J . OF RECEPTOR & SIGNAL TRANSDUCTION RESEARCH, 15(1-4), 427-442 (1995)

a-MSH RECEPTOR AUTORADIOGRAPHY ON MOUSE AND HUMAN MELANOMA TISSUE

SECTIONS AND BIOPSIES+

Claudia Bagutti, Marc Oestreicher#, Walter Siegrist, Martin Oberholzer# and Alex N. Eberle*

Department of Research (ZLF) and #Institute of Pathology University Hospital and University Children's Hospital

CH-4031 Basel, Switzerland

ABSTRACT

MSH receptors and their binding characteristics of [125I]-labelled derivatives of a-MSH have been studied extensively on various mouse and human melanoma cell lines in culture. The aim of this study was t o determine the binding characteristics of a-MSH radioligands to MSH receptors occurring in experimental mouse and human melanoma tumours as well as in human melanoma biopsies. For this reason, solid tumours were grown on experimental animals by inoculation of murine B16-F1 and human D10 and HBL melanoma cells. After excision and cryosectioning of the tumours, frozen tissue sections were incubated with [(125I)Tyr2]-a-MSH or [(1251)Tyr2,Nle4,0-Phe71-cr.-MSH and specific a- MSH binding sites were visualized by subsequent autoradiography. The presence of increasing concentra-tions of unlabelled a-MSH during incub- ation with tracer led to a dose-dependent displacement of the radioligand. Quantitative analysis of the autoradiograms produced dissociation constants which were comparable with those obtained with cell binding assays: KD = 1.87 and 1.31 nmolA for B16 tumours and cells, respective- ly; 0.32 and 0.33 nmol/l for D10, and 2.24 and 1.36 nmol/l for HBL tumours and cells, respectively. This indicates similar bindmg properties

+This paper is dedicated to Professor R. Schuppli, former Director of the Department of Dermatology, University Hospital Basel, on the occasion of his 80th birthday.

427

Copyright 0 1995 by Marcel Dekker, Inc

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428 BAGUTTI ET AL.

of a-MSH radioligands t o both cultured melanoma cells and tissue sections of melanoma tumours from experimental animals. Similar binding characteristics were also observed with human melanoma tissue sections originating from biopsies of melanoma patients.

INTRODUCTION

The tridecapeptide a-melanocyte-stimulating hormone (a-MSH) is derived from proopiomelanocortin (POMC) which occurs in the pars intermedia and the pars distalis of the pituitary, in the brain and in many peripheral organs where it serves as precursor for a family of peptide hormones, the melanocortins and endorphins. a-MSH, besides its melanogenic effect on pigment cells, participates in the regulation of neurons and many tissues and cells in the periphery [l]. These divese actions are mediated by interaction of the peptide with specific MSH receptors expressed in the different tissues. Although several types of melanocortin receptors have recently been cloned [Z-71 and PCR techniques are now available for the identification of receptor mRNA [8], direct visualization of receptors is still of great interest, in particular the mapping of functional receptors in tissues with marked regional variation of receptor density. For example, MSH receptors could be identified in distinct regions of the brain (hypothalamus, hippocampus, epithalamus, thalamus, olfactostriatum, septa1 area and midbrain) using tritiated and iodinated radioligands in combination with receptor autoradiography: [(3H2)Tyr2]-a-MSH [91 and [(3H)Nle4,D-Phe7I-a-MSH(4-11) [lo], and [(125I)Tyr2,Nle4,~-Phe7l-a-MSH 1111.

Receptor autoradiography has been widely used as a technique for the identification and localization of many neurotransmitter and neuropeptide receptors [12, 131, especially in the brain where binding sites, e.g., for dopamine [141, y-aminobutyric acid (GABA) [151, acetyl- choline [16] as well as for somatostatin [17], endothelin [18] and TRH [ 191) have been visualized. Quantitative receptor autoradiography was shown to be a suitable tool for analyzing differences in the expression of receptor in patients with brain diseases as compared t o normal

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CX-MSH RECEPTOR 429

individuals [ 131. For example, Parkinson's disease is manifested not only by a significantly lower hypothalamic dopamine content compared t o normal tissue but also by a different expression of D1 and D2 subtypes of dopamine receptors [20].

The autoradiographic mapping of particular peptide receptors in excised tumour tissue was found to be an indispensable step with regard to a subsequent application of the peptide to tumour diagnosis and treatment. For example, the demonstration of a large number of somatostatin receptor expressed in pancreatic tumours by receptor autoradiography [21] was the basis for the application of a potent and metabolically stable [1111n]-labelled somatostatin analogue for tumour imaging [221.

Extensive binding analyses and photocrosslinking experiments demonstrated the presence of MSH receptors on almost all mouse and human melanoma cells [23-271. Following the recent cloning of MSH receptor subtypes 12-71, the melanoma MSH receptors have been classified as MC1R which were shown t o be exclusively expressed on melanoma cells (and melanocytes). MSH receptor identification on melanoma tumours has been extended on tissue sections originating from xenografts of mouse B16 tumours where MSH receptors were vizualized [28] and quantified [29] by receptor autoradiography. Similarly, human melanoma biopsies were analyzed for the incidence of MSH binding sites [301.

In a recent paper [311 we demonstrated the potential of short [111In]-DTPA-coupled (r.-MSH derivatives for melanoma tumour targeting in experimental animals. A future clinical application of such compounds is however dependent on the presence of a high enough number of MSH receptors on melanoma tumours which must be unequivocally demonstrated on the tumour tissue itself. Although there has already been a clinical imaging trial using [111In]-labelled a-MSH tridecapeptide [32], its failure to detect small melanoma lesions in vivo shows the need for more information about the MSH receptor status in

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430 BAGUTTI ET AL.

primary melanoma tumours and metastases of different tumour variants. However up to now, it has not been possible to quantify the expression of functional MSH receptors on human melanoma tumours. In this study, we demonstrate the quantitative determination by autoradiography of MSH receptors on both mouse and human melanoma tumours and compare the results with a binding analysis using cultured melanoma cells.

MATERIALS AND METHODS

Peptides a-MSH, [NledI-a-MSH and [Nle4,D-Phe7]-a-MSH were purchased

from Bachem AG (Bubendorf, Switzerland). All peptides were iodinated enzymatically with Na125I using Enzymobeads (Biorad, Richmond, CA) and subsequently purified in two steps by reversed-phase mini-column (Spherisorb ODS/lOpm RP-Silica, Phase Separation Inc., Norwark, CT), using a step-wise gradient of 1% TFA/ methanol, and reversed-phase HPLC, as described earlier [23,331.

Cell binding assay

Murine B16-F1 cells were cultured according to the procedure described by Siegrist et al. [24]. For this study, the human HBL line was grown in Ham F10 medium containing 10% foetal calf serum, 2 mM L-

glutamine, 50 units/ml penicillin, 50 pg/ml streptomycin and 100 pg/ml kanamycin. All media and supplements were purchased from Gibco (Paisley, U.K.). The cells were maintained in 75 cm2 Falcon tissue culture flasks at 37°C in a humified 95% air/5% C02 atmosphere. The receptor binding assay was performed as reported [23,24]. Briefly, 50 p1 10-10 M [(125I)Tyr2]-a-MSH or [(125I)Tyr2,Nle4]-a-MSH and 50 pl a- MSH (10-12 t o 10-6 M) were incubated with 5x106 B16-F1, D10 or HBL cells in 500 p1 binding medium (modified Eagle's medium supplemented with 25 mM HEPES, 0.2% BSA and 0.3-1 mM 1,lO-phenanthroline) for 3 h at 15°C (B16-F1) and for 2 h at 37°C (D10, HBL), respectively. The reaction was stopped by oil centrifugation of the cell suspension at 4°C. The radioactivity of the pellet and the supernatant was measured in a y-

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a-MSH RECEPTOR 43 1

counter (Packard, Meriden, CT). Binding curves were calculated with the computer fitting program Ligand [341.

Experimental melanoma tumours in mice Melanoma tumours were generated by subcutaneous injection of

2x105 B16-F1 or 1 .2~107 D10 or HBL cells into both flanks of C57B1/6J or Balb/c n d n u mice. After 8-12 days (B16-F1), 64-70 days (D10) and 26-30 days (HBL) the mice were killed and the tumours were dissected, embedded in Tissue-Tek@ (Miles Lab. Inc., Naperville, U.S.A.) and frozen for cryosectioning.

Human melanoma biopsies Human melanoma biopsies originated from our own Hospital o r

were kindly supplied by Prof. J .-0. Gebbers, Institute of Pathology, Kantonsspital Lucerne, Switzerland. They were histopathologically diagnosed as melanomas. Of the three biopsies used in this paper (numbered #1, 2 and 3), #1 and #2 were metastatic tumours whereas #3 derived from a primary melanoma.

Receptor autoradiography The tumours were cut into sections of 14 pm using a cryotome at

-13°C and the sections were thaw-mounted onto slides which had been gelatinized with 0.5% gelatine in 0.05% KCr(S04)z. 12H20. All tumour- containing slides were preincubated in binding medium for 30 min at 4°C. The B16-F1-derived tumour sections were incubated with 7.5 x 105 cpm of [125I]-a-MSH for 90 min at room temperature in the absence or presence of 10-12 to 10-6 M a-MSH. The D10- and HBL-derived sections as well as the biopsy sections were incubated in the same way using 6x104 cpm of [( 125I)Tyr2,Nle4,o-Phe7]-a-MSH as radioligand. All peptides and tracers were diluted with binding medium. The slides were then washed on ice, three times in binding medium and once in H20, before being dried under a cold air-stream. The sections were exposed to a high-resolution film (Hyperfilm omax, Amersham, U.K.) at -70°C for 5- 14 days.

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432 BAGUTTI ET AL.

Quantification of autoradiograms Densitometric analysis of the autoradiograms was performed with

a video-connected image analyzing system WIDAS, Kontron, Switzerland). The optical density per area for each object was corrected by subtraction of the background optical density of the film to give the integrated optical density per object. By applying the same measure- ments and calculations to [ 125II-polymer standard strips (Amersham, U.K.), which were simultaneously exposed with the thin sections, standardization of the radioactivity of the probe sections was achieved.

RESULTS

The binding affinity of a-MSH to the MSH receptor has been determined for three cultured mouse or human melanoma cell lines in vitro. The mean dissociation constants (KO) obtained in competition binding assays using radioiodinated a-MSH as tracer and unlabelled a- MSH as displacer (Fig. 1) were 1.31 ? 0.3 nmolA for B16-F1 mouse cells, 0.33 k 0.1 nmol/l for human D10 cells and 1.36 ? 0.4 nmol/l for human HBL cells, respectively.

In order to obtain information on the expression of functional MSH receptors on melanoma tumours grown in a semi-natural environment in vivo, we inoculated all three melanoma cell lines into mice and examined the binding properties of the MSH receptors by receptor autoradiography using thin sections of the tumours. Specific receptor binding was demonstrated with the dose-dependent displacement by unlabelled a- MSH of the radioligands, [125I]-a-MSH used for B16-F1, and [(125I)Tyr2,

Nle4,D-Phe7)-a-MSH used for D10 and HBL tumour sections. Densito- metric analysis of the autoradiograms of the individual thin sections presented in Figure 2 revealed dose-response curves similar to those obtained for binding with isolated cells (Fig. 3). Calculation of the corresponding dissociation constants resulted in KD values which hardly differend from those obtained for isolated cells (KD = 1.87 k 1.6 nmoVl for B16-F1, 0.32 k 0.1 nmol/l for D10, and 2.24+ 0.4 nmol/l for HBL

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a-MSH RECEPTOR

100-

a0 - 0) c D

L3 0 c 0

v)

._

._ c 60-

.-

2 40-

s 20 -

0 -

433

I Isolated cells

t I I I I -10.5 -9.5 -8.5 -7.5 -6.5 -5.5

Concentration (log M)

FIG. 1. Log dose-response curves from competition binding assays with B16-F1 mouse (01, human D10 (A) and HBL ( 0 ) melanoma cells using [(1251)Tyrz]-a-MSH (B16, D10) and [(125I)Tyr2, Nle41-a-MSH (HBL) as radioligand. Mean values f standard deviation were calculated from three different experiments.

tumours), thus demonstating the similarity of the bindmg properties of the MSH receptors in tumour sections and isolated cells.

By performing receptor autoradiography on human melanoma biopsies it became evident that the conservation of functional MSH receptors was a very critical factor. Crucial elements were promptness of freezing as well as the method of coating of the slides. In addition, the melanoma biopsies showed some heterogeneity within the melanoma nodule with respect to melanoma cells and t o MSH receptor expression, respectively. Figure 4 displays a representative example of an a-MSH receptor autoradiogram showing total and non-specific binding of [( 1251) Tyr2,Nle4,D-Phe7)-a-MSH. The ratios of total to non-specific binding of

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434 BAGUTTI ET AL.

FIG.2. Examples of autoradiograms of B16-F1, D10 and HBL melanoma sections incubated with [(125I)Tyr2]-a-MSH (B16) and [( 125I)Tyr2,Nle4,~-Phe7l-a-MSH (D 10, HBL) in the presence of increas- ing concentrations of cold a-MSH. (Concentrations of a-MSH for B16 and D10, 1: 0; 2: 0.63 nM; 3: 18.7 nM; 4: 1.9 pM, and for HBL, 1: 0; 2: 18.7 nM; 3: 1.9 pM).

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a-MSH RECEPTOR 435

80 - cn c U ._ C 60- 0 c 0

v)

.-

n .-

40-

8. 20 -

0 -

Tumour sections

I I I I I I

-11 -10 -9 -8 -7 -6 Concentration (log M)

FIG. 3. Quantitative analysis of series of sections incubated in the presence of increasing concentration of a-MSH. Competition binding curves obtained with B16-F1 mouse (O), human D10 (A) and HBL (0 ) experimental melanomas. Mean values k standard deviation were calculated from three t o four hfferent experiments.

three human biopsies were compared with those of the experimental human melanoma, indicating that the binding capacity was only slightly higher for melanomas deriving &om cultured cells (Fig. 5).

DISCUSSION

Although human melanoma tumours deriving from biopsies have been analysed for the incidence of MSH binding sites [30], the quantification of the expression of MSH receptors on human melanoma tissue has never been attempted. We undertook a comparative determination of MSH receptors expressed on mouse and human cell

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436 BAGUTTI ET AL.

TABLE 1

Dissociation constants (KD) of a-MSH binding to melanoma cells and experimental melanoma tumours

~

Cell line Dissociation constants/nmol/l

Isolated cellsa Tumour sectionsb

B16-F1 1.31 _+ 0.3 1.87 f 1.6

n 10 0.33 _+ 0.1 0.32 f 0.1

HBL 1.36 k 0.4 2.24 * 0.4

Binding experiments using a isolated melanoma cells and b tumour sections from melanomas generated in mice. KD values of the latter were calculated by densitometric analysis of the autoradiograms.

FIG. 4. Representative example of an MSH receptor autoradiogram of a human metastatic melanoma thin section (biopsie #1). 'tt' represents total binding of [( 125I)Tyr2,Nle4,D-Phe7l-a-MSH and 'ns' non-specific binding in the presence of 1.9 pM unlabelled a-MSH.

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CX-MSH RECEPTOR 437

B16-Fl

HBL

D10

# I

#2

#3

rA

I I I I

0 0.5 1 1.5 2 2.5 3 3.5 Binding capacity (totalhon-specific)

FIG.5. Quantitative analysis of the ratio of total to non-specific binding of three human biopsies (#l , 2, 3 ) and B16-F1, HBL and D10 experimental melanomas (see Figures 2 and 3) . Total and non-specific binding of [(125I)Tyr2,Nle4,D-Phe7]-a-MSH was determined in the absence or presence of 1.9 pM a-MSH. Mean values k standard deviation were calculated from three to four different experiments.

lines in vitro and on thin sections of mouse and human melanoma tumours grown in animals.

The dissociation constant (KD) for the a-MSH radioligand with cultured B16-F1 cells was only marginally lower than the value obtained with experimental B16 tumour grown in mice (KD = 1.31 and 1.87 nmol/l, respectively). Similar results were obtained with human D10 and HBL cells and their homologous tumour grown in vivo (KD = 1.36 and 2.24 nmol/l, respectively, for DlO, and KD = 0.33 and 0.32 nmol/l, respectively, for HBL). This indicates that the binding properties of the MSH receptor are the same in isolated cells and in tissue sections.

We have then applied this method to human melanoma biopsies. The total-to-nonspecific binding was only slightly lower as compared t o that observed with experimental melanoma (see Fig. 4), indicating a

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43 8 BAGUTTI ET AL.

relatively similar binding capacity of the human melanoma biopsies and experimental melanomas. The quantitative determination of the receptor status and its functionality could be very important for the analysis of different tumour variants with respect to differences in expression of the MSH receptor. There are controversial reports as to whether MSH receptors are present on all human melanomas or not. Tatro et al. [30] hypothesized that receptors for MSH may not be present on all human melanomas. Conversely, Wraight and colleagues [32] reported that failure to detect melanoma lesions in a clinical imaging study using a radiolabelled a-MSH derivative was most probably due to the small size of the tumour and not to a lack of MSH receptors. In addition, the general consideration that amelanotic lesions may be deficient of MSH receptors was negated by the findings of Pawelek et al. [351. The stage of tumour progression of an individual melanoma tumour could be another factor influencing the receptor density. This information could prove useful for the diagnosis and therapy of melanoma. Since various approaches in hagnosing metastases are based on the binding affinity of a-MSH to its receptor by targeting radionuclides to the tumours, it is of crucial interest t o know whether malignant melanomas express MSH receptors in all stages of progression and in an abundance high enough for tumour imaging.

ACKNOWLEDGMENTS

We are grateful t o Prof. J.-0. Gebbers, Institute of Pathology, Kantonsspital Lucerne, for the supply of melanoma tumour probes, and to Mrs. Marlis Kaspar, Institute of Pathology, University Hospital Basel for the preparation of the cryosections. We are indepted to Dr. G. Ghanem, Brussels, for the kind gift of human HBL cells. This work was supported by the Swiss Cancer League and its Robert Wenner Fund and the Swiss National Science Foundation.

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