Ex Vivo” Study Giovanni Bottiroli, PhD, Anna C. Croce, PhD, Donata Locatelli, BSC,

Renato Marchesini, PhD, Emanuele Pignoli, PhD, Stefan0 Tomatis, PhD, Carolina Cuzzoni, MD, Silvana Di Palma, MD, Marco Dalfante, MD, and

Pasquale Spinelli, MD

Center for Histochemisrry, CNR (G.B., A.C.C., D.L.) and Department of Surgery, University of Pavia (C.C.), Pavia and Division of Health Physics (R.M., E.P., S.T.) and

Divisions of Pathology and Cytology (S. D. f?) and Endoscopy (M. D., P. S.), National Cancer Institute, Milan, Italy

Background and Objective: A microspectrofluorometric analysis on “ex vivo” samples from normal tissue and adenocarcinoma of the human colon has been performed to characterize the histological, biochemical, and biophysical bases of the autofluorescence. Study DesignlMaterials and Methods: Differences between normal and tumor tissues are found that concern both the intensity distribution and spectral shape of the autofluorescence emission. The different pattern of the fluorescence intensity can be related to the histological organization of the tissue, and involves mainly the arrangement of the submucosa, the most fluorescent layer. Results: The most evident differences in the spectral shape found in the 480-580 nm range involve the stromal compartment, seem to be due to the presence of different fluorochromes, and are possibly related to the host response to the tumor. Conclusion: The nature and the extent of the autofluorescence modification between normal and tumor tissue in sections explain at least partly the evidence of the “in vivo” analysis and highlight the importance of excitation for full exploitation of the potentials of autofluorescence in diagnosis. Q 1995 Wiley-Liss, Inc.

Key words: microspectrofluorometry, fluorescence imaging, endogenous fluorophors, adenocarcinoma, tissue organization

INTRODUCTION

Gastrointestinal neoplasia is a pathology that receives particular attention owing to its high and increasing incidence in Western indus- trialized countries. Gastrointestinal malignant cancer is supposed to arise in a premalignant lesion, since various stages of tumor progression can be found in the same individual. A sequence of events is proposed, based on genetic and envi- ronmental factors as initiating and promoting agents of the malignant progression, causing a field defect of colonic mucosa that can evolve from flat dysplasia to adenomatous polyp and finally to carcinoma [1,21. The detection of early dysplastic

changes and the identification of the stage of the “adenoma-carcinoma” sequence are of potential clinical importance in the screening and treat- ment of patients at high risk for cancer. Such a goal is difficult to attain through a colonoscopic analysis because it is based on gross architectural and morphological changes of the tissue. More specific techniques, exploiting cytological and bio-

Accepted for publication March 7 , 1994. Address reprint requests to Dr. Giovanni Bottiroli, Centro di Studio per l’Istochimica, CNR, Dipartirnento di Biologia Anirnale, Universita di Pavia, Piazza Botta, 10-27100 Pavia, Italy.

0 1995 Wiley-Liss, Inc.

Microspectrofluorometry of Human Colon 49

chemical alterations, are time-consuming and cannot be accomplished directly “in vivo,” because they require biopsy. For some years there has been a growing interest in detection of tumors through laser-induced fluorescence analysis, a non-invasive technique in which fluorescent drugs, mainly porphyrin derivatives, are used, because of their tumor-localizing properties [31.

More recently, the intrinsic photophysical properties of biological tissues have been considered as a parameter to discriminate diseased from normal tissues, In fact, when excited at a suitable wavelength, most of the biological components, either related to the tissue organization or involved in metabolic and functional processes, give rise to a fluorescence emission over a wide spectral range in the UV-visible region. Since the fluorescence emission is strictly dependent on the biochemical and physicochemical properties of the substrate, tissues with different morphofunctional conditions are in principle discriminated on the basis of their spectral properties.

The literature reports evidence of different fluorescence properties of normal and pathologi- cal conditions of human colon, concerning adenocarcinoma, adenoma, hyperplastic, and normal tissue. Cothren et al. [41 showed that, under excitation at 370 nm, the “in vivo” fluorescence intensity in the visible range of adenomas is significantly lower than that of non-neoplastic tissues. Results obtained by Kapadia et al. [5] in a study on specimens removed at colonoscopy indicated that adenomatous polyps can be distinguished from hyperplastic polyps and normal tissue by a quantitative analysis of the autofluorescence excited at 325 nm. Schomacker et al. [61 observed that, under excitation at 337 nm, the fluores- cences of the hyperplastic and adenomatous tissues differ from that of the normal tissue, primar- ily in the 450 nm spectral component, attributed to NADH. The authors showed that at endoscopy, bnder excitation at 405 nm, neoplasias differ from normal tissues in fluorescence intensity, and that lesions with different degrees of malignancy are distinguished by their spectral shape [71.

“Ex vivo” studies confirmed that the spectroscopic properties of the overall fluorescence emission of malignant, premalignant, and non-neoplastic tissue are sufficiently different to ensure a reliable differentiation in oncological diagnosis 181. In addition, spectrof luorometric analysis based on methods using fluorescence excitation- emission matrices evidenced the heterogeneous nature of the autofluorescence, resulting in a

number of excitation and emission bands [91. Dif- ferences between the fluorescence pattern observed in the overall emission of normal and in that of neoplastic tissue confirmed the validity of the spectroscopic approach and allowed diagnostic algorithms to be proposed. The importance of the morphological in addition to the biochemical features of the tissues in determining the discrimi- nating characteristics has been suggested by Schomacker et al. [6], who attributed the relatively low fluorescence signal of neoplastic tissues to a reduced involvement of the collagenous layers. The complexity, however, of the biological substrate makes it difficult to ascertain the rela- tionship between the spectroscopic evidence and the biochemical and histological features of the tissues that could guide the choice of the experimental parameters of fluorescence spectroscopy suitable for an optimal diagnostic scheme.

The purpose of this study is to define the intrinsic properties of the colon tissues and to iden- tify the biochemical and histological aspects that result in spectroscopic characteristics sufficiently different for malignant lesions to be discriminated from the corresponding normal tissue. To this end, a microspectrofluorometric study has been performed on “ex vivo” samples from normal tissues and adenocarcinoma, and the intrinsic photophysical properties of the biological components have been related to the morphofunctional characteristics of the tissues.

MATERIALS AND METHODS Tissue Processing and Handling

Samples of non-neoplastic human colon and human colonic tumors were obtained from seven patients who underwent oncological surgery. Nor- mal controls were obtained from uninvolved areas in resection specimens. Tumors were histologi- cally diagnosed as moderately differentiated adenocarcinoma. Soon after surgical resection, the biopsy was divided into two parts: One of them was immediately frozen, paying attention to the ori- entation of the sample. Colon tissue longitudinal sections of 20 p,m thickness were then obtained at cryostat and immediately submitted to microspectrofluorometric analysis. The remaining part of the biopsy was homogenized in a buffer solution at moderate ionic strength, where most proteins are maximally soluble [lo]. Phosphate buffer solution (pH 7.4; 0.05 M) containing anti-oxidant (dl-dithiothreitol; 20 mM), proteolytic inhibitor (phenylmethane sulphonyl fluoride; 1 mM), che-

50 Bottiroli et al. lating agent (EDTA; 5 mM), and detergent for protein solubilization (Triton X-100; 0.01% w/v) was used. Pieces of tissue, always kept wet with buffer solution, were disrupted into small frag- ments by means of a surgical blade and then ground three times for 15 s each time with an Ultra Turrax T25 tissue disintegrator (Janke & Kunkel-IKA Labotechnik, Staufen, Germany), keeping the tube in an ice bath. The homogenate was centrifuged for 15 min at 5,OOOg. The super- natant fraction was collected, diluted 10 times with the phosphate buffer solution, and centrifuged again until a clear solution was obtained, to be submitted to fluorometric analysis.

The possible interferences of extraction me- dium were evaluated by measuring the fluorescence of the pure solutions of single components. Triton X-100 only turned out to be fluorescent, in the spectral range 400-500 nm, under excitation at 310 nm. At, however, the dilution reached in the final extraction solution, its contribution to the tissue extract fluorescence did not exceed 3% at 440 nm, and was quite negligible at 540 nm.

The pellet was submitted for further extraction of lipid fluorophores, using a mixture of chloroform, methanol, and water (2:l:O.E by vol.) according to Armstrong et al. [ l l] .

Chemicals Fluorescence analysis was performed on

pure solution of the following compounds: bovine albumin, elastin, collagen type I11 (concentration 0.01 mg/ml), pyridoxine, pyridoxal-5-phosphate, 4-pyridoxic acid, flavin-mononucleotide, flavin- dinucleotide, and protoporphyrin IX disodium salt (concentration of M). All the compounds were directly dissolved in phosphate buffer solution (pH 7.4,0.05 M), except elastin and collagen, dissolved in acetic acid (0.1 M), and then neutral- ized in phosphate buffer. All the compounds were purchased from Sigma (St. Louis, MO).

Lipopigment solutions were obtained from neurons and non-neuronal cells of rat brain by extraction according to Armstrong et al. [ll].

Experimental Apparatus Fluorescence analysis in solution was per-

formed by means of a spectrofluorometer (Applied Photophysics, London, UK; mod SP-2), equipped with a Xenon 300 W lamp (O.R.C., Azusa, CA), a photomultiplier tube RCA 8850, and a photon counting system (EG & G, Ortec, Princeton, NJ). Excitation and emission light was selected by means of monochromators.

Microspectrofluorometric analysis on tissue sections was performed by means of a Leitz mi- crospectrograph (Wetzlar, Germany), equipped with an optical multichannel analyzer (EG & G, Princeton Applied Research, Princeton NJ). Flu- orescence emission was spectrally dispersed along the horizontal axis of the exit plane of the poly- chromator and imaged into 512-element intensi- fied linear diode array detector (mod. 1420/512), digitalized, and processed by the Optical Multi- channel Analyzer, under computer control.

A high-pressure 100 W Mercury lamp ( 0 s - ram, Berlin, Germany) was used as an excitation source, in combination with KG1 and BG38 an- tithermic filters, and UG1 band filter. Either 366 nm (HBw = 10 nm; T = 30%) or 405 nm (HBw = 10 nm; T = 35%) interference filters were used to select the excitation wavelengths, corresponding to the 366 and 405 nm lines of the lamp. Excita- tion was performed under epi-illumination conditions, using a TK405 dichroic mirror. The trans- mittance curve of the dichroic mirror (T400 = lo%, T410 = 50%, T,,, = 90%) induces some dis- tortions in the short-wavelength region (410-430 nm) of the spectra, which are constant in all measurements.

Leitz objectives, 25x (N.A. 0.50) and 40x (N.A. 0.651, were employed for all the measurements.

Fluorescence of Tissue

Microspectrofluorometric scan-analysis was performed on longitudinal sections of normal and tumor tissues, from the region exposed to the lumen toward the inside, with steps of 43 pm. A fixed diaphragm, with a 300 x 30 pm2 area on the objective plane, was used to delimit the tissue region under measurement.

A variable iris diaphragm, with an area ranging from a few to about 1,000 pm2 on the objective plane, was used to perform microspectrofluorometric analysis on selected histological structures.

RESULTS Fluorescence Scan-Analysis of Longitudinal Tissue Sections

A typical fluorescence pattern of the longitudinal section of normal human colon is shown in Figure la. Differences in fluorescence intensity make the histological organization of the tissue visible: the superficial epithelium, which in- vaginates to form mucosal glands extending into


Fig. 1. Fluorescence microphotographs of colon tissue sec- ltions obtained under excitation a t 366 nm. a: Non-neoplastic tissue. b: A detail of a showing glands and lamina propria. c: b detail of a showing the dense collagenous connective tissue of submucosa. d adenocarcinoma tissue. e: A detail of adeno-

carcinoma tissue showing the neoplastic cells of a degener- ated gland. f: A detail of the stroma: Yellow-fluoresceing lipopigment granules associated ta infiltrating cells are visible. a, d, bar = 90 pm; b,c,e,f, bar = 22.5 pn.

Uhe lamina propria, the muscularis mucosa, and Uhe strongly fluorescent submucosa. Details at higher magnification are shown in Figure lb,c,

illustrating portions of mucosa with glands and lamina propria, and of submucosa, respectively.

An example of fluorescence patterns of lon-

52 Bottiroli et al.

170n -

Fig. 2. Patterns of microspectrofluorometric scan-analysis performed along the axis of longitudinal tissue sections. Each curve corresponds to the emission spectrum recorded on a diaphragmed area of 300 x 30 pm2 on the tissue section. Spectra shown are measured every 170 km. Excitation wavelength: 366 nm. a: Non-neoplastic tissue. b: Adenocarcinoma lesion.

gitudinal sections of moderately differentiated human colon adenocarcinomas is shown in Figure Id. The hyperplastic condition of the neoplasia modifies drastically the organization of the tissue. The tumor has infiltrated deeply through the submucosa and muscularis, so that these two layers are no longer observed. Figure le,f shows, at higher magnification, an area of neoplastic cells, and a portion of stroma, respectively.

Typical examples of the fluorescence pattern obtained through microspectrofluorometric scan- analysis performed on longitudinal sections of both non-neoplastic and neoplastic colon, under excitation at 366 nm, are shown in Figure 2a,b, respectively. In order to avoid the reabsorption effect due to the presence of heme-based mole- cules 1121, only section regions not affected by the presence of blood, arising from tissue trauma dur- ing resection, were selected.

The patterns of the tumors differed from those of the non-neoplastic tissues in both the intensity and the spectral shape of the fluorescence emission. The non-neoplastic tissues exhibited patterns that were very consistent in all samples

considered and were characterized by a strict dependence on the tissue depth. From the lumen inward, the fluorescence intensity at first showed a slight decrease, followed by a marked increase. The average curve of the fluorescence intensity as a function of the tissue depth (Fig. 3) indicated that the slight decrease occurred at a depth less than 50 p,m, passing from superficial epithelium to mucosa, while the marked increase appeared at about 450 Fm, corresponding to the depth where the muscolaris mucosa and the fibrous and dense connective tissue, submucosa, are known to be located.

In tumor tissue, the fluorescence pattern was found to vary among samples. In each sample the values of the fluorescence intensity, although somewhat variable, were comparable to those of the mucosa layer of normal tissue over almost all the scanned region. Highly fluorescent regions, corresponding to submucosa, were observed only in tumors with a low degree of invasiveness, at a depth greater than 800 pm. The intensity values were comparable to those of submucosa of normal tissue. Examples of curves of the fluorescence in-

53 Microspectrofluorometry of Human Colon

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20.000 h

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6 15.000

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0 '- 0 200 400 600 800 1 .ooo 1.200 1.400

TISSUE DEPTH @rn)

Fig. 3. Curves of fluorescence intensity at 440 2 10nm vs. tissue depth measured in sections of non-neoplastic and adenocarcinoma tissues. Excitation wavelength 366 nm. (A) Non-neoplastic tissue. The curve is the average of measure-

tensity as a function of the tumor depth are shown in Figure 3.

As well as the signal amplitude, the fluorescence emission measured on the longitudinal sections of normal and tumor tissue differed in the spectral shape. In particular, a broadness of the emission band in the green-yellow region was recorded in the spectra of the tumor lesion, along with the occasional appearance of a narrow band at about 630 nm.

Microspectrofluorometric Analysis of Histological Structures

To investigate the possibility of a correlation between the spectral properties of the tissues and their morphofunctional characteristics, a spectral analysis was performed on selected and diaphragmed regions corresponding to the histological components of both normal and tumor tissues. Typical visible-emission spectra of normal tissue, excited at 366 nm and filtered through a 405 nm dichroic mirror, are shown in Figure 4a. They

ments performed on different regions of sections from four samples of non-neoplastic tissue. (*) Adenocarcinoma tissue. The curves shown were recorded on different regions of sections from three samples of adenocarcinoma.

consisted of a poorly structured emission band in the 440-580 nm region, with significant modifications depending on the histological component considered. The less structured profile was observed in the case of submucosa, which exhibited a narrow emission band centered at about 440 nm. A red-shift of the peak position, along with a broadening of the emission band toward longer wavelengths, was measured in the superficial epithelium and in the mucosa. In this latter case, some degree of variability in the spectral shape was observed when the measurements were performed on the lamina propria. A shape closely re- sembling that of submucosa was recorded in the region characterized by the presence of a fluorescent net, possibly attributable to fibres of collagen in the loose connective tissue. A shoulder at about 520 nm was measured in relation to the presence of green-yellowish granules. These granules, which were observed in relatively small numbers in all samples considered and were unevenly dis- tributed in the tissue, appeared associated with

54 Bottiroli et al.

m

10 -

440 460 480 500 520 540 560 580 600 620 640 660 680 700

WAVELENGTH lnml Fig. 4. Emission spectra recorded on regions of tissue sections selected by means of a diaphragm with a dimension variable from a few to about 1,000 pm2, depending on the histological component to be measured. Excitation wavelength = 366 nm. The curves are normalized to the peak values. Average values of fluorescence intensity a t the emission peak (FI, arbitrary units, f SD), normalized to the area under measurement, arc reported for each histological com-

cells infiltrating the connective tissue and resembled closely, in both fluorescence and morphol- ogy, particles attributed by some authors to lipopigment-loaded lysosomes [13,141.

As to the emission intensity, the signal amplitudes of the histological components, normalized to the area under measurement, are reported in the legend of Figure 4a. When the fluorescence intensity of submucosa is compared to that of mucosa, a higher relative amplitude is found than that observed in the microspectrofluorometric scan-analysis. This can be understood taking into account that, owing to the dimension of the measuring diaphragm, the scan-analysis possibly involves non-f luoresceing regions produced by the action of the knife on the fibrous tissue.

In the neoplastic tissue the emission spectra recorded on the submucosa layer, when present, exhibited shapes quite similar to those of the corresponding layer of normal tissue, as spectra of neoplastic cells resembled those of the normal

440 460 480 500 520 590 560 580 600 620 640 660 680 700

WAVELENGTH lnml ponent (n = 10 for each of the seven patients). a: Non-neoplastic tissue: (--.--.-) superficial epithelium, FI = 160 5

glands, FI = 119 k 14.2; (. . . .) submucosa, FI = 1,430 f 480. b: Adenocarcinoma tissue: (- ) neoplastic cells, FI = 105 f 13.2; (- - - -1 stroma, FI = 139 +- 21.7; (. . . .) submucosa, FI = 1,250 I 371; (--.--.-) yellow granules FI = 721 2 247.

6.4; (-- - - -) lamina propria, FI = 132 2 16.5; (- 1

glands. As to the other histological components, the typical superficial epithelium is hardly recog- nizable in the tumor lesion. Microspectrofluoro- metric analysis was performed on the surface area exposed to the lumen: The spectra, although somewhat affected by a certain variability, exhibited a relative amplitude at longer wavelengths higher than in non-neoplastic epithelium. The spectra recorded on tumor stroma were characterized by the presence of an emission band at about 510-520 nm that appeared more evident than that of normal lamina propria and that was pref- erentially observed in the regions close to the areas exposed to the lumen. The microscopical ob- servation of these regions did not allow a well defined attribution to any histological component, although staining with the lipophilic dye Nile Red [151 showed the presence of lipid microdroplets dispersed into the tissue. Shoulders at about 520-560 nm were also measured in some regions where an enrichment was found in the green-yel-


I I 300 350

100

5 0 .

300 350 400 450

WAVELENGTH (nm) Fig. 5. Average excitation spectra of buffer extracts from adenocarcinoma (A), and non-neoplastic (B) tissue. Emission wavelengths: 440 nm (a) and 540 nm (b). The curves are normalized to the peak values. The fluorescence intensities

lowish granules already described in normal tissue. Limited areas of some sections exhibited a narrow emission band at 630 nm that can be attributed to the presence of endogenous porphy- rins, possibly related to either an altered metab- olism of the cancer tissue [161 or a microbial synthesis [171.

The emission intensities, normalized to the area, are reported in the legend of Figure 4b. The fluorescence intensity of submucosa of neoplastic tissue is comparable to that of the corresponding layer of normal tissue, as was that of neoplastic cells to that of the normal glands. In this latter case, it must be noted that necrosis, when present, resulted in an appreciable increase in the fluorescence intensity. As to the stroma, normal tissues exhibited fluorescence intensities slightly higher than tumor, when the measurements were performed in regions without yellow granules. The presence of the granules, which are characterized by a high fluorescence intensity, resulted in a signal variability that appeared particularly marked in the tumor.

When the histological structures were excited at 405 nm, a remarkable decrease of the emission amplitude was observed in all the cases with respect to the excitation at 366 nm that can be only partially ascribed to the reduced excitation intensity in the experimental conditions employed. As to the spectral shape, no significant modification was detected in the case of the sub-

(FI, arbitrary units) normalized to the tissue weight are reported. Non-neoplastic tissue: FI(exc. 280 nm, em. 440 nm) - 5,000, FI(exc, 330 nm, em, 540 nm) = 3,500; tumor tissue:

-

‘‘kx~. 280 om, em. 440 nml = 7,500; F1(exc. 330 nm, em. 540) = 2,800’

mucosa in either normal tissue or tumor lesion. A slight increase in the relative amplitude of emission in the green-yellowish region was observed in the spectra recorded on glands and lamina propria. The modifications appeared more evident in the case of tumor lesion, mainly for the stroma, where the appearance of the 510-520 nm shoulder was favored (data not shown).

Spectrofluorometric Analysis of Tissue Extracts In an attempt to characterize the excitation

properties of the endogenous fluorophores, a spectrofluorometric study has been performed on extract solutions of both normal and tumor tissues. Data have been normalized to both the tissue weight, in order to estimate the total amount of the solved fluorophores, and to the signal amplitude, in order to evaluate the differences in the relative concentration of the f luorophores between normal and tumor tissues.

Peak amplitude-normalized excitation spectra of extracts obtained from both the lesion and its normal surrounding tissue are shown in Fig- ure 5. The measurement wavelength ranges cho- sen were 440 ? 5 and 540 ? 5 nm, approximately corresponding to the main emission band and to the shoulders of the emission spectra recorded on tissue sections, respectively.

The spectra of Figure 5a indicate that the fluorescence emission at 440 nm on both normal and tumor tissues is mainly favoured upon exci-

56 Bottiroli et al.

250 300 350 400 450 500

WAVELENGTH (nm)

Fig. 6. Excitation (a) and emission (b) spectra of pure solution of endogenous fluorophores, possibly involved in the autofluorescence. Spectral shapes are shown for the best relative excitationiemission conditions. Fluorescence emission intensity values (FI, arbitrary units) are reported for each compound, normalized to the weight of .01 mgiml, exc. 366 nm, emission wavelength corresponding to 380 nm for collagen, albumin, and elastin, and at the emission peak for all the other compounds. A: collagen FI = 840. B: Tryptophan-con-

tation at about 280 nm and in the 325-350 nm range. Apart from the relative amplitudes of the two bands, the spectra are quite similar, thus sug- gesting that the fluorescence emission at 440 nm can be ascribed to the same fluorophores in both normal and tumor tissues. When compared to the excitation spectra of pure solutions (Fig. 6a) and to the data reported in the literature on endogenous fluorophores, the 280 nm excitation band can be attributed to the aromatic aminoacids of proteins, namely tryptophan and tyrosine. The excitation band at 325-350 nm can be ascribed to fluorophores such as pyridoxine derivatives and NAD(P)H, along with the solved fraction of collagen, that fluoresces at longer wavelengths than proteins, because of the presence of cross links involving tyrosine residues [181. In both normal and tumor tissues the excitation band at 280 nm exhibited a greater amplitude than that at 330- 350 nm. This can be explained taking into account that collagen, one of the fluorophores contributing to the 330-350 nm excitation band, is poorly soluble. In fact, it must be noted that after extraction an amount of unsolved pellet was

taining protein, albumin FI = 26. C: Elastin FI = 64. D: Pyridoxal-5-phosphate FI = 22. E: Pyridoxine FI = 590. F: 4-pyridoxic acid FI = 7,960. G: NADH FI = 950. H: Lipo- pigments obtained by chloroformimethanol extraction from rat brain tissue FI = 523. I: IX protoporphyrin FI = 25,400. L: flavin-mononucleotide FI = 17,272 or flavin-adenin-dinucleotide FI = 1,960. All curves are normalized to the peak values.

found that appeared like a fibrous material. The microspectrof luorometric analysis performed on pellet smears confirms the connective nature of the unsoluble residuals.

When the fluorescence intensity values were normalized to the tissue weight, the normal tissues gave a signal about 35% less than that of the lesions under excitation at 280 nm, thus indicat- ing a larger amount of easily soluble proteins in the lesions, in agreement with the fact that the unsolved pellet is significantly higher in normal than in tumor tissue. On the contrary, when excited at 330-350 nm, the fluorescence signal was about 20-25% greater in normal than in tumor tissues. This difference is to be attributed to a larger amount of NAD(P)H, as indicated by the emission spectra performed under excitation at 340 nm, that exhibited an emission band at 460 nm larger in normal than in tumor tissues (data not shown). The larger amount of connective components in normal than in tumor tissues accounts only partly for the difference, because of its low solubility.

The excitation spectra measured at 540 nm


were well structured in the 300-450 nm range. In particular, they appeared as a convolution of a number of excitation bands centered at about 330, 360, 380, and 440 nm. A minimum was observed B t about 410 nm that can be partially ascribed to the reabsorption effect resulting from the presence of blood, as confirmed by evaluating the amount of hemoglobin in the absorption spectra.

The excitation spectra indicated that the emission at 540 nm could reasonably be ascribed to the simultaneous contributions of different fluorophores, in agreement with the variability of the emission spectrum shape observed in tissue sections (Fig. 4a,b). After normalization to the tissue weight, the fluorescence intensity under excitation in the 330-350 nm range was found to be greater in normal than in tumor tissue by about

The comparison with the spectra of pure solutions indicated that pyridine derivatives, NAD(P)H and flavins, which are substances mainly related to metabolic processes, are respon- sible for the autofluorescence at 540 nm, along with the collagen. As expected, owing to their physicochemical nature, lipopigments, which are present to a greater extent in tumor tissue, were extracted in a negligible quantity in a polar solvent, so that they were not significantly detect- able in the excitation spectra shown in Figure 5a,b. The presence of lipopigments was evidenced by performing the extraction with organic sol- vents. Figure 7 shows the excitation and emission spectra recorded on chloroform-methanol extracts obtained from tumor tissues. These spectra appeared somewhat different from those obtained from purified endogenous fluorophores (Fig. 6). It must, however, be noted that lipopigments are a highly heterogeneous group of substances that are found in several tissues, mainly in brain, and that are supposed to be derived from lysosomal material through both oxidation and polymerization processes. They differ in basic structure, in degree of polymerization, and in the nature and amount of non-lipid material included in the po- lymerizing mass [ 191.

15 -25%.

DISCUSSION

The microspectrofluorometric analysis performed on histological structures and along the longitudinal section of “ex vivo” samples indicated that tumor differs from normal tissue both in the spectral shape and in the intensity distribution of the autofluorescence.

5 z W

z W 0

Y 0

I 3 0

D 350 400 450 500 550 600

WAVELENGTH (nm)

Fig. 7. Excitation (observed wavelength: 510 nm) and emission (excitation wavelength: 370 nmj spectra of a solution obtained by extraction with ch1oroform:methanol:water solution (2:l:O.E by vol.) of the adenocarcinoma pellet, previously submitted to extraction by buffer solution. The curves are normalized to the peak values.

The autofluorescence, excited at 366 nm and analyzed in the 430-700 nm spectral range, appears characterized by a main emission band peaking at about 440-460 nm along with the occasional presence of shoulders in the 500-580 nm region. The less structured spectral shape is found in submucosa that is the collagenous connective layer acting as a base of the mucosa in the colon organization. As expected, because of its non-tu- moral nature, submucosa did not exhibit any appreciable difference between normal and tumor tissues. Only a narrow emission band centered at about 440 nm was observed, that, owing to the layer composition, can be ascribed to the contribution of constituent proteins such as collagen and elastin, With respect to the spectral shape recorded in submucosa, a slight broadening of the band toward longer wavelengths was observed in gland cells of mucosa, both normal and neoplastic. A shoulder at about 460-470 nm was detected, that can be attributed to the contribution of the coenzyme NAD(P)H, as suggested by the metabolic activity exherted by the cells and supported by the spectral evidence obtained in both pure solutions and tissue extracts. Actually, the contribution of NAD(P)H appears markedly less evident than that of the proteins. In this regard, it must be considered that the tissue processing re- quired by the extraction procedure possibly results in oxidative processes leading to an underestimation of the NAD(P)H contribution [6].

Shoulders in the 480-580 nm region were

Bottiroli et al. 58 observed in the emission spectra recorded in the stroma, their relative amplitudes varying between tumor and normal tissues. In particular, these shoulders appeared more evident and the spectral profile was better structured in the tumor tissue. The presence of fluorescing granules and lipid microdroplets in greater number than in normal tissue could at least partly explain the differences observed in that spectral region, as is suggested by the spectra of organic solvent extract.

Both the complexity of the stromal tissue and the processes occurring in this compartment as a consequence of the host-tumor response [20 - 221 could account for these differences. Areas of inflammation and granulation can be found, with accumulation of interstitial fluid and migrating cells, especially granulocytes containing large numbers of granules and lipid bodies when en- gaged in an inflammatory response [231. An ex- cess of lipid storage can also be found in tumor tissue, possibly related to an injury of mitochon- drial membranes consequent upon a hypoxic condition [241.

Other fluorophores, such as flavins, that are known to fluoresce at about 500-530 nm, could contribute to the modifications observed, owing to the unbalance of the natural redox equilibrium 1251.

The presence of the shoulders becomes more noticeable when the autofluorescence is excited at a longer wavelength, that is, 405 nm instead of 366 nm. This can be ascribed to a reduction of the relative amplitude of the main band at 440-460 nm, rather than to an actual enhancement of emission at longer wavelength. In fact, the emission band at 440-460 nm, attributable to the contribution of proteins, collagen and NAD(P)H, is particularly favored by exciting in the range 280- 360 nm, as is indicated by the excitation spectra measured in both pure solution and tissue extracts.

The differences in the fluorescence intensity pattern are related to the histological organization of the tissue and can be attributed mainly to the distribution of submucosa. This layer, which is about ten times more fluorescent than the remaining part, in normal tissue is usually located at a constant depth not exceeding 450-500 pm, while in neoplastic lesions, when present, it starts at a depth greater than 800 pm, depending on the tumor invasiveness.

The dependence of the autofluorescence properties on the histological composition and or-

ganization of the tissue could provide information useful for interpreting the results previously obtained in an “in vivo” study [71. In that study, adenocarcinoma was found to differ from the non- neoplastic surrounding tissue in terms of both fluorescence intensity and spectral shape. The difference of fluorescence intensity concerns the emission band at 440-460 nm, which exhibited a higher amplitude in normal than in tumor tissues. This band, which is the most important component of the overall autofluorescence of colonic tissue, was shown to be the major spectral component of the emission of all the histological structures, and in particular of the submucosa, in relation to the density of the strongly fluorescent collagen. Taking into account the tissue penetra- bility of the light in the range 300-410 nm [6,261, it is likely that the difference of location of submucosa between normal and tumor tissue results in a different involvement in the fluorescence excitation-emission process, thus contributing to the differences in the intensity of the overall fluorescence emission of the two tissues. This is in agreement with Schomacker et al. [6] who found that under excitation at 337 nm, a condition particularly favorable for collagen, the major change in autof luorescence in malignant progression is a relative decrease in 390 nm fluorescence, as a consequence of the screening of fluorescence from the collagenous submucosal layer by the thick- ened mucosa present in polyps. A contribution to fluorescence enhancement in normal tissue is also provided by the superficial epithelium, which was found more fluorescent than the surface area exposed to the lumen in the tumors. Although the differences found in the fluorescence intensity are rather low and concern a very short thickness, it must be considered that this layer is deeply involved in the excitation-emission process. A further contribution could be provided by the mucosa, which appears somewhat more fluorescent in normal than in tumor stroma, because of the presence of a fluorescent net not observed in the tumor stroma that is attributable to collagen fi- bers. This additional fluorescence, however, should be balanced by the presence of a larger amount of green-yellowish highly fluorescent granules in neoplastic than in normal stroma.

The fluorescence intensity ratio up to 5 found between normal and tumor tissues in the “in vivo” measurements, however, suggests that aspects other than the tissue organization might be involved. A different relative concentration of fluorophores, and, in particular, a change of


NAD(P)H fluorescence, according to the data obtained from tissue extracts, could be hypothesized in the two tissues. Lesser NAD(P)H fluorescence intensity in transformed than in normal cultured cells has already been observed in relation to the absolute coenzyme concentration [27]. Moreover, a reduction of binding sites for NAD(P)H in cancerous tissue has been observed 1281, resulting in a lower proportion of the coenzyme form with higher fluorescence efficiency 1291.

This hypothesis is not fully supported by the data obtained in tissue sections, which showed only a 10% higher fluorescence in the normal than in tumor gland cell compartment. It must be, however, considered that an underestimation of the NAD(P)H contribution occurs in measurements in tissue sections. Richards-Korthum et al. [91, by applying a method of investigation based on fluorescence excitation-emission matrices to “ex vivo” samples, evidenced a peak attributed to NAD(P)H twice as intense in normal as in adenomatous tissue. This result is consistent with the findings obtained in patients affected by adenomas, by Cothren et al. [41 and by some of the present authors [71, who observed a greater fluorescence intensity in surrounding normal tissue than in neoplastic lesion, under excitation at 370 and 405 nm, respectively.

The shape differences found in the 500-580 nm range between the emission spectra of stroma in tumor and those of normal tissue in sections could account for the spectral modifications measured “in vivo,” the spectral region involved being the same. No evidence of spectral modification in this range is reported in the literature on either “ex vivo” or “in vivo,” under excitation in the range 320-370 nm. In this context Shomacker et al. [61 suggested that by excitation at short wavelengths, where the contribution of highly fluorescent collagen from extracellular tissue is favored, differences in spectral shape between dysplastic and normal cells could be masked. Actually, this is supported by our measurements on tissue sections where a greater emission of the fluorophores related to the longer wavelengths is obtained under 405 nm excitation than at 366 nm. Moreover, the excitation spectra obtained on tissue extracts showed that the emission at 540 nm can be particularly influenced by the excitation wavelength.

It must, however, be noted that measurements performed by the authors “in vivo” on adenomas in the same excitation conditions used for adenocarcinomas evidenced only slight spectral

modifications between the pre-malignant lesions and the surrounding normal tissue. At present we cannot rule out that this different behavior may be attributed to a remarkable degeneration of the stroma in lesions passing from the pre-malignant to the malignant condition.

CONCLUSIONS

The results presented concerning the spectrof luorometric analysis of “ex vivo” samples sup- port the view that adenocarcinoma lesions can be distinguished from normal colonic tissue by both autofluorescence intensity and emission spectral shape. The differences observed on tissue sections can be related to the presence of various fluorophores in terms of both their relative concentration and distribution, in connection with the arrangement and, possibly, the composition of the histological components. The nature and the extent of the differences, which are consistent with the data previously obtained “in vivo,” highlight the role of the excitation wavelength in defining an optimal diagnostic scheme exploiting various properties of tissue autof luorescence. In particular, the results obtained suggest that excitation at short wavelengths (<350 nm), favoring the emission at 440 nm typical of the biological tissues, result in differences of fluorescence intensity depending on the organization of the colonic tissue (collagen) and, possibly, on the cell metabolic activity (NAD(P)H). Excitation at long wavelengths (>350 nm), involving fluorophores in some way related to degeneration processes, could enhance the differences in the spectral shape arising from the tumor host response.

An important factor in the validity of the excitation wavelength choice would be the morphological organization of the neoplastic tissue, a characteristic that is among the criteria for the histogenetic classification of tumors. The proportion of tumor cells to stroma, for instance, could influence the relative contribution of the tissue structures to the overall autofluorescence. In the perspectives of the foregoing considerations, great attention must be paid to the excitation-emission delivery system, which, depending on the probe geometry, can influence the sensitivity of the response toward the biochemical and morphological properties of the tissue.

The use of a Monte Carlo model and re- gression analysis, considering the histological organization and photophysical properties of the biological substrate, will allow us to verify the

80 Bottiroli et al. experimental conditions that enhance the modifi- bation of autofluorescence characteristics, thus approaching an “in vivo” histochemical analysis.

14. Goldfisher S, Villaverde U, Forschirm R. The demonstra- tion of acid hydrolase, thermostable reduced diphos- phopyridine nucleotide tetrazolium reductase and perox- idase activities in human lipofuscin Histochem Cytochem 1966; 14:641-652.

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ACKNOWLEDGMENTS 15. Greenspan P Mayer E, Fowler SD. Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell

This work was supported by the CNR Special Biol 1985; 100:975-973. 16. Leibovici L, Schoenfeld N, Yehoshua HA, Mamet R, Ra-

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Natural fluorescence of normal and neoplastic human colon