Proc. Nati Acad. Sci. USAVol. 79, pp. 2379-2383, April 1982Medical Sciences

Human lung small-cell carcinoma contains bombesin(radioimmunoassay/immunohistochemistry/peptide hormones/high-performance liquid chromatography)

MICHAEL D. ERISMAN*t, R. ILONA LINNOILA*t, OSCAR HERNANDEZ§, RICHARD P. DiAUGUSTINE*, ANDLAWRENCE H. LAZARUS*¶*Laboratory of Pulmonary Function and Toxicology, and §Laboratory of Environmental Chemistry, National Institute of Environmental Health Sciences,Research Triangle Park, North Carolina 27709

Communicated by George H. Hitchings, December 18, 1981

ABSTRACT The presence of immunoreactive bombesin in ahuman lung small-cell carcinoma grown in nude mice was estab-lished by several criteria: (i) Radioimmunoassay of tissue extractsfor bombesin revealed approximately 6.5 pmol/g of tissue; (ii)bombesin was found in 12-14% of the tumor cells by immuno-histochemical localization; (iii) gel filtration of small-cell carcinomaextract on Sephadex G-75 and Bio-Gel P-4 gave only a single peakofimmunoreactivity, which occurred at the elution volume ofbom-besin; and (iv) reverse-phase HPLC of acid-solubilized extractsseparated the immunoreactive material into three discrete peaks,one of which eluted with a retention time identical to that of syn-thetic bombesin. The presence of bombesin may represent theectopic expression of this peptide in small-cell carcinoma, becauseimmunoreactive bombesin was found in human fetal and neonatallung but apparently not in adult lung tissue [Wharton, J., Polak,J. M., Bloom, S. R., Ghatei, M. A., Solcia, E., Brown, M. R. &Pearse, A. G. E. (1978) Nature (London) 273, 769-770]. The im-munoreactive bombesin previously found in mammalian tissuesis considerably larger than amphibian bombesin; these data sub-stantiate the presence of a mammalian form of bombesin in a hu-man tumor that may have a structure similar to that of the am-phibian peptide.

Small oat-cell carcinoma accounts for 10-25% of identifiablehuman lung cancers (1). This neoplasm is characterized by arapid growth rate and a propensity to metastasize (1) and is fre-quently a source for "ectopic" hormone production (1-7). Manyof the clinical symptoms manifested by patients with small-cellcarcinoma (SCC) can be attributed to increased levels ofectopichormones (5-7). For example, the syndrome of inappropriatesecretion of antidiuretic hormone is apparently due to[Arg]vasopressin (5, 6, 8), whereas Cushing syndrome is con-sidered to be caused by ectopic adrenocorticotropin (ACTH)formation and secretion, which is nonsuppressible by dexa-methasone (3, 5, 7). Other symptoms may be due to the pres-ence ofpresently unknown or undiscovered peptide hormones.In this regard, bombesin, a tetradecapeptide from anuran skin,elicits numerous physiological and pharmacological responses(9, 10) that resemble some of the clinical symptoms observedfor SCC.(5, 11). Bombesin-like immunoreactivity was detectedin numerous mammalian tissues (12-20), including human fetaland neonatal lung, but is apparently absent from adult lung (21).Because many immunological and enzymological changes thatappear in tumors resemble those normally associated with fetaltissues, it seemed reasonable to evaluate the presence of bom-besin in a lung neoplasm noted for ectopic hormone production.Bombesin in a human lung SCC grown in nude mice was dem-onstrated by (i) immunohistochemical localization of this pep-

tide in the tumor, (ii) its quantitation in extracts by radioim-munoassay, and (iii) analysis by gel-filtration chromatographyand reverse-phase HPLC.

MATERIALS AND METHODSTumor. A SCC resected from a 51-year-old man with few

apparent clinical endocrinopathies was propagated subcutane-ously in athymic (nude) mice by Reid et al. (22) and generouslysupplied by G. Sato (University of California, San Diego). Tu-mors from two separate passages were analyzed separately. Thetumors, 8.71 and 6.36 g wet weight, were removed and sec-tioned for histology, and the remainders of each were mincedat 10C. They were homogenized in 5 ml of ice-cold 0.01 M so-dium phosphate buffer, pH 6.8, containing 1 mM phenylmeth-ylsulfonyl fluoride. The homogenate was slowly added to 25 mlof boiling water, boiled 30 min, and cooled on ice; then formicacid was added to a final concentration of 1.0 M (23, 24) and themixture was stirred for 60 min. The extract was clarified by cen-trifugation (37,000 X g for 30 min at 40C), and the supernatantwas freeze-dried. The yield was approximately 33 mg of dryextract per g of tissue.

Immunohistochemistry. Sections of tumor (0.1 cm3) werefrozen to the temperature of liquid nitrogen, lyophilized, andfixed with either diethylpyrocarbonate or parabenzoquinonevapors (25, 26); other sections (0.03 cm3) were fixed either in10% buffered formalin or Bouin's fluid and embedded in par-affin. Samples for electron microscopy (0.03 cm3) were fixed in2.6% glutaraldehyde/2.0% formaldehyde (wt/vol) and embed-ded in Epon. Immunohistochemistry used an improved im-munoglobulin-enzyme bridge technique (27) with antiseraagainst the following peptides at 1:1,000 dilution: ,B subunit ofhuman chorionic gonadotropin (hCGB; batch 2, Pituitary Hor-mone Distribution Program, National Institutes of Health),bombesin (see below), physalaemin (PS-1) (23, 24), ACTH (A7,midregion specific) (28), Mr 16,000 region of pro-opiomelano-cortin (B. Eipper, University of Colorado) and human /3-en-dorphin (V. Fang, University of Chicago). Sheep anti-rabbitgamma globulin was purchased from Antibodies, Inc. (Davis,CA), and rabbit anti-peroxidase serum and sheep anti-rabbitantiserum were from Cappel Laboratories (Cochranville, PA).Controls for the specificity of the interaction included incuba-tion of serial sections oftissues with antigen-inactivated antisera

Abbreviations: SCC, small-cell carcinoma; ACTH, adrenocorticotropin;RIA, radioimmunoassay; GH, somatotropin (growth hormone), hCG',,B subunit of human chorionic gonadotropin.t Present address: Institute for Cancer Research, Fox Chase, Phila-delphia, PA 19111.

t Present address: Laboratory of Pathology, NCI, NIH, Bethesda, MD20205.

¶ To whom reprint requests should be addressed.

2379

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-nent" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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FIG. 1. Immunohistochemical localization of bombesin in SCC. (A) Hematoxylin/eosin stain of a diethylpyrocarbonate vapor-fixed (26) sample.(x 120.) (B-F) Immunostaining (black areas denoted by arrows) on tissue fixed with parabenzoquinone vapor (25), which gives the photomicrographstheir grayish tone. (B-D) Consecutive serial sections (6 am) immunostained with hCGB antiserum (B), physalaemin antiserum (C), and ACTHantiserum (D) and counterstained with toluidine blue. (x 290.) (E andF) Serial sections (12 gim) immunostained with bombesin (E) and physalaemin(F) antisera. (x 290.)

(100-250 ug of peptide per ml) or normal rabbit serum. Ar-gyrophilia was determined by using the silver impregnationmethod of Grimelius (29) on Bouin's fixed paraffin sections.

Radioimmunoassays (RIAs). [Tyr8]Bombesin (donated by J.E. Rivier, the Salk Institute, La Jolla, CA) was iodinated andpurified according to published procedures (30). Antiserum to

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bombesin was raised in rabbits by injecting TPCK-trypsin(Worthington)-digested bombesin (des-<Glu'-Arg3-bombesin)coupled to keyhole limpet hemocyanin with carbodiimide (31).Physalaemin was immunoassayed as published (23, 24). Wethank C. Cooper for assays of calcitonin and gastrin and J. vanWyk for somatomedin C (both at the University of North Car-olina, Chapel Hill). The hCG/3 level was determined by En-docrine Sciences (Tarzana, CA), and human somatotropin(hGH, human growth hormone) was measured with RIA kitssupplied by CIS Radiopharmaceuticals (Bedford, MA).The bombesin RIA mixture contained 50 mM sodium phos-

phate at pH 7.6, 0.025% bovine serum albumin, 0.085% gammaglobulins, 1% Trasylol, 30 mM 2-mercaptoethanol, 25 mMNaCl, 5,000 cpm of "WI-labeled [Tyr8]bombesin, antiserum(BM-XII-165-4; 1:4,000 final dilution), and either bombesinstandards (Bachem Fine Chemicals, Torrance, CA) or extractin a final volume of 100 ,1. Incubation of assay mixtures in poly-propylene tubes was complete after 22 hr at 40C and was ter-minated by adding 0.05 ml of outdated human plasma and 1 mlof 15.6% polyethylene glycol 6000. After centrifugation of thesamples and aspiration of the supernatants, the radioactivitiesin the pellets were measured. The background in the absenceof antiserum or antiserum in the presence of excess bombesin(60 pmol) was 3.61 ± 1.54% of the total cpm (mean ± SD). Ourbombesin antiserum recognized the region between residuesLeu4 and Gln-7 due to the slight crossreactivity with litorin(0.14%) and substance P (0.00027%). Other peptides (physa-laemin, neurotensin, and bradykinin) were ineffective in com-petition in amounts as high as 10 nmol. The sensitivity of theassay (50% displacement) was 21.1 ± 9.6 pg (12.5 ± 5.7 fmol).

Chromatography. The lyophilized extract of SCC was redis-solved in 0.1 M acetic acid/i mM EDTA and clarified by cen-trifugation; 52% of the extract remained insoluble and was dis-carded. Aliquots of that solution were fractionated on aSephadex G-75 column (1.5 X 90 cm) equilibrated in acid/EDTA at4°C. Other samples were resolubilized in 1.0 M formicacid at 450C and clarified by centrifugation, and 150 ,ul of thissolution was chromatographed on Bio-Gel P-4 (100-200 mesh;0.7 x 49cm; Bio-Rad) (23, 24). Aliquots of the 0.25-ml fractionswere immunoassayed directly. Samples were injected intoeither a Spherisorb octadecylsilica (ODS) 5-,um or a WhatmanODS 5-gm column, using Laboratory Data Control HPLC unitsfitted with Rheodyne injectors, and were eluted with lineargradients of one of the following solvent systems: (a) 20-75%(vol/vol) methanol in 0.01 M Tris HCl, pH 7.0, (b) 0-80% (vol/vol) acetonitrile in 0.01 M heptafluorobutyric acid (32), or (c)0-60% (vol/vol) acetonitrile in 0.02 M sodium phosphate, pH6.97. After a 5-min delay, fractions were collected at 20-sec to1-min intervals at a flow rate of 0.8-1.0 ml/min. A blank col-umn run was performed before each analysis of tumor extract;these controls established a true background for the RIA andruled out any possible contamination of the injector, precol-umn, or metal/Teflon surfaces by either the extract or the syn-thetic bombesin standard.

RESULTSElectron microscopy of this human lung SCC propagated inathymic mice revealed the high nucleus-to-cytoplasm ratio typ-ical of these tumors (11, 33, 34). Very few, if any, small dense-cored vesicles were identified in the cells, and the cytoplasmwas essentially free ofrough endoplasmic reticulum. At the lightmicroscope level, SCC was composed of small fusiform cells(Fig. IA). Silver impregnation indicated scattered groups of2-10 argyrophilic cells that account for about 10% of the totalcell population in the tumor.

Immunohistochemistry. Previous studies showed that vapor-fixed tissues gave the best results for the immunohistochemicallocalization of bombesin-like (12, 15) and physalaemin-like im-munoreactivities in mammalian tissues (24). In the presentstudy positive immunostaining was observed after incubationwith antisera to several peptides and hormones: (i) hCGB wasfound in approximately 9-13% of the tumor cells in severalfields ofview (Fig. 1B); (ii) physalaemin was observed in 9-11%of the cells (Fig. 1 C and F); and (iii) bombesin occurred in12-14% of the cells (Fig. 1E). The specific immunoprecipitateformed by these antisera was located in either the same singlecells or groups of cells, on the basis of their localization in serialsections. Groups of 2-15 immunopositive cells were scatteredunevenly throughout the specimen. Cells containing ACTH-like immunoreactivity were infrequent and did not occur in thesame region as cells immunopositive for bombesin, physalae-min, or hCG3 (Fig. 1C). No immunostaining was detected byusing antisera to the Mr 16,000 region of pro-opiomelanocortinor to ,3-endorphin, or by using controls of nonimmune serumor antisera previously adsorbed with excess peptide.RIA of Peptides. Further characterization of the tumor in-

cluded RIAs for ectopic products normally associated with thisneoplasm (2, 5, 7, 8, 35, 36); e.g., the levels ofimmunoassayableACTH, calcitonin, and hCGJ3 were 10.5, 0.18, and 410 pmol/g of tissue, respectively. The amount of physalaemin-like im-munoreactivity was low, about 0.8 pmol/g; analyses of hGH,gastrin, and somatomedin C were consistently negative, inagreement with an earlier report (3). The level of immuno-reactive bombesin, 5.81 and 7.13 pmol/g in the SCC samples,on the other hand, was similar to that ofACTH. Bombesin im-munoreactivity from both the initial acid extract and partiallypurified material produced displacement curves parallel to thecurve for bombesin (Fig. 2).

Chromatographic Characterization of Bombesin Immuno-reactivity. An aliquot of resolubilized SCC was initially frac-tionated on Sephadex G-75 in order to detect high molecularweight forms of bombesin, as described for ovine hypothalami(14), rat stomach, intestine (13, 17), and plasma (13), porcinenonantral gastric mucosa (18), and chicken proventriculus (20);we found only a single species of immunoreactive material thathad the same elution volume as bombesin (data not shown).Further analysis on Bio-Gel P-4 confirmed the presence of animmunoreactive peptide that coincided with the elution volume

0

90

8070

o 60IrD- 50x 401- 3020

0.1Volume, ,4

1.0 10

Bombesin, pg

FIG. 2. RIA for bombesin. The initial SCC extract (A), column frac-tion (0), and a bombesin standard (e) were assayed. B, labeled bom-besin bound; Bo, labeled bombesin bound in the absence of competitor.

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O 1500

0'-

e 100

Un

00

50

50

C-t

20 40 60 80Fraction

xC.)

FIG. 3. Molecular sieving of SCC extract. A 150-,ul aliquot (5-24mg) was chromatographed on Bio-Gel P-4 (23, 24). The numbered ar-rows denote markers: void volume V., bovine serum albumin; 1, 125I-labeled [Tyr5]bombesin; 2, 125I-labeled physalaemin; 3, 125I-labeledphysalaemin-(1-8)-octapeptide. The bar graph is iodinated bombesinand o denotes immunoreactivity. The total volume Vt corresponds tofraction 142.

ofbombesin (Fig. 3). Reverse-phase HPLC, on the other hand,separated the bombesin immunoreactive material into threediscrete components. In solvent systems a and b with 1-minfractions, the retention times were 43, 45, and 49-50 min, and30, 33, and 36 min, respectively. In solvent c (Fig. 4) with 20-sec resolution, the three immunoreactive peaks emerged at36:20-36:40, 39:00-39:40, and 43:20-43:40 min, respectively.The last peak coincided with the appearance of the syntheticamphibian bombesin in the three solvents used.

DISCUSSIONThe major conclusions that can be drawn from our data are that(i) immunoreactive bombesin was present in a SCC resectedfrom a male patient at a level comparable to ACTH (refs. 2-4,our data), which is a common ectopic hormone in these tumors(1, 2, 5, 7) and (ii) this molecule appeared to be chromatograph-ically similar to the amphibian peptide. To verify these conclu-sions, we applied several of the criteria required for the as-sessment of ectopic tumor products (7): RIA, immunohis-tochemistry, gel filtration chromatography, and reverse-phaseHPLC.

RIAs detected nanogram levels ofboth bombesin and ACTHin extracts of the tumor. This was confirmed by immunohis-tochemical localization of the peptides in serial sections of thetissue, which suggested that the same cell, or group of cells,contain bombesin as well as hCGf3 and physalaemin-like im-munoreactivity, whereas ACTH was found in different cells(Fig. 1). The presence of multiple peptide hormones in the tu-mor could be accounted for by the apparent heterogeneity ofSCC of the lung (37), which contains numerous foci with theappearance ofcells at different stages ofdifferentiation (33). Thisdiversity in biochemical (8) and chromosomal variability (34),also observed during the establishment ofdistinct cell lines fromthe tumor (34, 38, 39), may account for the appearance ofectopicpeptides. Because the expression of hormones may also changeduring passage ofthe tumor in nude mice (4), this could possibly

I--

a04.)C.)

'4-4

axtUr.0

35 40 45Retention time, min

-

._

C.)

'UctJ

FIG. 4. Reverse-phase HPLC of SCC extract and synthetic bom-besin. An aliquot (50 .D) of formic acid-solubilized extract was injectedonto a Spherisorb octadecylsilica 5-/Am column in 0.02 M sodium phos-phate, pH 6.97, and eluted with a linear gradient of 0-60% acetonitrilein buffer at a flow rate of 1 ml/min, and fractions were collected at 20-sec intervals. Immunoreactivity in the blank column, between 25- and50-min retention time, averaged 17.7 pg per fraction, with no peakscorresponding to those observed in either the tumor extract or the bom-besin standard peptide.

account for the immunopositive staining ofbombesin in a smallpercentage of the tumor cells. Alternately, the absence of im-munostaining may indicate that a peptide is either below thelevel of the threshold for detection by immunohistochemicaltechniques or absent altogether.

Molecular sieving confirmed that the immunoreactivity inthe tumor extracts resided in a relatively small peptide (Fig. 3).HPLC analyses demonstrated that these extracts contained apeptide that appeared to be very similar to amphibian bom-besin. The superposition of the retention profiles on HPLC ofone peak ofimmunoreactive bombesin from the tumor with thatof synthetic bombesin argues against any major differences instructure because HPLC offers a high degree of resolution oflimited differences in a peptide, such as the separation of pep-tides differing by only one D amino acid isomer replacement(40) or its sulfoxide derivative (41). The heterogeneity of im-munoreactivity in the tumor extracts revealed by HPLC maybe due to the presence of an oxidized methionine residue (un-published data).

It is also interesting from an evolutionary viewpoint that amammalian tissue would contain a peptide resembling an am-phibian peptide and that this peptide, expressed only in fetal/neonatal lung tissue (21), would reappear in an adult lung neo-plasia. The reappearance of fetal isozymes in cancerous adult

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tissues is a well-established observation. The tumor might rep-resent a reversion to a biochemical state present in prenataltissue by one of several mechanisms (7), including the process-ing of an inactive precursor, thereby enabling the reexpressionof a peptide hormone.

In mammals, bombesin elicits numerous pharmacologicaland physiological effects (9, 10), several of which resemble theclinical symptoms reported for patients with SCC (1, 2, 5, 11).For example, the marked reduction in the absorption of waterand electrolytes in the intestine due to bombesin (10) couldcontribute to hyponatremia (5, 11), generally assumed to becaused by inappropriate secretion of antidiuretic hormone. Sus-tained clinical effects could then result from the constant low-level secretion of a bioactive form of bombesin, such as foundduring infusion studies in human subjects (42, 43), as well asother peptide hormones from the tumor. Once in plasma, bom-besin could interact with tissue receptor sites (44), therebymediating intracellular changes that stimulate secretion of en-zymes (44-46), or affect electrical potentials (10, 46), or bringabout the release of other peptide hormones, such as insulin(47, 48), glucagon (48), gastrin (9, 10, 42, 43, 49), and prolactinand GH (50). With the growing evidence of ectopic secretionof peptides by SCC and other neoplasms (6, 7, 11), we suggestthat bombesin should be considered among the various peptidescausing endocrinologic abnormalities in patients with lung tu-mors. Furthermore, bombesin might then serve as a potentialpeptide marker in the screening of patients with SCC.

After submission of this article, Moody et al. (51) and Woodet aL (52) presented data that cell lines derived from SCC andtumor samples from patients, respectively, contain bombesin-like immunoreactivity.

We acknowledge and appreciate the skilled technical assistance ofJ. Frushstick, G. D. Jahnke, C. M. Soldato, and F. Talley, and the in-valuable comments of the reviewers. M.D.E. was supported by theAmerican Lung Association.

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