Secondary Ion Mass Spectrometry (SIMS) Microscopy:A New Tool for Pharmacological Studies in HumansPHILIPPE FRAGU1* AND EDMOND KAHN2

1Equipe de Microscopie Ionique INSERM, Institut Gustave-Roussy, Villejuif France2INSERM U66 Hopital de la Pitie, Paris France

KEY WORDS SIMS microscopy; human pharmacology; 5-fluorouracil; 48-iododeoxyrubicin;cancer

ABSTRACT The secondary ion mass spectrometry (SIMS) microscope has opened new fields inbiological investigation because of its ability to map chemical elements that are either naturallypresent in tissue or introduced for diagnostic or therapeutic purposes. In this review, we willdescribe our attempts to localise and quantify antitumor drugs in histological sections to betterevaluate successful early cancer treatment. Detection is dependent on the presence of chemicalelements in the drug structure, for example halogens (F, Br, I, At) which are imaged and quantifiedwithin the nuclei. Our methodological approach combines the results obtained with ionic andphotonic microscopes on serial sections. Thus, the different structures in tumor tissue (blood vesselsand cells) can be identified and drug localisation visualized. Using embedded samples, wedemonstrate that both fluorine (19F) in 5-Fluorouracil and iodine (127I) in 48-iododeoxyrubicin can bemapped in human biopsy material obtained after in vivo chemotherapy. Microsc. Res. Tech.36:296–300, 1997. r 1997 Wiley-Liss, Inc.

INTRODUCTIONThe identification, localization, and quantification of

intracellular chemical elements is an area of scientificendeavour which has developed continuously over thepast 30 years (Ingram et al., 1989). The Secondary IonMass Spectrometry (SIMS) microscope (Castaing andSlodzian, 1962) has opened new fields in biologicalinvestigation because of its ability to map chemicalelements which are either naturally present in tissuesections or are introduced for diagnostic or therapeuticpurposes. As reviewed recently (Fragu et al., 1994), thismicroscopic quantitative imaging technique has beenused to study plant and animal physiology. In biomedi-cine, SIMS microscopy has been shown to be themethod of choice for the evaluation of tissue distribu-tion of radiopharmaceuticals (Clerc et al., 1993, 1995;Fourre et al., 1992). Another area in which SIMS can beused in medicine is the localisation of drug markers intumor tissue. Detection depends on the presence ofchemical elements in the drug structure such as halo-gens (F, Br, I and At). Provided the chemical elementhas not been released during sample preparation, it canbe localized on human biopsy material (Fragu et al.,1991, 1992). In this latter case, ion microscopic imagesneed to be compared to optical microscopic images todeterminewhether the drugs present are in the neoplas-tic cells, the cells of normal residual tissue or stromaltissue. In the present review, we will describe ourattempts to evaluate successful early cancer treatmentwith this new technique.

MATERIAL AND METHODSBiological specimen

Biopsies of gastric mucosa (7 patients) were obtainedduring endoscopy 15 minutes after the beginning of

chemotherapy with 5-Fluorouracil (5-FU: 1 g/m2/day),an antagonist of pyrimidine metabolism involved inDNA and RNA synthesis. Cutaneous metastases in 7patients with squamous cell carcinoma were also biop-sied 10minutes after the administration of 48-iododeoxy-rubicin (IDX: 80mg/m2, 10min i.v. infusion), an interca-lating agent which impairs DNA template functionrequired for DNAand RNAsynthesis.

Sample preparationThe direct observation of cryofixed tissue on a cooled

microscope stage is actually impossible. Ion mapping ofall the linked or diffusible elements and molecules canonly be achieved by using cryotechniques (Kellen-berger, 1991) which allow their in situ immobilization(Stelly et al., 1995). This physical procedure was used todetect 5-Fu in the cytoplasm because this small mol-ecule was reported to be highly diffusible in the organicsolvents used in the chemical procedure.With cryometh-ods, tissues are cryofixed by ultra rapid immersion(5000K sec21) in liquid propane, subcooled (77K) byliquid nitrogen, cryosubstituted in acetone (183K) andthen embedded in Lowicryl K11M (Chemische WerkeLowi, Germany), at 213K.However, chemical fixation methods currently used

for histology are very suitable for the analysis ofelements bound to macromolecules (Rognoni et Simon,1974). These methods were used for the detection ofboth DNA and drug interaction in cell nuclei. Tissues

Received 8 February 1995; accepted in revised form 8March 1995.*Correspondence to: Docteur Philippe Fragu, Service de Medecine Nucleaire,

Institut Gustave-Roussy, 94805 Villejuif Cedex, France.Contract grant sponsor: Caisse Nationale d’Assurance Maladie des Travail-

leurs Salaries; Contract grant number: CNAMTS 998583.

MICROSCOPY RESEARCH AND TECHNIQUE 36:296–300 (1997)

r 1997 WILEY-LISS, INC.

were fixed in a solution containing 1 g/l glutaraldehydeand 20 g/l paraformaldehyde in cacodylate buffer (0.1MpH 7.4). Fragments were dehydrated in ethanol andembedded in Historesin (Pharmacia, Upsalla, Sweden).In both cases serial semi-thin sections (3 µm and 1.5

µm) are cut at room temperature and laid, respectively,on ultrapure gold holders for SIMS analysis and onglass slides for histological controls.

SIMSmicroscopeThe instrument used is the IMS 3F microscope

(Cameca Courbevoie, France) fitted with a Cs1 sourcefor halogen detection. The SIMSmicroscope is based onthe sputtering phenomenon induced by the bombard-ment of a biological sample surface with an energetic‘‘primary ion’’ beam. Part of the sputtered matter iscomposed of positive or negative, single or polyatomicions which are characteristic of the atomic compositionof the analyzed area. These ‘‘secondary ions’’ are thencollected and separated according to their mass in amass spectrometer.A multichannel plate, a fluorescent screen and a SIT

camera linked to an image processing system arecombined for the visualization of the analytical imageof the selected element. The system is also connected toa photonic microscope used for controls on serial sec-

tions (Olivo et al., 1989). Original software permitsimage digitisation (512 3 512 pixels, 8 bits), summa-tion (25 images/second) and final storage on a digitaloptical disk (800 MB). We developed an on- and off-linesuperimposition program in which a previously ac-quired image is overlaid with a new one, so thatreal-time control is available to ensure optimal imagingconditions. Two tasks can be achieved with such aprogram, namely, the analysis of distribution, and thesuperimposition of several elements in the same ana-lyzed field.The selected ion beam intensity can also be measured

with an electron multiplier. In SIMS, there is a directrelationship between the secondary ion beam currentand the local elemental concentration of the specimen.Using an internal reference such as carbon and aniodine standard curve established with an iodized resin(Jeusset et al., 1995) the results are expressed in ng/mgof tissue within the analysed area. Cell nuclei wereselected with an adapted aperture (8 µm diameter).Each quantitative result is the mean of 10 measure-ments on the same nucleus.Sample Analysis. Section flatness and adherence

to gold holders, accessible with resin embedded mate-rial, is essential for analysis. It avoids relief effects andminimizes charge effects, at least in microscope mode.A

Fig. 1. Opical (a) and ionic images (b–d) of human gastric mucosa (normal part). The phosphorusdistribution (b) gives the histological structure of the gastric gland in which elements of interest such assuphur (c) and chlorine (d) of mucus can be evidenced. Image field: 150 µm.

297SIMS AND PHARMACOLOGICAL STUDIES, IN HUMANS

low primary ion beam intensity is necessary (5–30 nA),especially on account of the insulating properties ofembedded biological material.The complexity of the biological matrix, rich in

carbon, hydrogen, oxygen, nitrogen and phosphorus,leads to the emission of a large number of polyatomicions partially composed of these major elements. Amajor drawback is that most of these polyatomic ionsgenerate artifactual signals which may interfere withthe selected element: the greater the mass, the morenumerous the polyatomic ions, and the higher the massresolution needed. Furthermore, whatever the mass,when the element to be analyzed is present at a lowconcentration and/or when the ion yield is inadequate,the polyatomic ion distribution is superimposed on thatof the element of interest under low mass resolutionconditions. High mass resolution (M/DM . 2000) isthen indispensible to separate the specific signal (Fourreet al., 1992) and has been shown to be as easilyaccessible with the IMS 3F in imaging mode as inquantitative or spectral mode.

RESULTSFigure 1 shows the images of serial sections of human

gastric mucosa with optical (A) and ionic (B–D) micro-scopes. The main advantage of SIMS microscopy is itsability to preserve elemental mapping in relation to thehistological structure. Here this structure (B) is definedby the distribution of phosphorus (31P) associated withcellular nuclear DNA and the phosphorylated cytoplas-mic molecules. Thus, it is possible to localize sulfur andchlorine bound to macromolecules in the same field;both elements are distributed at the apical pole of theglandular cell where mucus is present. In these treatedpatients, the fluorine of 5-Fu is mainly observed in thenuclei of normal and neoplastic gastric cells (Fig. 2A).When cryotechniques were used for sample prepara-tion, fluorine was also evidenced in the cytoplasm ofgastric cells (Fig 2B).Figure 3 shows the mapping of iodine of IDX in a

cutaneous metastasis of a squamous cell carcinoma.Our methodological approach combined the resultsobtained on serial sections with the ionic and photonicmicroscopes. Thus, the different structures of tumortissue (blood vessels and tumor cells) can be identifiedand screened for drug localisation. The drug is seen tobe distributed in the nuclei of both endothelial andtumor cells (Fig. 3 A–B). 127I emission appears to bevariable among the tumor cells (Fig. 3 C–D).Since the images failed to reflect element concentra-

tion accurately, fluorine and iodine were evaluatedquantitatively on the phosphorus image. The overallresults of concentrations of these two elements aresummarized in Figure 4. The 5-Fu concentrationsmeasured in the nuclei of the specimens obtained from7 patients ranged from 5–25 ng per mg of wet tissue(Fig. 4A) while that of IDX was more scattered andranged from 0.4 to 400 ng per mg of wet tissue (Fig. 4B).

DISCUSSIONLocalizing drug markers in tumor tissue seems to be

the main application of SIMS in medicine. It is awell-known fact that the penetration of cytotoxic agentsin the cells of neoplastic tissue is critical for theiractivity although the in vivo distribution between nor-

mal and tumor cells remains enigmatic. Most of theresults reported by other groups were obtained onisolated cells cultured on gold holders and observeddirectly without embedding (Adovelande et al., 1994;Berry et al., 1994; Hindie et al., 1989, 1992). However,section flatness is not present when isolated cells areobserved directly without embedding. This leads todifferential erosion rates between the cytoplasm andthe nucleus (Hallegot et al., 1990), especially when cellsare observed in microscope mode.Our results, obtained with embedded samples, dem-

onstrate the interest of SIMS microscopy for the local-ization of halogeneous antitumor drugs after in vivoadministration in humans. It should be noted that thisapproach could be extended to all molecules afterlabeling with a stable isotope (Hindie et al., 1992) aswell as to the intracellular localisation of boronateddrugs in tumor tissue for neutron therapy (Bennet etal., 1992). One of the major advantages of SIMSmicros-copy is its capacity to quantitatively measure elements

Fig. 2. 5-FU mapping. Computerised image of fluorine (white-purple) superimposed onto that of phosphorus showing the histologi-cal structure of human gastric mucosa obtained by biopsy duringtreatment and fixed either chemically (a) or physically (b). Theseimages demonstrate that the drug had reached its target but onlycryopreparation will allow drug visualization within the cytoplasmicarea. Image field: 60 µm.

298 P. FRAGU AND E. KAHN

in histological specimens without cell disruption. Thisraises the question of the interest of quantitative drugmappingwith regard to clinical response. Such informa-tion would complement that given by HPLC plasmaevaluation.Numerous chemoresistance mechanisms have been

identified (Taperio et al., 1989), one of the most impor-tant is the absence of drug penetration. This can now beexplored at the cellular level through SIMSmicroscopy,in vivo and in clinical studies. Our methodologicalapproach combines the results of the ionic and photonicmicroscope. Here is a means by which inadequate drugconcentration in a given tissue can be correlated withthe expression of the multidrug resistance gene re-vealed either by immunohistochemistry and/or by insitu hybridization. Using cryotechniques for samplepreparation enables the imaging methods of the differ-entmicroscopes to be used on serial sections of the sameembedded specimens (Fragu et al., 1990). Thus, theadequacy of drug distribution and its corollary, success-ful targeting and treatment, could be better evaluatedwith limited requirements in specimen material. Fur-thermore, bromodeoxyuridine (BrdU), which is a thymi-dine analog involved in DNA synthesis during the S

phase of the cell cycle, can also be detected after in vivoinjection (Casiraghi et al., 1993; Clerc et al., 1993). Thesimultaneous injection of an antitumor drug and BrdU,used as a radiosensitizer in humans (Mitchell et al.,1986), will allow a better appraisal of drug penetrationin relation to the cell cycle.Specifications regarding the lateral resolution of SIMS

is of critical importance for biologists. The IMS 3Fmicroscope is well adapted to the study of elementdistribution at the tissue level. It permits elementlocalization in the cytoplasm or the nucleus but cannotanalyze the chemical content of cellular organelles.This characteristic may dissuade some pharmacolo-gists from using SIMS microscopy in research. How-ever, with the current developments in instrumenta-tion (Levi-Setti et al., 1992; Slodzian et al., 1992) a newgeneration of instruments specifically designed toachieve high lateral resolving power (,0.1 µm) andhigh mass resolution with efficient transmission couldbe manufactured in a not too distant future. It shouldbe kept in mind that sample preparation remainscrucial if the best resolution is to be obtained (Brianconet al., 1994). Many pharmacological problems willbenefit from the unique potential of SIMS microscopy

Fig. 3. IDX mapping. Optical and ion images obtained from two serial cross-sections of cutaneousmetastasis of squamous cell carcinoma. In the section observed with the optical microscope (a–b) thearchitecture of the tissue can be easily identified. 127I in IDX is mainly detected inside the nuclei (c–d).Image field: 150 µm.

299SIMS AND PHARMACOLOGICAL STUDIES, IN HUMANS

as new ion probes become capable of quantitativechemical mapping at the ultrastructural level.

ACKNOWLEDGMENTSThe authors express their gratitude to Lorna Saint

Ange for editing the manuscript and Josette Jeusset forthe preparation of the illustrations.

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Fig. 4. SIMS quantification of 5-Fu (A) and IDX (B). Each reportedvalue is the mean of 10 consecutive measurements on the same point.The results of the different patients are pooled.

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