ExpressionandGrowthDependencyoftheInsulin...

Expression and Growth Dependency of the Insulin-Like GrowthFactor IReceptor in Craniopharyngioma Cells:ANovelTherapeutic ApproachElfar Ulfarsson,1Alexandra Karstr˛m,4 Shucheng Yin,5 Ada Girnita,5 Daiana Vasilcanu,5Marja Thoren,2

Gunnar Kratz,6 Jan Hillman,7 Magnus Axelson,3 Olle Larsson,5 and Leonard Girnita5

Abstract Craniopharyngioma is a rare benign intracranial epithelial tumor that, however, often recurs andsometimes kills the affected patients, one-third of which are children. In many cases, the patientsacquire growthhormone deficiencyandpostoperatively need substitution. Generally, growthhor-monepromotes local release of insulin-like growth factor I (IGF-I),which in turnactivates the IGF-Ireceptor (IGF-IR) if present. Together, these circumstances raise the question whether IGF-IRmay be involved in craniopharyngioma growth.To address this issue, we analyzed phenotypicallywell-characterized primary low-passage craniopharyngioma cell lines from nine different patientsfor IGF-IRexpression and IGF-Idependency.Two of the cell lines showedno/very low expressionof the receptorandwas independenton IGF-I,whereas five cell lines exhibiteda strongexpressionandwas clearly contingent on IGF-I.The two remaining cell lines had low receptor expression andIGF-I dependency. Upon treatment with an IGF-IR inhibitor, cells with high IGF-IR expressionresponded promptly with decreased Akt phosphorylation followed by growth arrest. Theseresponses were not seen in cells with no/very low receptor expression. Growth of cell lineswith low IGF-IRexpressionwas only slightly affectedby IGF-IR inhibition.Taken together, our datasuggest that IGF-IR may be involved in the growth of a subset of craniopharyngiomas and pointsto thepossibilityof the involvementof IGF-IRinhibitors as a treatmentmodality to obtaincompletetumor-free conditions before growthhormone substitution.

Craniopharyngiomas are benign epithelial neoplasms mostoften found in the intra/suprasellar region. They representone of the most difficult benign intracranial lesions to treat,because of their close relation to the surrounding structuresand high tendency to recur. They are classified into twohistopathologic subtypes: adamantinomatous type and squa-mous papillary type (1). The adamantinomatous types areconsidered to be derived from an enamel anlage in Rathke’spouch, and squamous papillary types are assumed tooriginate in the metaplastic squamous cell nests of theadenohypophysis (1).

The incidence of craniopharyngioma is about 1.3 permillion, with one-third occurring in children under age 15

(2). Little is known about the natural course of the disease butcontinuing tumor growth after partial tumor removal isreported to be 63% to 90% (3–10), with only 20% to 42%10-year survival rates (6, 7, 11, 12). The main treatmentoptions, microsurgery and radiation, render a majority of thepatients growth hormone–deficient (5, 13–15). The use ofgrowth hormone to promote growth in children withcraniopharyngioma has been in practice for more than twodecades and is now a part of the standard treatment for thispatient group (16, 17). The fact that growth hormone ismitogenic (18–21) either directly or indirectly via the insulin-like growth factor I (IGF-I) has raised the question of a possiblerole for growth hormone in inducing the recurrence ofcraniopharyngiomas in children (8, 21, 22). The overallopinion based on the few clinical studies addressing thisrelationship is still that growth hormone treatment does notincrease the recurrence rates (8, 21–23). However, thosestudies have some weaknesses, and thus far, no experimentalstudies have been done to confirm this statement. In theory,one can expect growth hormone to stimulate growth ofcraniopharyngioma cells. The craniopharyngioma cells arehistologically similar to basal epidermal cells (24), and it iswell known that the main stimulatory effects of growthhormone on the growth of normal epithelial cells are exertedthrough the IGF-I system (25–28).

The specific aim of our study is to evaluate the expressionstatus and possible dependency of IGF-I receptor (IGF-IR) inpromoting the growth of craniopharyngioma cells.

Human Cancer Biology

Authors’ Affiliations: Departments of 1Neurosurgery, 2Endocrinology andDiabetology, and 3Clinical Chemistry, 4Institution for Surgical Sciences,5Department of Oncology and Pathology, Cancer Center Karolinska, KarolinskaUniversity Hospital, Stockholm, Sweden; Departments of 6Plastic Surgery, and7Neurosurgery, University Hospital, Link˛ping, SwedenReceived1/18/05; revised 3/29/05; accepted 3/31/05.Grant support: Swedish Children’s Cancer Society, the Swedish Cancer Society,the Cancer Society in Stockholm, the Jubilee Fund of King Gustaf V, Alex and EvaWallstr˛m’s Foundation, and the Karolinska Institute.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Requests for reprints: Department of Oncology and Pathology, Cancer CenterKarolinska, R08:04, Karolinska University Hospital, Stockholm 17176, Sweden.Phone: 46-8-5177-5242; Fax: 46-8-321047; E-mail: [email protected].

F2005 American Association for Cancer Research.

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Materials andMethods

Compounds and antibodies. Picropodophyllin (99.97% purity) wasprepared as described (29). A monoclonal antibody against phospho-tyrosine (PY99), polyclonal antibodies to the h-subunit of IGF-IR (C-20), h-subunit IGF-IR (H-60), pAkt1 (Ser473) and Akt1 were from SantaCruz Biotechnology, Inc. (Santa Cruz, CA).

Cell cultures. Primary cultures of human craniopharyngioma cellswere isolated and prepared from tumor samples in a similar manneras for keratinocytes according to the methods described (30). Briefly,after surgical removal, a portion of the tumors was immediately put inDMEM with 10% FCS and stored at 4jC. Each tumor specimen wascut down to small pieces and washed in PBS + penicillin/streptomycinsolution. The tumor cells were dispersed by treatment with trypsin for30 minutes at 37jC and 5% CO2. The growing cells were thencultured on mitomycin-pretreated 3T3 cells in DMEM/Ham’s F12medium (3:1; Life Technologies, Gaithersburg, MD) containing insulin(5 Ag/mL), transferrin (5 Ag/mL), T3 (2 � 10�9 mol/L), hydrocorti-sone (0.4 Ag/mL), cholera toxin (2 � 10�10 mol/L), antibiotics(penicillin 50 Ag/mL and streptomycin 50 units/mL) and 10% FCS(Life Technologies; referred to as complete keratinocyte mediumwithout epidermal growth factor). After 2 days of culture, epidermalgrowth factor (10 Ag/mL, Sigma, St. Louis, MO) was added to theculture medium. The obtained cultures were used for experiments.

Immunocytochemical stainings. Immunostainings of tumor tissuesand cell cultures for cytokeratin-7 and IGF-IR were done using thestandard avidin-biotin complex technique as previously described (31).We classified the results of IGF-IR stainings as negative (�) when <10%,

weakly positive (+) when 10% to 30%, intermediately positive (++)

when 30% to 60%, and strongly positive (+++) when >60% of the

epithelial cells were positive.Assay of cell growth. Measurement of de novo DNA replication was

done by assessment of [3H]thymidine incorporation, mainly as

described by Kratz et al. (32). Craniopharyngioma cells were seeded

in 96-well plates (Corning Life Sciences, Schipol-Rijk, Netherlands, 3 �103 cells per well) and were allowed to grow for 24 hours before

12 hours of serum starvation. Cells were then incubated for 24 hours in

DMEM with IGF-I, T3, and growth hormone at indicated concen-

trations. Controls were incubated with DMEM only. To all the media,

[3H]thymidine (Amersham Biosciences, Umea, Sweden, 5 Ci/mmol)

was added to a final concentration of 0.5 Amol/L (0.5 ACi/mL). After

24 hours, the cells were rinsed twice with PBS, lysed with 0.1% Triton X-

100 in distilled water, and harvested with a Scatron cell harvester.

Radioactivity was measured in a scintillation counter (Beckman,

Fullerton, CA). Results are expressed as means F SD of percentages of

increase in quadruplicate cultures.

Cell proliferation determinations were also done using the Cell

Proliferation Kit II (Roche, Inc., Indianapolis, IN), which is based on

colorimetric change of the yellow 2,3-bis[2-methoxy-4-nitro-5-sulfo-

phenyl]-2H-tetrazolium-5-carboxanilide inner salt in orange formazan

dye by the respiratory chain of viable cells (33). All standards and

experiments were done in triplicate.Expression and phosphorylation of IGF-IR. Exponentially growing

cells were serum-depleted for 20 hours, after which they were treatedwith desired concentrations of picropodophyllin for 60 minutes. Afterthis treatment, IGF-I (100 ng/mL; control cells with 0 ng/mL) was added

Table1. Clinical information on patients fromwhich cell lines were derived

Age (y) Growth hormone ^ deficiencybefore operation

Operation(y, m)

Tumorresection

Cellline

Growth hormoneafter operation

Recurrence(y, m)

32 no 2000, 01 subtotal C1 no 2002, 068 unknown 2000, 04 radical C2 yes no45 unknown 2000, 05 subtotal C3 yes 2001, 0311 yes 2000, 09 subtotal C4 no no9 yes 2000, 09 subtotal C5 yes 2004, 0635 yes 2000,12 radical C6 no no14 yes 2001, 03 radical C7 yes no8 probably 2001, 04 radical C8 yes 2002,116 unknown 2001, 07 radical C9 no no

Table 2. Immunohistochemical analysis of craniopharyngioma cell lines and corresponding tumor specimens

Cell line/tumorsample

Cytokeratin 7in cell lines

Cytokeratin 7immunoreactivityin tumors

IGF-IR grade immunoreactivityin cell lines

IGF-IR grade immunoreactivityin tumors

C1 Pos. ND +++ NDC2 Pos. Pos. � �C3 Pos. Pos. +++ +++C4 Pos. ND +++ ++C5 Pos. ND +++ NDC6 Pos. ND ++ NDC7 Pos. ND + NDC8 Pos. Pos. � �C9 Pos. ND +++ ND

NOTE:�, negative; +, weakly positive; ++, intermediately positive; +++, strongly positive; ND, not determined; Pos., positive without quantification.

IGF-IR in Craniopharyngioma

www.aacrjournals.org Clin Cancer Res 2005;11(13) July1, 20054675



to the medium and the cells were incubated for another 5 minutes. Cellswere then harvested for assay of IGF-IR phosphorylation usingimmunoprecipitation and Western blotting as described (29, 34). Themembranes were probed with PY99 and after stripping with an IGF-IR h-subunit antibody (loading control). Determination of pAkt/Akt wasdone directly by Western blotting using specific antibodies.

Results

Establishment and characterization of primary craniopharyng-ioma cell lines. Primary cell lines obtained from nine tumorspecimens from nine different craniopharyngioma patientswere included in the study. Clinical information for each of thecases is listed in Table 1. All nine tumors exhibited adaman-tinomatous histology. From each tumor specimen, a primarycraniopharyngioma cell line was prepared according toestablished procedures to selectively raise epithelial cells (30)(see Materials and Methods). It was proven that all investigatedcells of each cell line expressed cytokeratin-7 (Table 2), which isa well-documented immunomarker for craniopharyngiomacells irrespective of histological subtype (35–37). Based onthe selective cell culture procedures employed, together withthe epithelial cytomorphology and the cytokeratin-7 pheno-

type, it is evident that primary cell lines represent homogenouscultures of craniopharyngioma cells.

Table 2 also shows the level of immunoreactivity of the ninedifferent cell lines (denoted C1-C9) to IGF-IR, as assayed by

immunostaining. Five of the craniopharyngioma cell lines (C1,C3-5 and C9), exhibited strong immunoreactivity, whereas C2and C8 were negative. For three of the cell lines (C2, C3, andC8), we had access to paraffin-embedded materials from thecorresponding tumor specimens. In agreement with the celllines, immunohistochemical stainings with antibodies to IGF-IR show that the C2 and C8 tumors were IGF-IR-negative,whereas C3 was positive (Table 2). Figure 1A shows thehistology of the tumors in cases 2 and 3. Both samples arepredominated by an epithelial component. Several cysts areshown in case 2. Figure 1B shows microphotographs of thecytokeratin-7 expression (localized in the cytoplasm) of the twocell lines, C2 and C3. Figure 1C shows immunoreactivity toIGF-IR in case 2 (negative) and case 3 (positive) tumors. Thepositive epithelial cells in case 2 show brown staining in thecytoplasm/cell membrane components. This staining is notshown in the epithelial cells in case 2. For further details, seelegend of Fig. 1.

Fig. 1. Histology and IGF-IR expressionof craniopharyngioma cases. A , histology oftwo craniopharyngioma tumor specimens(C2 and C3) stained routinely with H&E(�200 magnification). Epithelial tumortissue is indicated by E. Cysts appearing incase 2 are indicated by (C). B, cytokeratin-7immunostainings of cell lines C2 and C3(�200 magnification). Arrows, somepositive cells (brown staining). C, IGF-IRimmunostainings of two craniopharyngiomatumors specimens (C2 and C3;�200magnification).The epithelial component ofcase 3 shows IGF-IR-positive cells (brownstaining).





Growth dependence of IGF-I. All nine cell lines, atindicated passages (passages 2-6), were investigated forgrowth-stimulatory effects of IGF-I in comparison with T3,and growth hormone as well as T3 and IGF-I in combina-tion. Serum-depleted cells were treated with these com-pounds for 24 hours, whereupon [3H]thymidine uptake wasdetermined (Fig. 2). As can be seen, IGF-I was the mosteffective stimulant and caused alone significant growthresponse in four of the cell lines (C1, C3, C4, and C9)and together with T3 in five cell lines (C1, C3, C4, C5,and C9).

As assessed by Western blotting, strong IGF-IR expression wasfound in five of the cell lines (C1, C3-5, and C9; Fig. 3). Thefour other cell lines showed low (C6 and C7) or no/very low(C2 and C8) IGF-IR signals. These data are well consistent withthe growth stimulatory effects of IGF-I (cf. Fig. 2). Four of thecell lines with high IGF-IR expression were responsive to IGF-I.Also, the data fit with the IGF-IR immunostaining dataobtained from the corresponding immunocytostainings as wellas the three available immunohistostainings (cf. Table 2).

Phosphorylation of IGF-IR and effects of inhibition. Two IGF-IR-positive (C1 and C9) and two cell lines with no/very lowIGF-IR expression (C2 and C8) were selected for analysis ofIGF-I induced phosphorylation of IGF-IR and Akt (Ser473).Figure 4A shows that IGF-I stimulates IGF-IR phosphorylationin the C1 and C9 cells. In contrast, no such effects were seenin C2 and C8. It is also shown that treatment withpicropodophyllin, administered at concentrations of 0.5 or2.5 Amol/L, ablates the IGF-I-stimulated phosphorylation ofIGF-IR. Picropodophyllin is a cyclolignan compound thatinhibits tyrosine phosphorylation of IGF-IR selectively(29, 38).

The phosphatidylinositol-3 kinase/Akt (protein kinase B)branch constitutes a major pathway mediating IGF-IR-depen-dent intracellular signaling for cell proliferation and survival(39). After IGF-I-induced phosphorylation of the receptorphosphoinositide-3-kinase becomes activated and phosphor-

ylates Akt at Ser473 (39). This is also shown to occur incraniopharyngioma cell lines with high expression of IGF-IR(C1 and C9; Fig. 4B). In the C2 cell line, being without IGF-IRactivity (see Fig. 4A), the ligand did not induce Aktphosphorylation. Surprisingly, in serum-depleted C8 cells (alsoIGF-IR negative), there was a high basal Akt phosphorylation,which was neither increased by IGF-I nor inhibited bypicropodophyllin (Fig. 3B). This means that Akt is constitu-tively activated and serum growth factor–independent in thiscell line. The underlying mechanism for this is unknown andnot relevant to this study, but could be due to PTEN(phosphatase and tensin homologue gene) mutation.

Fig. 3. Expression of IGF-IR in craniopharyngioma cells. C1-C9 lines were analyzedfor the expression of the h-subunit of IGF-IRusingWestern blotting (top). Actinwas used as loading control.The signals were quantified by densitometry and theIGF-IR expression calculated for each cell line as (absorbance of IGF-IR) /(absorbance of actin)�100. Bottom, means and SDs of IGF-IR expression of fourdeterminations.

Fig. 2. The effect of different supplementson growth of primary craniopharyngiomacells.The nine cell lines C-C9 were culturedin 96-well plates.The cells were allowed toattach and grow for 24 hours.They werethen serum-starved for12 hours, and thenstimulated with the following compounds inthe absence of serum for 24 hours:T3(20 nmol/L), IGF-I (10 ng/mL), growthhormone (10 ng/mL), IGF-I (10 ng/mL) + T3(20 nmol/L) or no supplement (CTL). Cellswere labeled with [3H]thymidine. After24 hours, cells were harvested and assayedfor incorporation of [3H]thymidine per wellinTCA insoluble material. Means and SDof quadruplicates are shown. Any significantstimulatory effects are indicated.Theexperiment was repeated thrice with similarresults.





Effect of IGF-IR inhibition on growth of craniopharyngiomacells. All nine craniopharyngioma cell lines were assayed foreffect of IGF-IR inhibition on proliferation. Cells were treatedwith picropodophyllin at different concentrations (50-2,500nmol/L) for 48 hours and assayed by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner saltfor cell growth. As shown in Fig. 5A, proliferation of C1, C3to C5, and C9 was markedly reduced with IC50 values ofV0.5 Amol/L. The maximal inhibitory effect (80%) after 48 hoursof treatment was seen at the highest concentration. To achievea complete inhibition of cell growth, the picropodophyllintreatment had to be continued for another 24 to 48 hours.This delayed response of picropodophyllin could be due toautocrine IGF-I/II expression. Growth of C5 and C6 cells wereslightly reduced but C2 and C8 were hardly affected at all.Table 1 and Fig. 5B summarize the proliferation data andrelate them to expression levels of IGF-IR for each cell line(cf. Fig. 2). As shown, responsiveness to IGF-IR inhibition

correlates well with IGF-IR expression. For closer details, seelegend of Table 3.

Discussion

One of the major issues regarding treatment of craniophar-yngioma patients, is not only the ability to accomplish tumor-free survival but at what price in terms of quality of life.Morbidity and mortality in this patient group results frominjury to the critical structures surrounding the tumor,inflicted by the tumor itself and the treatment. Recent seriesreports 81% to 91% 10-year recurrence-free survival rates aftersubtotal tumor removal followed by radiotherapy, with 46%to 58% of the survivors living a normal independent life(40, 41). Still, a substantial part of the patients are carryingconsiderable morbidity. Ten years after diagnosis, mostcraniopharyngioma patients can be expected to have panhy-popituitarism, and many to have serious visual disturbances,

Fig. 4. Phosphorylation of IGF-IR.Two cell lines with highIGF-IR expression (C1and C9) and twowith low IGF-IRexpression (C2 and C8)were serum-depleted overnight andthen treated with picropodophyllin (0, 0.5, and 2.5 Amol/L)for1hour, whereupon the cells were stimulatedwith IGF-I(100 ng/mL) for 5 minutes. Effects on phosphorylation ofIGF-IR (A) and Akt (s473) (B) were determined byimmunoprecipitation and/orWestern blotting as described inMaterials andMethods. As loading controls, IGF-IRh-subunit and Akt were detected.

Fig. 5. Effect of IGF-IR inhibition of growthof craniopharyngioma cells.A, all nine celllines were grown in complete medium.Picropodophyllin was added at differentconcentrations (0-2,500 nmol/L) for 48hours. Cells were analyzed for proliferationusing the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay. Means andSDs of triplicates are shown. B, relationbetween response to picropodophyllin(0, no;1, slight; and 2, strong, as defined inTable 3) and level of IGF-IR expression(from Fig. 2, bottom).





morbid obesity, memory disturbances, lack of libido, etc. Muchof this morbidity is caused by the treatment and the impactdiffers between surgery and radiation. Although the morbidityfrom surgery does arise directly after treatment, the effect ofradiation can unfold much later. Studies analyzing the lateeffects of radiation in craniopharyngioma patients are few. Onesuch study reports radiation-related complications in 58% of thechildren and 46% of the adults (42). Of the adult patients whodied without tumor growth, 41% of the deaths were related tocomplications from radiation treatment.

Because the clinical course of a craniopharyngiomas isknown to vary greatly and there are historical reports onsubgroups of patients living for many decades with mildsymptoms and a slowly progressing disease (43, 44), we needto find methods predicting the long-term outcome at the timeof diagnosis to enable individual treatment strategy. No suchmethods are known today.

This is the first study to show how craniopharyngioma cellscan be cultured systematically to produce cell lines. Not onlycan this method be used as an experimental tool for studyingthe versatile biological behavior of craniopharyngiomas but itcan also be helpful for the patients from whom the cultures areretrieved. One could establish an in vitro tumor profile for eachcraniopharyngioma patient, which takes into account thesensitivity of the tumor cells to different treatment modalities.By such means, one can hopefully get one step closer inestimating future prognosis of patients with a newly diagnosedcraniopharyngioma, and make an individualized treatmentstrategy, based on tumor biology, possible.

In this study, we show for the first time that craniophar-yngioma cells express IGF-R both in cell culture and in availableparaffin-embedded material and that expression varies betweenpatients. Craniopharyngioma cell cultures from five out of ninepatients showed high expression of the IGF-IR and that IGF-Ialone promoted growth in four of them. The fifth cell line (C5)with high IGF-IR expression was not responsive to IGF-I alone,but in combination with T3, this cell line was growth-stimulated. T3 alone had no mitogenic effect. Inhibition ofphosphorylation of IGF-IR resulted in marked reduction inproliferation of all five cell lines (also C5) with high expression.This suggests that IGF-IR is also critical for C5 growth, even

though this cell line requires permissive factors (e.g., T3) inaddition to IGF-I for proliferation.

According to our present results, one could expect thatpatients harboring viable craniopharyngiomas with highexpression of IGF-IR may have higher incidence of tumorrecurrence during growth hormone therapy because most ofthe growth stimulatory effect of growth hormone is mediatedvia the IGF-I/IGF-IR pathway in vivo (18, 20). A reported studyshowing that growth hormone therapy might increase thelikelihood of tumor recurrence support this notion (22, 45).On the other hand, three other clinical studies addressing thisissue are not in concordance with this assumption (8, 21, 23).However, all these three studies are associated with somelimitations. Most important, they do not report adequatecontrol groups for the treatments given before or during thegrowth hormone therapy, nor do they note the growth pace ofthe tumor before starting growth hormone therapy (8, 21, 23).To exemplify the impact of these variables, patients having nosigns of tumor remnants and patents receiving radiotherapybefore or during the study should be excluded or analyzedseparately because the recurrence rates in those groups ofpatients are much lower (40, 41, 46–50) compared withpatients harboring viable tumor after surgery (8). Also,because the growth potential of craniopharyngiomas differsbetween individuals, one has to note the growth pace of thetumor in each patient both before and after start of growthhormone treatment. It is difficult to evaluate the possibleeffects of growth hormone therapy on craniopharyngiomasthat are growing rapidly because they are likely to continue togrow irrespective of growth hormone treatment or not.

To summarize, our data show that craniopharyngioma cellswith high IGF-IR expression are IGF-I-dependent and thatattenuation of IGF-IR activity blocks their growth. Theseobservations suggest a functional impact of IGF-IR in a subsetof craniopharyngiomas. Further studies on larger collectionsof cases are needed to evaluate the expression level anddistribution of IGF-IR and its correlation to clinical variables.

Our study also points to the possibility of using IGF-IRinhibition as a treatment complement to radiotherapy in thefuture and in this way reduce the long-term complications forcraniopharyngioma patients.

Table 3. Relationship between IGF-IR expression and growth inhibition

Cell line IGF-Rexpression level* Picropodophyllin IC25c Picropodophyllin IC50

c Level of picropodophyllin responseb

C1 88 <50 <500 highC2 10 >2,500 >2,500 noC3 113 <50 <500 highC4 106 <500 500 highC5 96 <500 500 highC6 30 <2,500 >2,500 lowC7 23 <2,500 >2,500 lowC8 11 >2,500 >2,500 noC9 96 <50 <500 high

*Relative values (obtained from Fig. 3).cIC25 and IC50 (nmol/L picropodophyllin resulting in 25% and 50% inhibition of cell proliferation, respectively). Data obtained from Fig. 5A.bPicropodophyllin responses defined as follows: no, IC25 > 2,500 nmol/L; low, IC25 < 2,500. But IC50 > 2,500 nmol/L; high, IC50 < 500 nmol/L. Data obtained fromFig. 5A.





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2005;11:4674-4680. Clin Cancer Res Elfar Ulfarsson, Alexandra Karström, Shucheng Yin, et al. Novel Therapeutic ApproachGrowth Factor I Receptor in Craniopharyngioma Cells: A Expression and Growth Dependency of the Insulin-Like

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