AUTOFLUORESCENCE SPECTROSCOPY OF A HUMAN

GASTROINTESTINAL CARCINOMA CELL LINE

Design of Optical Sensors for the Detection of Early Stage Cancer

D. S. Ferreira

, M. Henriques

, R. Oliveira

, J. H. Correia

and G. Minas

Algoritmi Centre, Universidade do Minho, Campus Azurém, 4800-058 Guimarães, Portugal

Department of Biological Engineering, Universidade do Minho, Campus Gualtar, 4710-057 Braga, Portugal

Keywords: Autofluorescence, Fluorophores, Cancer, Optical sensors.

Abstract: Human tissues show autofluorescence (AF) emission spectra when excited by ultraviolet or short-

wavelength visible light. The intensity and shape of these spectra are dependent on the tissues pathological

state and, therefore, its measurement gives information about the degree of malignant transformations that

could lead to cancer. In this article, it is characterized the AF spectra of one human gastrointestinal

carcinoma cell line (CACO-2). The obtained results showed significant AF signal for the presence of amino

acids. The spectral information obtained can be used for the design of fluorescence optical sensors that will

be incorporated on an endoscopic capsule, for measuring the AF emission spectra of normal and cancer

cells. This integrated optical system will innovate on the diagnosis of early stage cancer.

1 INTRODUCTION

Cancer of the gastrointestinal (GI) tract is the second

most common cause of cancer death in the United

States and industrialized countries. Patients with a

family history of colon cancer, familial polyposis,

long-standing inflammatory bowel disease, and

Barrett’s esophagus are at high risk for developing

this kind of cancer. The new innovations in

endoscopy, including capsule endoscopy, made the

GI tract one of the most frequently and completely

examined system. Despite the greater access, early

detection of neoplasia is difficult during routine

endoscopy due to the absence of typical

morphological structures and, as a result, is still

limited to blind random biopsies in patients at high

risk of developing cancer, in a both inefficient and

costly process. Therefore, more sensitive endoscopic

screening tools, which enable differentiation

between premalignant and benign lesions during

endoscopy, are of scientific and clinical interest

(Prosst, 2002; Banerjee, 2004; Georgakoudi, 2006).

When human tissues are illuminated with

ultraviolet or short wavelength visible light, they

emit fluorescence light of a longer wavelength

(Figure 1). This tissue ‘autofluorescence’ (AF) arises

from endogenous molecules within the tissue, called

fluorophores (Haringsma, 1999; DaCosta, 2002).

The GI tract tissues are composed by a complex

combination of several fluorophores that occur in

different concentrations and at different depths. The

mucosa, submucosa and muscularis propria have

distinct fluorophore compositions, so that even

though each fluorophore has a distinct fluorescence

spectrum, the total fluorescence measured comprises

contributions from the fluorophores in the various

layers. Hence, excitation and fluorescence emission

wavelength bands are often broad, relatively

featureless and overlap with one another, so that

identifying individual fluorophores in a given tissue

spectrum is difficult (Haringsma, 1999;

DaCosta, 2002).

Some research groups have explicitly studied the

AF emission of established cell lines (DaCosta,

2005). These studies, by the use of single living

cells, allow the isolation of the contribution of

intracellular fluorescence in the tissular emission,

through the elimination of the extracellular matrix

emission and the influence of light absorption and

scattering. Therefore, such studies provide a better

understanding of the biochemical changes associated

with malignant transformation. Also, cellular

Ferreira D., Henriques M., Oliveira R., Correia J. and Minas G. (2009).

AUTOFLUORESCENCE SPECTROSCOPY OF A HUMAN GASTROINTESTINAL CARCINOMA CELL LINE - Design of Optical Sensors for the

Detection of Early Stage Cancer.

In Proceedings of the International Conference on Biomedical Electronics and Devices, pages 61-66

DOI: 10.5220/0001539000610066

 SciTePress

reference spectra are needed for the modeling of the

tissue fluorescence, in view of spectral

quantification which should be very useful for

diagnostic purposes. To quantify the relatively weak

AF created by one single fluorophore, sophisticated

algorithms have been developed (Stepp, 1998;

Prosst, 2002; Villette, 2006).

Mucosa

Submucosa

Muscularis propria

Fluorophore

Chromophore

Specular

Re f lectance

Incident

Light

Diffuse

Re flectan c e

Scattering

Absorption

Fluorescence

Emis sion

Figure 1: Light–tissue interactions include reflectance,

scattering, absorption and fluorescence. Tissue

fluorescence is originated by the absorption of light by

fluorophores.

Among the significant fluorophores in the GI

tract are: tryptophan and tyrosine (aromatic amino

acids present in cells); NADH and NADPH (cellular

metabolism related coenzymes), which assist

oxidation and reduction processes and are found

mainly in mitochondria; and flavins and flavin

nucleotides, which are mostly bound to enzymes and

concentrated in the mitochondria (DaCosta, 2005).

Each of these fluorophores has its characteristic

excitation and emission spectrum (Table 1). Thus,

different excitation wavelengths result in the

activation of different fluorophores.

Table 1: Excitation and emission maxima of some

endogenous fluorophores (DaCosta, 2002).

Endogenous

fluorophores

Excitation

maxima (nm)

Emission

maxima (nm)

Amino acids

Tryptophan 280 350

Tyrosine 275 300

Phenylalanine 260 280

Metabolic cofactors

FAD, Flavins 450 515

NADH 350 450

NADPH 336 464

Over the last decade a number of optical

techniques have been developed to try to detect early

GI neoplastic lesions. These optically-based

methods have the potential to detect the very earliest

mucosal changes at the micro structural and

molecular levels (Haringsma, 1999). Light induced

autofluorescence spectroscopy, for example, is based

on the analysis of the fluorescence emission of

endogenous fluorophores, providing information that

can be used to characterize changes that take place

as tissues become diseased. It involves delivery of

the excitation light via placement of a fiber-optic

probe in direct contact with tissue. The fluorescence

is collected by the same probe and delivered to the

input of a spectrometer. Achieving discrimination

between diseased and healthy tissue relies upon the

identification of differences either in emission

intensity, spectral distribution, fluorescence lifetime,

or crucially, a combination of these (Stringer, 2004;

Georgakoudi, 2006).

The capsule endoscopy is a relatively recent

method for GI tract evaluation that uses an

endoscopic capsule (EC). Since, presently, some of

the most promising methods for detecting cancer at

an early stage are based on tissue’s fluorescence, the

EC can play an important role in this kind of

diagnosis, once it can be equipped with miniaturized

fluorescence CMOS optical sensors. These sensors

should be incorporated in the EC for measuring the

AF emission spectra of normal and cancer cells, and

should have the higher quantum efficiency in a

defined spectral range (Delvaux, 2006; Dias, 2007).

This integrated system will certainly innovate in the

diagnosis of GI cancer.

It is important to notice that

current endoscopic capsules do not have added

diagnostic functions, such as spectroscopic analysis,

which are commonly available in conventional

endoscopes. Therefore these developments will be a

major step towards creating a new platform for

diagnosis.

In this article, it is characterized the AF spectra

of one human GI carcinoma cell line (CACO-2). The

results obtained showed significant AF signal for the

presence of amino acids, namely tryptophan. The

resulting spectral information of the CACO-2 cell

line is a good initial approach for the design of

fluorescence optical sensors, to be incorporated on

an EC. This study was performed using

spectrofluorimetry on cell monolayers, with several

excitation wavelengths, resulting in several

fluorophores emission spectra. Some of the reported

results appeared promising, being in accordance

with previously published results.

BIODEVICES 2009 - International Conference on Biomedical Electronics and Devices

2 EXPERIMENTAL

2.1 Human Carcinoma Cell Line

Culture

The experimental studies were performed using one

human GI carcinoma cell line - CACO-2 - that was

purchased from the European Collection of Cell

Cultures (ECACC). These cells were grown in 88%

Minimum Essential Medium Eagle’s (EMEM),

supplemented with 10% Fetal Bovine Serum (FBS),

1% Non Essential Amino Acids (NEAA), and 1%

Glutamine (2mM) in a humidified atmosphere, at

37ºC and 5% CO

. Cells were grown in 25 cm

flasks to 70 – 80% confluence and their morphology

was routinely inspected.

2.2 Preparation of CACO-2 Cell Line

for Spectrofluorimetry

The flasks of cultured cells were examined under the

inverted microscope to observe cell confluency.

After that, the old medium was removed and

discarded and cells were washed with warm (37ºC)

phosphate-buffered solution (PBS). A small volume

of trypsin (~1 mL) solution was added to the flasks

and the flasks were placed back in an incubator at

37ºC. Five to ten minutes after, cells were examined

under the inverted microscope to confirm that most

of the cells had rounded up and were detaching from

the flask surface. Finally, it was added 4 mL of fresh

growth medium to the cell suspensions. The number

of viable and dead cells/mL was determined using a

Neubauer haemocytometer and the trypan blue dye

exclusion method.

A total of eight experiments were performed for

the CACO-2 cell line. To support the cells for these

experiments it was used glass cover slips placed

inside 6-well plates; after that the final cell

suspensions were divided and placed within these 6-

well plates. The cells used were grown as

monolayers over the cover slips. After 24, 48 or 72

hours (depending on the experiment), cover slips

were collected from the 6-well plates and packed in

Petri dishes, with 3 mL of PBS solution, for the

experiments. Spectral analysis was then performed

within less than half an hour after cells were placed

in Petri dishes.

2.3 Spectrofluorimetry of CACO-2 Cell

Line

Fluorescence spectra were performed on human

carcinoma cell monolayers. The samples were

measured using a SPEX

FluoroLog

- a high

sensitivity spectrofluorometer with a SPEX1680

(0,22m) Double Spectrometer. The excitation light

was provided by a Xenon lamp of 450W with a DC

450RAM power supply, from EUROSEP

Instruments. The equipment was connected to a

computer to control and collect the data. The slit

widths of both spectrometers were adjusted in order

to provide a good spectral resolution in the

excitation and emission path.

2.4 Fluorescence Spectra of CACO-2

Cell Line

Emission spectra were recorded from 270–690 nm,

for an excitation wavelength ranging from 260 to

350 nm. These excitation wavelengths were chosen

in order to allow a comparison with data obtained

from literature. Reference spectra were obtained

with pure PBS solutions. All the spectra were

normalized subtracting the reference spectra from

the fluorescence spectra. The fluorescence emission

spectra were also corrected for the spectral response

of the spectrofluorometer. The recordings were

performed for several cell concentrations, depending

on the experiment (24, 48 or 72 hours). These cells

were maintained in PBS during the measurement,

which could in some way put in risk cell viability.

All measurements were performed under the same

experimental conditions.

3 RESULTS AND DISCUSSION

The analysis of the two epithelial cell lines AF was

performed using spectrofluorimetry on cell

monolayers. The measurements were performed on

cell monolayers to use epithelial cells as close as

possible to their in vivo tissular physiological

conditions, as cells grown as monolayers undergo

less stress than cells in suspension. This was

confirmed on previous studies on normal cells from

primary cultures, which easily grow as adherent

monolayers looking like an epithelium structure

while showing a poor viability in suspension.

Furthermore, cell monolayers can be considered as

an optically thin medium, while the acquired spectra

from cell suspensions are strongly affected by

AUTOFLUORESCENCE SPECTROSCOPY OF A HUMAN GASTROINTESTINAL CARCINOMA CELL LINE -

Design of Optical Sensors for the Detection of Early Stage Cancer

scattering, inducing spectral distortion (Villette,

2006).

The experiments started measuring the AF signal

of the CACO-2 cell line, with 24, 48 and 72 hours of

growth, placed inside a 6-well plate and slightly

immersed in PBS. The fluorescence measurements

were made for different excitation wavelengths (λ

)

- 260, 270, 280, 335, and 350 nm - and the emission

was collected in the range 270-690 nm. The average

number of viable and dead cells was determined in

each experiment (Table 2).

Table 2: Number of viable and dead CACO-2 cells, after

24, 48 and 72 hours in culture.

Time in culture

(hours)

Live Dead

Number of

cells/mL

24 16,5x10

1,25x10

48 22,0x10

1,40x10

72 63,5x10

2,50x10

In all experiments, for λ

of 260, 275 and

280 nm the emission spectra are very similar in

shape (Figures 2, 3 and 4) - all of them exhibit a

broad emission band from 310 to 370 nm, with a

peak around 340 nm, which is primarily caused by

amino acids, namely tryptophan.

However, the AF intensity diverges among all

the acquired spectra, being related with culture time.

The maximum AF intensity is achieved for a 48

hours cell culture, decreasing then as time elapses.

This can be explained by the number of viable and

dead cells. From 24 to 48 hours there is a

considerable increase in the number of viable cells,

which is translated by AF signal intensification.

From 48 to 72 hours, despite the increase in the

number of viable cells, there is a reduction in AF

signal intensity. This intensity drop (for the

minimum value) can be justified not only by the

raise in the number of dead cells, but also by an

increase in cell fragility, as cells are in culture for a

longer time, supplied by the same growth medium

(it’s important to notice that the culture medium was

not discarded and replaced by fresh growth medium

in the period of 72 hours).

For other spectra acquired using λ

of 350 and

335 nm, for the detection of NADH and NADPH,

respectively, the resulting fluorescence intensity was

approximately zero, when compared to the amino

acids AF intensity (Figures 5 and 6). This may be

justified by the fact that the AF emission from these

fluorophores, although existing, is very weak and

doesn’t superimpose over the PBS signal (reference

signal). Probably these molecules are not present in

significant amounts to contribute to a high quality

and strong signal. It may also be possible that some

other molecules partly affect the measurements by

absorbing the fluorescence or by emitting

fluorescence in the same bandwidth. These results

were very similar for all the experiments performed

(24, 48 and 72 hours growth).

0.00E+00

2.00E+10

4.00E+10

6.00E+10

8.00E+10

1.00E+11

270 310 350 390 430 470 510 550

Emissionwavelength(nm)

Autofluorescenceintensity(a.u.)

260nm

275nm

280nm

Figure 2: Autofluorescence emission spectra of a CACO-2

cell line, with 24 hours growth, obtained for different

excitation wavelengths: 260, 275 and 280 nm.

0.00E+00

5.00E+10

1.00E+11

1.50E+11

2.00E+11

2.50E+11

270 310 350 390 430 470 510 550

Emission wavelength(nm)

Autofluorescenceintensity(a.u.)

260nm

275nm

280nm

Figure 3: Autofluorescence emission spectra of a CACO-2

cell line, with 48 hours growth, obtained for different

excitation wavelengths: 260, 275 and 280 nm.

0.00E+00

1.00E+10

2.00E+10

3.00E+10

4.00E+10

5.00E+10

270 310 350 390 430 470 510 550

Emissionwavelength(nm)

Autofluorescenceintensity(a.u.)

260nm

275nm

280nm

Figure 4: Autofluorescence emission spectra of a CACO-2

cell line, with 72 hours growth, obtained for different

excitation wavelengths: 260, 275 and 280 nm.

BIODEVICES 2009 - International Conference on Biomedical Electronics and Devices

Recent studies have investigated the spectra of

human intestinal mucosa using a spectrofluorometer.

The measurements were performed using short

wavelengths for excitation, and revealed utility in

detecting neoplasia. The AF intensity increased with

neoplasia and had the spectral line shape of

tryptophan, indicating that AF emission from

tryptophan might represent a viable approach to the

detection of malignancy within colonic tissue

(Banerjee, 2000, 2002). A different study had

confirmed that the amount of amino acid related

fluorescence (emission between 300 and 380 nm) is

greater in adenomatous polyps than in the normal

tissues (DaCosta, 2003). Therefore, it can be said

that the results obtained, namely for amino acids AF

emission, are a good initial approach for the design

of optical sensors for the detection of GI early stage

cancer.

‐2.50E+09

‐1.50E+09

‐5.00E+08

5.00E+08

1.50E+09

2.50E+09

360 400 440 480 520 560 600 640

Emissionwavelength(nm)

Autofluorescenceintensity(a.u.)

350nm

Figure 5: Autofluorescence emission spectra of a CACO-2

cell line, with 48 hours growth, obtained for an excitation

wavelength of 350 nm.

‐2.50E+09

‐1.50E+09

‐5.00E+08

5.00E+08

1.50E+09

2.50E+09

345 385 425 465 505 545 585 625

Emission wavelength(nm)

Autofluorescenceintensity(a.u.)

335nm

Figure 6: Autofluorescence emission spectra of a CACO-2

cell line, with 48 hours growth, obtained for an excitation

wavelength of 335 nm.

4 CONCLUSIONS

There is a great interest in developing

autofluorescence-based spectroscopic systems for

the detection of early stage cancers. Understanding

the autofluorescent tissue components and how these

components change in concentration and distribution

with disease is essential in terms of optimizing

diagnostic techniques. Several endogenous

fluorophores have been identified and alterations in

concentrations of these are suggested for

discrimination of normal and malignant tissues.

In this article, it is characterized the AF spectra

of one human GI carcinoma cell line (CACO-2),

using several different excitation wavelengths,

which targeted different molecular species. The

obtained results showed significant AF signal for the

presence of amino acids, namely tryptophan. The

resulting spectral information of the CACO-2 cell

line is consistent with previously published results of

malignant colonic tissues, and is a good initial

approach for the design of fluorescence CMOS

optical sensors, to be incorporated on an EC, for the

differentiation of normal and malignant tissues. It is

important to notice, however, that when comparing

fluorescence spectra of cell monolayers with those

from in vivo or ex vivo tissues is has to be considered

the possible changes due to the physicochemical

microenvironment.

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BIODEVICES 2009 - International Conference on Biomedical Electronics and Devices