(CANCER RESEARCH 51. 2438-2443. May 1, 1991|

Dependence of a Human Squamous Carcinoma and Associated ParaneoplasticSyndromes on the Epidermal Growth Factor Receptor Pathway inNude Mice1

I'oshi) uki Yoneda,2 Maria M. Alsina, Kazuya Watatani, FrançoiseBellot, Joseph Schlessinger,

and Gregory R. MundyDivision of Endocrinology and Metabolism, Department of Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 [T. K,M. M. A., K. W., G. R. M.J; Rarer Biotechnology Inc., King of Prussia, Pennsylvania 19406 [F. B.J; and Department of Pharmacology, New York University,New York. New York 10016 /J. .V./

ABSTRACT

Increased levels of epidermal growth factor receptor (EGFR) havebeen shown on squamous cell carcinomas. Recently, we described asquamous cell carcinoma (MH-85) derived from the oral cavity whichwas associated with several paraneoplastic syndromes including hyper-calcemia and cachexia. This tumor induced the same paraneoplasticsyndromes in nude mice (BALB/c, nu/nu, male, 4-6 weeks old). Scat-chard analysis revealed that there are two classes of EGFR in MH-85.The dissociation constant and number of binding sites for the high affinityreceptors were 38 p\i and 5 x 104/cell, respectively, and 2.2 UMand 6 x10*cell, respectively, for the low affinity receptors. Growth of MH-85 in

culture was stimulated by epidermal growth factor (EGF) and inhibitedby monoclonal antibody 108 to human EGFR, which recognizes theextracellular domain of the EGF receptor. Surgical removal of subman-dibular glands from male nude mice resulted in a dramatic decrease inplasma EGF levels and a significant reduction of tumor growth, hyper-calcemia, and cachexia. When EGF (5 ¿ig/mouse,every 2 days for 6weeks, i.p.) was administered to these sialoadenectomized animals, tumorgrowth increased, with a parallel increase in hypercalcemia. When monoclonal antibody 108 (1 mg/mouse, i.p.) was given 1, 5, and 10 days afterMH-85 tumor implantation, tumor formation was retarded, which resulted in delayed onset of hypercalcemia and cachexia. Moreover, whenthe antibody was injected 6 times in nude mice exhibiting large tumorsand profound hypercalcemia and cachexia, there was a striking decreasein tumor growth, which was accompanied with a reversal of hypercalcemiaand cachexia. These results indicate that growth of the human squamouscell carcinoma MH-85 is dependent on the EGFR pathway and thatsubsequent development of hypercalcemia and cachexia is dependent ontumor growth. They also suggest that agents which interfere with theEGFR pathway may have therapeutic potential as anticancer agents insome human tumors.

INTRODUCTION

It is well established that the EGFR1 signal transduction

pathway is important in the growth of many human tumors (1,2). Overexpression of EGFR is common in a variety of humanepithelial malignancies (3), including breast (4-6), esophagus(7), bladder (8), lung (9), female genital tract (10), brain (11,12), and head and neck (13-16). Expression of EGFR in thesetumors is related to cell growth and tumorigenicity (17), and

Received 8/27/90; accepted 2/19/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by Grants DE-08526 and CA-40035 from the NIH.2To whom requests for reprints should be addressed, at Division of Endocri

nology and Metabolism, Department of Medicine. The University of TexasHealth Science Center at San Antonio. 7703 Floyd Curl Drive. San Antonio, TX78284-7877.

3The abbreviations used are: EGFR, epidermal growth factor receptor; TGF-n, transforming growth factor «;«MEM,a minimal essential medium; FCS, fetalcalf serum; PBS, phosphate-buffered saline; mAblOS, monoclonal anti-epidermalgrowth factor receptor antibody 108; mAb7H01. monoclonal anti-human hepatitis B virus antibody; EGF, epidermal growth factor.

blocking of this pathway with monoclonal antibodies againstEGFR inhibits tumor growth (18-24). TGF-a, which is also aligand for the EGFR, is frequently produced by malignant aswell as normal cells and may stimulate their own proliferation(25-27); thus, it has been implicated as an autocrine factor intumor growth in vivo (28, 29). However, less is known aboutthe role of EGF itself in oncogenesis. EGF is rarely if everproduced by human tumors and, therefore, is unlikely to be anautocrine factor (29). In this report, we have identified a humansquamous cell carcinoma, derived from the maxillary sinus,which was dependent on the EGFR signal transduction pathwayfor its growth both in vitro and in vivo. The cell line (MH-85)established from this tumor overexpressed EGFR. EGF stimulated and inAh IOS inhibited MH-85 growth in culture. Innude mice which underwent surgical removal of the subman-dibular glands, a major source of EGF in male mice, tumorgrowth was diminished, which was reversed by injection ofEGF. Furthermore, mAblOS inhibited MH-85 tumor growthin nude mice. We conclude that growth and associated paraneoplastic syndromes of this human squamous cell carcinomain nude mice are under the control of EGF and the EGFRpathway.

MATERIALS AND METHODS

MH-85 Tumor and Cells. The MH-85 tumor was isolated from apatient with a squamous cell carcinoma in the maxillary sinus, whomanifested marked leukocytosis (180,000/mm1, >90% mature granu-

locytes), cachexia (loss of body weight, muscle, and fat), and hypercalcemia (13.0 mg/dl) (30). Nude mice transplanted with this tumor alsodevelop profound hypercalcemia, cachexia, and leukocytosis 6 weeksafter transplantation (31).

MH-85 cells were grown in «MEM(Hazleton Biologies, Inc., Le-nexa, KA) supplemented with 10% heat-inactivated FCS (HycloneLaboratories, Inc., Logan, UT) and 1% penicillin-streptomycin solution(GIBCO, Grand Island, NY).

125I-EGF Binding Assay. Confluent MH-85 cells in 24-well plateswere placed on ice, washed twice with ice-cold binding medium [«MEMsupplemented with 2 mg/ml bovine serum albumin and 25 mM 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid, pH 7.4], and incubatedin the binding medium for more than 2 h at 4"C. The cells were thenincubated with different concentrations of ' "I-EGF (76.6 ^Ci/^ig; ICNRadiochemicals, Irvine, CA), in the presence or absence of 200-foldexcess unlabeled EGF, for 2 h at 4°C.At the end of the incubation, the

cells were washed 5 times with ice-cold binding medium and solubili/edwith lysis buffer (0.01 MTris-HCl, pH 7.4, 1 mM EDTA, 0.5% sodiumdodecyl sulfate), and radioactivity was counted.

Colony Assay for MH-85 Growth. MH-85 cells (100/17-mm well)were seeded in plastic tissue culture plates (24-well plates; CorningGlassware, Corning, NY) and cultured overnight in «MEM supplemented with 10% FCS. The cells were then cultured in «MEMsupplemented with 0.2% FCS, in the presence or absence of varying concentrations of EGF (Collaborative Research, Bedford, MA) and mAblOS,for 8-10 days. At the end of culture, the plates were washed with Ca/

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EGFR PATHWAY AND HUMAN SQUAMOUS CANCER GROWTH

Mg-free PBS, fixed with 4% (v/v) formaldehyde in Ca/Mg-free PBSfor 15 min, and stained with hematoxylin as described (22). The numberof colonies containing more than 20 cells was counted under themicroscope.

MH-85 Tumor Implantation into Nude Mice. MH-85 tumor (3 mmin diameter) was implanted s.c. in the right dorsal area of 4- to 6-week-old male BALB/c nu/nu mice (HaríanSprague-Dawley, Indianapolis,IN). Animals were fed, ad libitum, an autoclaved diet (Tekland-LH-485; calcium, 0.83 mg/kg; phosphorus, 0.65 mg/kg; HaríanSprague-Dawley) and tap water (acidified to pH 2.5). The body weights of MH-85 tumor-bearing animals and their tumor sizes were measured usinganesthesia with ether. Tumor size was calculated as 4/3 ?r|(Iong axis +short axis-l)/2p, as described previously (32).

Determination of Blood Ionized Calcium Levels. Thirty «1of bloodwere drawn from the orbital plexus with calcium-titrated sodium he-parinate-coated tubes (Corning, Halstead, Essex, England), using anesthesia with ether. Blood ionized calcium levels were determined usinga Ciba-Corning calcium-pH analyzer (model 634; Corning, Medfield,MA).

Sialoadenectomy. Bilateral submandibular glands of 4- to 6 week oldmale nude mice were aseptically removed after vertical midline incisions, approximately 1 cm in length, were made on the skin of the neckregion, using anesthesia with Nembutal (0.05 mg/g body weight).Control animals received the same cutaneous incision. Cutaneous incisions were closed by Autoclips (Clay Adams, Parsippany, NY).

Determination of Plasma EGF Levels. Blood was drawn by cardiacpuncture and centrifuged to isolate plasma. EGF in the plasma wasextracted according to the methods described (33), with slight modifications. Plasma was acidified with acetic acid at a final concentrationof 3%, diluted with 0.1% trifluoroacetic acid by 5-fold, and applied todisposal Sep-Pak Cig columns (Waters Associates, Milford, MA), whichwere equilibrated with 10% acetonitrile and 0.1% trifluoroacetic acid.EGF was eluted with 40% acetonitrile containing 0.1% trifluoroaceticacid. The élûteswere concentrated to dryness in a Speed-Vac (modelRH40-II; Savant Instruments Inc., Farmingdale, NY) and reconstitutedin 100 n\ of Ca/Mg-free PBS. EGF concentration was measured byradioimmunoassay using a commercial kit (Amersham, ArlingtonHeights, IL), according to the manufacturer's instructions.

Injection of mAblOS into Nude Mice. mAblOS of the IgG2a isotypewas prepared from ascitic fluids purified by ammonium sulfate precipitation, followed by affinity chromatography on a Sepharose CL-Pro-tein A column, as described (22). The antibody was raised against theextracellular domain of human EGFR and does not cross-react withmouse EGFR (34). One mg mAblOS in 0.1 ml/mouse was given i.p.,using 27-gauge needles. As a control. Protein A affinity-purified ascitescontaining mAbTHOl of IgG2a isotype, raised against human hepatitisB virus (22), were used.

RESULTS

'"I-EGF Binding to MH-85 Cells. MH-85 demonstrateddose-dependent and saturable binding of EGF (Fig. la). Scat-chard analysis of the binding data indicated the presence of twopopulations of receptor, with different affinities for EGF. Thereceptor with higher affinity has a dissociation constant of 38pM and an estimated number of binding sites of 5 x 104/cell

(Fig. \b). The receptor with lower affinity has a dissociationconstant of 2.2 nM and an estimated number of binding sites of6 x 105/cell. These values are comparable to those of other

human epidermal cancer cell lines such as A431 (35) and KB(36) cells, which are known to possess overexpressed EGFRwith high affinity.

Effect of EGF and mAblOS on MH-85 Growth in Culture.EGF as low as 10 ng/ml significantly increased colony formation of MH-85 (Fig. 2). On the other hand, mAblOS decreasedcolony formation of MH-85 in a dose-dependent manner, bothin the presence and in the absence of EGF (50 ng/ml) (Fig. 3a).

1234'•'VEUFAdded (nM)

0 50 100•"Õ-EGFBound(fmol/10'cells)

Fig. 1. a, '"I-EGF binding lo MH-85 cells. Confluent MH-85 cells wereincubated with '"I-EGF. in the presence or absence of 200-fold excess unlabeledEGF, for 2 h at 4°C.O, specific binding; •.total binding: A, nonspecific binding.

/>,Scatchard analysis of the binding.

Control 1 10 50 100

EGF (ng/ml)Fig. 2. Effect of EGF on colony formation of MH-85 cells. MH-85 cells (100/

well, 24-well plate) were cultured in «MEMsupplemented with 0.2% PCS, in theabsence (D) or presence (•)of EGF, for 10 days, *, significantly greater thancultures without EGF (P < 0.01, Student's t test).

Equal concentrations of control mAb7H01 showed no effect(Fig. 30).

Effects of Sialoadenectomy on MH-85 in Nude Mice. Theresults described above indicated that MH-85 growth in cultureis stimulated by EGF, probably through overexpressed EGFR.We next examined whether MH-85 growth in vivo is alsomodulated by EGF. To study this, submandibular glands ofmale nude mice were removed 7 days prior to MH-85 implantation. It is known that more than 95% of EGF is produced insubmandibular glands in male mice (37). Consistent with theseprevious reports, plasma EGF levels of sialoadenectomizedmale nude mice at the time of MH-85 tumor implantation (7days after surgery) dramatically decreased (0.09 ±0.08 ng/ml),compared to those of sham-operated animals (2.01 ±1.4 ng/ml) (Fig. 4). Plasma EGF levels of sialoadenectomized animalswith established MH-85 tumors at 10 weeks after tumor implantation slightly increased but were still much lower thanthose of sham-operated animals (sham-operated = 2.8 ±1.2,sialoadenectomized = 0.23 ±0.14 ng/ml, n = 6 and 7, respectively). The data also demonstrate that carrying MH-85 tumorsdid not significantly change plasma EGF levels in sham-operated (week 0 = 2.01 ±1.4, week 10 = 2.8 ±1.2 ng/ml, n = 13and 6, respectively) and sialoadenectomized (week 0 = 0.09 ±0.08, week 10 = 0.23 ±0.14 ng/ml, n = 8 and 7, respectively)nude mice. Non-tumor-bearing nude mice with no treatmentdid not show significant changes in plasma EGF levels at week0 (2.4 ±1.3 ng/ml, n = 4) and week 10 (2.1 ±0.9 ng/ml, n =4).

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EGFR PATHWAY AND HUMAN SQUAMOUS CANCER GROWTH

Colony No/well(Mean iS.E., n = 4)

100

80

60

40

20

0.1 05 25

EGFRMoAb ( jj.g/ml)

12.5

100-

80-

60-

40-

20-

nCo

(MealonyNo/wel

n »S.E, n=Õ

T4)1idin

Hurt*

TlAb

7H01Ab

108Õ*TÕb*T

0.1 0.5 2.5 12.5

MAb Concentrations (ng/ml)

Fig. 3. a, effect of mAblOS on colony formation by MH-85 cells in the presence(D) or absence (D) of EOF (50 ng/ml). b, effect of mAblOS (D) and mAbVHOl(control ascites) (D) on colony formation of EOF (50 ng/ml)-stimulated MH-85cells. Experiment was performed in the same way as described in the legend toFig. 2. *. significantly different than cultures without antibodies (P< 0.01).

4-1

3

2

1

EOF (ng/ml)

B)

Sham Sialoadenectomy

Fig. 4. Plasma EGF levels in sham-operated (D) and sialoadenectomized (•)nude mice at the time of MH-85 tumor implantation. Plasma EGF was determined as described in "Materials and Methods." Values are mean ±SE. Numbersin parentheses indicate the number of animals examined. Sham-operated animalsdemonstrated significantly higher levels of plasma EGF (/>< 0.05).

As shown in Fig. 5a, MH-85 grew to approximately 20 cm1

in size in sham-operated animals 10 weeks after tumor implantation. When MH-85 was implanted into sialoadenectomizedmale nude mice with decreased plasma EGF levels, there was amarked decrease in tumor growth (P < 0.01). However, injection of 5 ¿igof EGF every 2 days for 6 weeks into sialoadenectomized animals completely restored MH-85 growth. Plasma

EGF levels in these animals at the time of sacrifice (5 days afterlast injection of EGF) were 3.6 ±1.8 ng/ml (n = 5).

Sham-operated animals bearing MH-85 tumor showed aprofound decrease in body weight due to cachexia, comparedto non-tumor-bearing animals (P < 0.01) (Fig. 5b). However,there was less decrease in body weight in sialoadenectomizednude mice bearing MH-85 tumor than in sham-operated nudemice with MH-85 tumor (P < 0.05). Although EGF restoredMH-85 tumor growth in sialoadenectomized nude mice, theseanimals did not manifest cachexia to the same degree as sham-operated animals.

Sham-operated animals bearing MH-85 tumor manifestedmarked hypercalcemia (Fig. 5c). The hypercalcemia was significantly diminished in sialoadenectomized animals (P < 0.01),due to decreased tumor burden. When EGF was injected intosialoadenectomized animals for 6 weeks, marked hypercalcemiaidentical to that seen in sham-operated animals developed,resulting from restored tumor growth. The same dose of EGFdid not increase blood ionized calcium levels in non-tumor-bearing animals (data not shown).

Effects of mAblOS on MH-85 in Nude Mice. In the first setof experiments, nude mice were given injections of mAb 108 ormAb7H01 (1 mg/mouse, i.p.) at 1,5, and 10 days after tumorinoculation. With mAblOS, tumor growth was markedly delayed (Figs. 6, center, and la). Tumor were not palpable until60 days after tumor inoculation, compared with 20 days foranimals given injections of mAb7H01. Because of this delay ofMH-85 tumor development, the onset of hypercalcemia wasretarded (Fig. Ib) and no cachexia occurred (Figs. 6, center,and le). Dependency of these paraneoplastic syndromes onMH-85 growth was previously demonstrated (31).

In the second set of experiments, cachectic and hypercalcémienude mice bearing large MH-85 tumors were given injectionsof mAblOS or mAb7H01 60 days after tumor inoculation. WithmAblOS, continued tumor growth was halted, and there was aslight regression in tumor growth (Figs. 6, right, and 8a). As aresult of the changes in tumor growth, blood ionized calciumlevels started to decrease (Fig. SA) and the body weight of theanimals started to increase (Figs. 6, right, and 8c).

In both sets of experiments, nude mice given injections ofmAblOS showed more activity in their behavior and food intakethan did nude mice given injections of mAbTHOl (data notshown), suggesting that mAblOS at this dose (1 mg/mouse) isnot harmful to tumor-bearing nude mice.

Tumor Size(cm3)

Body Weight

(g)

Ca2»

(mmol/L)

Weeks After Tumor ImplantationFig. 5. Effects of sialoadenectomy on MH-85 tumor size (a), body weight of

MH-85 tumor-bearing nude mice (o). and calcium levels (c). O. sham-operated;•.sialoadenectomized: •.sialoadenectomized plus EGF (5 pg/mouse, every 2days for 6 weeks, i.p.): A. non-tumor-bearing. Values are mean ±SE of twoseparate experiments (n = 9 for each treatment group).

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EGFR PATHWAY AND HUMAN SQUAMOUS CANCER GROWTH

Fig. 6. Photograph of MH-85 tumor-bearing animals injected with mAb7H01 (controlascites) (left), mAblOS at 1 mg/mouse on daysI, 5, and 10, i.p. (center), or mAblOS 1 mg/mouse, every 2 days 5 times from day 40, i.p.(right). The picture was the most representative one and was taken 60 days after tumorimplantation. Note (a) large tumor formation,decrease in body weight (21 g). and loss ofmuscle and adipose tissue in the animal treatedwith mAb7H01 (control ascites) (left); (b)marked decrease in tumor growth, no decreasein body weight (31 g). and no loss of muscleand adipose tissue in the animal treated withmAblOS on days 1, 5, and 10 (center): and (c)significant decrease in tumor size due to necrosis and recovery from decrease in body weight(30 g) and loss of muscle and adipose tissue inthe animal treated with mAblOS from day 40(right). Blood ionized calcium levels in left,center, and right animals were 2.49, 1.21,and 1.43 mmol/L (normal range. 0.95-1.28mmol/L), respectively.

Tumor Size (mm'x 103) Ca!* (mmol/L)

1.50-

1.00

0 AooSoo,

Bodyweight(g) treatment of nude mice with mAblOS not only delays MH-85tumor formation but also inhibits progressive tumor growth.These results indicate that growth of MH-85 in nude mice ismodulated by EGF and the EGFR pathway.

More recently, it has also been shown that tyrphostins, smallmolecular weight compounds which inhibit EGF-dependenttyrosine kinase, are capable of influencing tumor cell proliferation in vitro (38-41). In a preliminary study, we have found

0 40 80 120 0 40 80 120 0

Days After Tumor Implantation

Fig. 7. Effects of mAblOS tumor size (a), blood ionized calcium levels (ft), andbody weight (c). mAblOS (1 mg/mouse) was given on days 1. 5. and 10 aftertumor implantation, as indicated by arrows. •,animals given injections ofmAb7H01 (control ascites); O, animals given injections of mAblOS. Values shownare mean ±SE of six animals for each treatment group. *. significantly differentfrom corresponding control (P < 0.05).

DISCUSSION

In the present study, we have examined an established cellline, MH-85, derived from a human squamous cell carcinoma,both in vitro and in vivo. We have found that the cells overex-press EGFR and they are responsive to EGF and mAblOS. Wehave shown that in male nude mice removal of the submandib-

ular glands decreases plasma EGF levels, which is accompaniedby significant diminution of MH-85 tumor growth. The decreased tumor growth in these sialoadenectomized animals canbe reversed by injection of EGF. We have also shown that

Tumor Size (mm3x 103) Ca!°(mmol/L) Body Weight (g)

Ab

Days After Tumor Implantation

Fig. 8. Effects of mAblOS on tumor size (a), blood ionized calcium levels (ft),and body weight (c). mAblOS (1 mg/mouse) was given every 2 days 4 times andevery 3 days twice from day 60. when MH-85-bearing animals manifested largetumor, marked hypercalcemia. and cachexia. as indicated by »mmv •.animalsgiven injections of mAbVHOl (control ascites): O. animals given injections ofmAblOS. Twenty-four animals were inoculated with MH-85 tumor at day 0 anddivided into two groups (mAblOS- and mAb7H01-treated groups) at day 60.Values are mean ±SE of three separate experiments (n = 24 until day 60 and n—12 for each treatment group thereafter). *. significantly different from corre

sponding control (P < 0.05).

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EGFR PATHWAY AND HUMAN SQUAMOUS CANCER GROWTH

that one of these compounds (38) has growth-inhibitory effectson MH-85.4 Together with data which show that transfection

of cells with mutated EGFR influences their growth in vivo,depending on the capacity of these receptors to increase tyrosinekinase activity (42), this shows that the EGF signaling pathwayis clearly important in MH-85 tumor growth.

Our findings indicate the dependence of MH-85 tumors onEGF or factors which interact with the EGFR. In the presentstudy, mAb 108 decreased the growth of unstimulated as wellas EGF-stimulated MH-85 cells, suggesting that this antibodyinterferes with the binding to the EGFR of autocrine growthfactor(s) secreted by MH-85. We could not detect significantlevels of EGF in MH-85 culture supernatants by using theradioimmunoassay described above (data not shown). This isconsistent with previous findings that very few human tumorsproduce EGF (28, 29). As another likely candidate for anautocrine growth factor, we measured TGF-a levels by usingan enzyme-linked immunosorbent assay kit (Triton Biosciences,Alameda, CA). We found 5-35 pg/ml TGF-a in MH-85 culturesupernatants. Although we do not know if these levels of TGF-a are sufficient to cause MH-85 tumor growth, TGF-a producedby MH-85 may play a role as an autocrine or paracrine growthfactor in this tumor.

Previous reports have shown that anti-EGFR monoclonalantibodies markedly inhibit tumor formation when administration is started on the day of tumor implantation (18, 22).However, in these experiments the antibodies are not able todecrease growth of established tumors, although they inhibitthe further growth of the tumors. This is largely due to clonalheterogeneity of established tumors, which are composed ofcells dependent or independent on EGF or TGF-a. In ourstudy, mAb 108 given only 3 times immediately after tumorinoculation dramatically suppressed tumor formation. Furthermore, six injections of mAb 108 to animals with establishedtumors not only inhibited further enlargement of the MH-85tumor but also decreased its size. This result suggests that amajor population of cells in the MH-85 tumor is dependent onthe EGFR signal transduction pathway, which makes the tumorhighly sensitive to the growth-inhibitory effect of the monoclonal antibodies, mAb 108 has already been reported to inhibitgrowth of the human epidermoid cancer KB (22) and the breastcancer MDA-468 (24). In the present study, we have shownthat mAb 108 can also suppress growth of another humansquamous cancer, MH-85, both in vitro and in vivo.

Our results clearly demonstrate that mAb 108 alone has inhibitory effects on MH-85 tumor growth in vivo. The antibodyis also useful for targeting of cytotoxic drugs or toxins to tumorswith overexpressed EGFR. Recently, it has been shown that,when the cytotoxic drug doxorubicin is conjugated to mAb 108,doxorubicin is delivered specifically and efficiently to the tumorsite, where it can exert its cytotoxic effect on the target tumorcells (43). The conjugates were more effective than either thefree drug or antibody given to tumor-bearing nude mice alone.Similarly, it is also reported that immunotoxins made by conjugating monoclonal antibodies against EGFR to toxins suchas recombinant ricin A chain (44) or gelonin, a ribosome-inactivating protein (45), are specifically cytotoxic for tumorswith increased levels of EGFR expression in vivo and thecytotoxicity is correlated with the numbers of EGFR on targettumors. Thus, monoclonal antibodies to EGFR not only exert

4T. Toneda, R. M. Lyall, M. M. Alsina, P. E. Persons, A. P. Spada, A.Levhzki, A. Zilberstein, and G. R. Mundy. The antiproliferative effects of tirosinekinase inhibitor tyrphostin on a human squamous cell carcinoma in vitro and innude mice, manuscript in preparation.

anticancer action by themselves but also can be utilized toincrease the specificity and efficacy of other anticancer agents.

We have shown that MH-85 has two populations of receptor,with different affinities (dissociation constants of the receptorswith high and low affinity are 0.038 and 2.2 HM, respectively).Defize et al. (46) and Bellot et al. (34) have used monoclonalantibodies (including mAb 108) to demonstrate that the highaffinity binding sites are crucial for EGFR signaling, mAb 108,used in the present study, has been shown to interact specificallywith high affinity EGFR, which are essential for eliciting severalearly responses, such as EGFR autophosphorv lation, hydrolysisof phosphatidyl inositol, release of calcium from intracellularstores, and elevation of cytoplasmic pH (34).

The MH-85 tumor is associated with several common para-neoplastic syndromes, in the patient from whom the tumor wasoriginally derived as well as in nude mice bearing this tumor.The two most common paraneoplastic syndromes are hyper-calcemia and cachexia. In the MH-85-bearing nude mice treatedwith niAblOS or by removal of the submandibular glands, amajor source of EGF, progress of these paraneoplastic syndromes was markedly suppressed. These results raise the possibility that modulation of the EGFR signal transduction pathway is directly responsible for hypercalcemia and cachexia. Infact, TGF-a, which binds to EGF receptors and shares similarbiological activities with EGF, has a powerful hypercalcémieaction (47), and we have recently found that Chinese hamsterovary cells which have been transfected with the TGF-a genecause hypercalcemia and cachexia in nude mice. However, wethink that this possibility is unlikely in MH-85-bearing nudemice. As we described previously (31), surgical excision of MH-85 resulted in complete recovery from hypercalcemia, leukocy-tosis, and cachexia. In the present study, inhibition of MH-85growth by treatment with mAb 108 caused impairment of hypercalcemia and cachexia. It seems most likely that development of hypercalcemia and cachexia in MH-85 tumor-bearinganimals is intimately dependent on the growth of the tumors.

ACKNOWLEDGMENTS

We are grateful to Nancy Garre« and Thelma Barrios for theirskillful secretarial assistance.

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1991;51:2438-2443. Cancer Res   Toshiyuki Yoneda, Maria M. Alsina, Kazuya Watatani, et al.   Receptor Pathway in Nude MiceParaneoplastic Syndromes on the Epidermal Growth Factor Dependence of a Human Squamous Carcinoma and Associated

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