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Cancer Therapy: Preclinical |
Authors' Affiliations: Departments of 1 Head and Neck Surgery, 2 Cancer Biology, and 3 Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas
Requests for reprints: Jeffrey N. Myers, Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, Unit 441, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone: 713-792-6920; Fax: 713-794-4662. E-mail: jmyers{at}mdanderson.org.
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Abstract |
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Experimental Design: The expression and activation of the IGF-IR and some of its downstream signaling pathway components were evaluated in both human thyroid cancer specimens and thyroid cancer cell lines. The therapeutic potential of a humanized monoclonal antibody (A12) directed against IGF-IR was assessed in vitro and in vivo in an orthotopic model of ATC. Tumor volume and overall survival time were analyzed to evaluate the efficacy of A12 in vivo.
Results: IGF-IR was overexpressed in 94% of the thyroid cancers. Blockade of IGF-IR with A12 was effective in attenuating IGF-IR signaling both in vitro and in vivo. However, the inhibitory effects of A12 on cell proliferation were cell line dependent, as those ATC cell lines that had detectable levels of pIGF-IR were more sensitive to A12 treatment. A12 was equally effective in vivo, where it brought 
57% (P = 0.041) inhibition in tumor volume. The concomitant use of A12 and irinotecan produced additive effects and resulted in a 93% (P < 0.001) reduction in tumor volume. Blocking IGF-IR blocked Akt phosphorylation and decreased proliferation and microvessel density but increased apoptosis within the tumor xenografts. Our results also highlighted a previously undefined IGF-IR-mediated antiangiogenic effect on tumor-associated endothelium in thyroid cancers.
Conclusion: Blocking the IGF-IR with A12 seems to be a potential avenue for treating patients with ATC by its direct antitumor effects and its effects on the tumor vasculature.
30,180 cases in 2006. Although anaplastic thyroid carcinoma (ATC) constitutes only 1.6% of all thyroid cancers, it accounts for 30% of thyroid cancer deaths. Even when patients with ATC receive aggressive multimodal therapy (surgery, radiotherapy, and chemotherapy), 80% die within 7 months as a result of local invasion and distant metastasis, especially those patients >60 years old (2, 3). Thus, novel therapeutic approaches are needed to improve the outcome of these patients (4).
Insulin-like growth factor-I receptor (IGF-IR) is a ubiquitous transmembrane tyrosine kinase composed of two extracellular 
subunits and two intracellular ß subunits (5–10). IGF-IRß transmits a ligand-induced signal by phosphorylating its substrates, mainly insulin receptor substrate-I (IRS-I). IGF-IR has been proven to mediate the mitogenic and antiapoptotic properties of its ligands, IGF-I and IGF-II, principally through the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase (MAPK) pathways, in a variety of human cancers (11–25).
Targeted therapy consisting of IGF-binding proteins with human monoclonal antibodies (mAb) and small-molecule tyrosine kinase inhibitors against IGF-IR have been developed, and studies of these have yielded encouraging results both in vitro and in vivo. These anti-IGF-IR-targeted treatments have been shown to significantly decrease migration, invasion, metastatic spread, and angiogenesis in tumor models. The inhibition of IGF-IR can also enhance the sensitivity of cancer cells to radiation or chemotherapeutic agents (26–36). These data suggest that targeted therapy directed at the IGF-IR given in combination with chemotherapy, radiotherapy, or both could be an attractive new treatment strategy for cancer patients (13).
A12 is a high-affinity, fully human IgG1
mAb that specifically binds to human IGF-IR and blocks IGF-I and IGF-II signaling but does not block the binding of insulin to the insulin receptor. It has been shown to inhibit the growth of breast, colon, and pancreatic cancer cell lines both in vitro and in s.c. tumor nude mouse models by antibody-mediated blockade of ligand binding to IGF-IR. In a study of a xenograft tumor model, A12 produced a marked increase in apoptosis with minimal toxicity (27).
Given previous studies showing the up-regulation of components of the IGF-I signaling pathway, we evaluated human thyroid tissue microarrays and found consistently higher IGF-IR levels in thyroid tumors than in normal thyroid tissue. These findings led us to hypothesize that targeting the IGF-IR could be an effective strategy for the management of ATC. We tested our hypothesis both in vitro and in vivo by treating ATC tumor cells with the A12 antibody alone and in combination with the cytotoxic chemotherapeutic drug irinotecan.
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Materials and Methods |
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Animals and maintenance. Male athymic nude mice, 8 to 12 weeks old, were purchased from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD). The mice were housed and maintained in laminar flow cabinets under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care in accordance with current regulations and standards of the U.S. Department of Agriculture, the U.S. Department of Health and Human Services, and the NIH. The mice were used in accordance with the Animal Care and Use Guidelines of The University of Texas M.D. Anderson Cancer Center (Houston, TX) under a protocol approved by the Institutional Animal Care and Use Committee.
Reagents. A12 antibody was generously provided by ImClone Systems, Inc. (New York, NY). For in vitro administration, A12 was dissolved in PBS to a concentration of 10 mg/mL and further diluted to an appropriate final concentration in RPMI 1640 with 2% FBS or without FBS. For in vivo testing, A12 was dissolved in PBS to achieve a final concentration of 4 mg/mL just before giving it to the mice. Irinotecan (Camptosar, Pharmacia and Upjohn Co., Kalamazoo, MI) was diluted in PBS to a concentration of 5 mg/mL for i.p. injections. Propidium iodide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were both purchased from Sigma-Aldrich Corp. (St. Louis, MO).
The following antibodies were used: anti-IGF-IRß (C-20; Santa Cruz Biotechnology, Santa Cruz, CA); anti-pIGF-IR (Tyr1131)/IR (Tyr1146), anti-IRS-I, anti-pIRS-I (Ser307), anti-Akt, anti-pAkt (Ser473), anti-MAPK mouse mAb (p42), and anti-pMAPK (Tyr42/Tyr44; Cell Signaling Technology, Beverly, MA); anti-human IGF-I mAb (R&D Systems, Inc., Minneapolis, MN); anti-ß-actin (Sigma-Aldrich); mouse anti–proliferating cell nuclear antigen (PCNA) clone PC-10 (DAKO A/S, Copenhagen, Denmark); rat anti-mouse CD31/platelet-endothelial cell adhesion molecule-1 and rat anti-mouse CD31 peroxidase-conjugated rat anti-mouse IgG1 (PharMingen, San Diego, CA); peroxidase-conjugated goat anti-rabbit IgG and peroxidase-conjugated goat anti-rat IgG1 (Jackson ImmunoResearch Laboratories, West Grove, PA); peroxidase-conjugated rat anti-mouse IgG2a (Serotec; Harlan Bioproducts for Science, Inc., Indianapolis, IN); Hoechst dye 3342 molecular weight 615.9 (Hoechst, Warrington, PA); and Alexa Fluor 594–conjugated goat anti-rat IgG and Alexa Fluor 488–conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR).
A terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay was done with a commercial apoptosis detection kit (Promega Corp., Madison, WI).
Western blotting. To study IGF-IR expression and autophosphorylation in different thyroid cancer cell lines, extracts of total cell proteins were obtained from the ARO, DRO, Hth74, KAT4, KAT18, WRO, NPA187, TPC-1, and TT cells. Cells were washed with PBS and lysed in buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 2% (v/v) NP40, 100 nmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 20 mmol/L aprotinin-leupeptin-trypsin inhibitor, and 2 mmol/L sodium orthovanadate. The samples were diluted in sample buffer [10% SDS, 0.5 mmol/L Tris-HCl (pH 6.8), 1 mol/L DTT, 10% (v/v) glycerol, 1% bromophenol blue] and boiled. Proteins (50 µg) were resolved by PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 1% bovine serum albumin (Sigma-Aldrich) in 0.1% (v/v) Tween 20 in TBS for 30 minutes, probed with anti-pIGF-IRß antibody (1:1,000), anti-IGF-IRß antibody (C-20; 1:4,000), and anti-ß-actin antibody (1:5,000) diluted in 1% bovine serum albumin overnight, and incubated with secondary horseradish peroxidase–conjugated goat anti-rabbit IgG antibody (1:3,000) in 1% bovine serum albumin for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (Pierce, Rockford, IL).
Western immunoblotting was done to show that A12 is able to inhibit the phosphorylation of IGF-IRß, IRS-I, Akt, and MAPK in vitro. For these studies, ARO cells were incubated in serum-free medium for 24 hours and either treated with 0.1 to 100 nmol/L A12 for next 2 hours or incubated with A12 at the same concentration (50 nmol/L) from 30 minutes to 6 hours before the addition of IGF-I or IGF-II (10 nmol/L; R&D Systems) for 15 minutes. Briefly, proteins (50 µg) were resolved by PAGE and transferred onto nitrocellulose membranes as described earlier. The membranes were blocked with 1% bovine serum albumin for 30 minutes; probed with anti-pIGF-IRß antibody (1:1,000), anti-IGF-IRß antibody (C-20; 1:4,000), anti-pIRS-I antibody (1:1,000), anti-IRS-I antibody (1:1,000), anti-pAkt antibody (1:2,000), anti-Akt antibody (1:2,000), anti-pMAPK antibody (1:3,000), anti-MAPK mouse antibody (1:2,000), and anti-ß-actin antibody (1:5,000); and incubated with secondary horseradish peroxidase–conjugated goat anti-rabbit IgG antibody (1:3,000) for 1 hour at room temperature. For anti-MAPK immunoblotting, the blots were incubated with a goat anti-mouse IgG secondary antibody that was conjugated with horseradish peroxidase (1:3,000) for 1 hour at room temperature.
Measurement of cell proliferation and cell death. We used a MTT assay to test the ability of A12 to inhibit the proliferation of TC cell lines in vitro. Approximately 2 x 103 cells per well were grown in RPMI 1640 supplemented with 10% FBS in 96-well tissue culture plates. After 24 hours, the cells were treated with various concentrations of A12 (0.1-100 nmol/L) in RPMI 1640 supplemented with 2% FBS. To measure the number of metabolically active cells after a 3-day incubation period, a MTT assay was done. Absorbance at 570 nm was recorded with a 96-well microtiter plate reader (MR-5000, Dynatech Laboratories, Inc., Chantilly, VA).
To measure the extent of cell death, ARO and DRO cells were plated at a density of 2 x 105 per well in 38-mm2 six-well plates (Costar, Cambridge, MA) and maintained for 24 hours before treatment with A12. After 24 hours, A12 was added in various concentrations (0-100 nmol/L) in RPMI 1640 supplemented with 2% FBS. After 48 hours of treatment with A12, the extent of cell death was determined by propidium iodide staining of hypodiploid DNA in Nicoletti buffer (50 µg/mL propidium iodide, 0.1% sodium citrate, 0.1% Triton X-100) for 20 minutes at 4°C. Cell cycle data were then analyzed by flow cytometry, and the sub-G0-G1 fraction was measured using Multicycle System (Phoenix Flow System, San Diego, CA).
Immunohistochemistry on human thyroid cancer tissue arrays and murine tumor tissue sections. Immunohistochemistry was done with the following antibodies: anti-pIGF-IRß, anti-IGF-IRß, anti-IGF-I, anti-pAkt, anti-Akt, anti-pMAPK, anti-MAPK, PCNA, CD31/platelet/endothelial cell adhesion molecule 1, and CD31/TUNEL. For IGF-I, IGF-IRß, and PCNA staining, paraffin-embedded sections were dewaxed and treated with pepsin for 20 minutes at 37°C. After blocking the endogenous peroxidases, the specimens were treated with a protein-blocking solution (5% horse serum with 1% goat serum) for 1 hour at room temperature. Primary antibody (1:50 dilution) treatment was carried out at 4°C overnight. The slides were blocked again with protein-blocking solution for 1 hour and incubated with horseradish peroxidase–conjugated anti-rabbit antibody at a 1:200 dilution for 1 hour at room temperature. The slides were then washed in PBS and incubated with 3,3'-diaminobenzidine for 10 minutes. After the excess 3,3'-diaminobenzidine was washed off, counterstaining was done with Gill's no. 3 hematoxylin.
For staining with antibodies against pIGF-IRß, pAkt, Akt, pMAPK, MAPK, and CD31/platelet/endothelial cell adhesion molecule 1, 8- to 10-mm-thick frozen sections were stained according to methods described previously (37). p-IGF-IR, pMAPK, and total MAPK antibodies were used at 1:100 dilution; pAkt, total Akt, and CD31/platelet/endothelial cell adhesion molecule 1 antibodies were used at 1:400 dilution. All antibody treatments were for 18 hours at 4°C.
For pIGF-IRß, pMAPK, total MAPK, pAkt, and total Akt, the samples were then incubated with Alexa Fluor 488–conjugated goat anti-rabbit IgG at 1:400; for CD31/platelet/endothelial cell adhesion molecule 1, the samples were incubated with Alexa Fluor 594–conjugated goat anti-rat IgG at 1:400 for 1 hour at room temperature in the dark. Samples were counterstained with 300 µg/mL Hoechst dye for 1 to 2 minutes at room temperature and then mounted using propyl gallate.
TUNEL staining was done according to the recommendations of the manufacturer. Red and green fluorescent images were acquired using a Zeiss Axioplan2 microscope (Carl Zeiss, Thornwood, NY) equipped with a 100-W HBO mercury bulb and the requisite filter sets (Chroma, Inc., Brattleboro, VT). Images were captured using a C5810 Hamamatsu color-chilled three-chip charge-coupled device camera (Hamamatsu, Tokyo, Japan) and digitized using imaging software (Optimas, Silver Spring, MD). The 3,3'-diaminobenzidine-stained, paraffin-embedded sections were examined under a Microphot-FX microscope (Nikon, Melville, NY) equipped with a three-chip charge-coupled device color video camera (model DXC990, Sony Corp., Tokyo, Japan).
For quantitative analysis of PCNA and CD31, the mean positive area and mean positive intensity were quantified in five random 0.159-mm2 fields (magnification, x100) per slide from a total of five slides per study group using the Image-Pro Plus software package (Media Cybernetics, Inc., Silver Spring, MD). For TUNEL staining quantification, the labeled cells were counted in five random 0.159-mm2 fields (magnification, x100) per slide from a total of five slides per study group.
The photomontages were prepared using Photoshop software (Adobe Systems, Inc., San Jose, CA).
Effects of A12 and irinotecan on the growth of orthotopic ATC xenografts in nude mice. Orthotopic xenografts in nude mice were established as described previously (37, 38). Briefly, ARO cells were harvested from subconfluent cultures by trypsinization. ARO cells (5 x 105) were then injected into the right thyroid lobe of each mouse in an injection volume of 5 µL using a 30-gauge needle. On the fifth day after their inoculation, five mice were euthanized to confirm establishment of the tumor mass with H&E staining. The orthotopically implanted tumors showed a 100% "take" rate in this pilot study (37). Consequently, for all subsequent studies, 5 x 105 ARO cells were implanted orthotopically and tumors were allowed to grow for the next 4 days and then randomized into different treatment groups.
For this study, they were randomized into four study groups of 10 mice each. The drugs were given as follows: (a) A12 via i.p. injection, 1 mg/injection, in 250 µL volume twice weekly; (b) irinotecan via i.p. injection at 50 mg/kg in 250 µL volume once weekly; (c) both A12 1 mg/i.p. injection twice weekly and irinotecan 50 mg/kg via i.p. injection in 250 µL volume once weekly; and (d) 250 µL PBS via i.p. injection once weekly as a placebo. The twice weekly injection regimen of 4 mg/mL A12 was determined following Burtrum et al. (27), who have shown that the half-life of A12 is 4 days in mice.4
A12 treatment on mice was continued for 3 weeks and their weights were recorded twice weekly. Our animal care and use protocol required that the animals be killed if they lost >20% of their body weight or if they became moribund. However, all the animals maintained their weights, and none had to be killed before the end of the treatment period. At the end of the 3-week treatment period, the mice were killed by CO2 asphyxiation, and necropsy was done. The cervical lymph node, the lungs, and the thyroid tumors were removed during the necropsy, sectioned and stained with H&E, and examined for the presence of metastasis. At the time of the necropsy, the tumors were measured in three dimensions. The volumes of the tumors were determined using the formula: V = 4 / 3(
)XYZ, where X, Y, and Z are the perpendicular radii of the tumors in each dimension. To ensure the immunohistochemical detection of pIGF-IR and pAkt, the last doses of A12 and irinotecan were given 2 hours before the mice were to be killed at the end of the 3-week treatment period. The percentage of tumor inhibition was calculated according to the formula: [1 – (T / C)] x 100, where T and C are the mean tumor volumes of the treatment group and the control group, respectively.
For immunohistochemical and routine H&E staining, one part of the tumor was fixed in formalin and embedded in paraffin. The other part was embedded in OCT compound (Miles Inc., Elkhart, IN), flash frozen in liquid nitrogen, and stored at –80°C.
Effects of A12 and irinotecan on the survival of nude mice bearing orthotopic ATC xenografts. Orthotopic ATC xenografts were established in nude mice as described above. Each group of mice (control, A12, irinotecan, and combination) was treated with PBS, A12, irinotecan, or both agents as described in the previous section. The mice were weighed twice weekly and killed if they showed a weight loss of >20% or appeared moribund. The mice were treated for 6 weeks.
Statistical analyses. Associations between normal and thyroid cancer for IGF-IR expression were tested with 
2 test. Best-fit curves were generated for the MTT and propidium iodide assays and used to determine the IC50. The nonparametric Kruskal-Wallis test was used to detect the difference in tumor volume between the four groups of mice. Pairwise comparisons between each treatment and control group and between treatments were assessed using the Wilcoxon rank-sum test. Survival was analyzed with the Kaplan-Meier method. Differences between the treatment and control groups were compared with the log-rank test. Quantified results of PCNA, CD31, and TUNEL staining were compared by an independent-samples t test. A two-tailed P < 0.05 was considered significant. All statistical analyses were done using Stata 9.0 (Stata Corp., College Station, TX).
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Having established that the components of the IGF pathway are indeed overexpressed in human thyroid cancer, our next objective was to choose a thyroid carcinoma cell line with which the antitumor activity of A12 could be tested.
Selected thyroid cancer cell lines have an intact IGF-I signaling axis. Western blot analysis was used to determine the expression of IGF-IR and its phosphorylation in nine human thyroid cancer cell lines. All of these cell lines expressed IGF-IRß (Fig. 1C). However, when we specifically assessed the ability of ATC cell lines to respond to IGF-I treatment, only three of the six ATC cell lines responded with phosphorylation of the IGF-IRß subunit (Fig. 1D). The remaining three cell lines (Hth74, KAT4, and ATC-A) did not respond to IGF-I stimulation, although they expressed measurable levels of the total receptor. Further studies were done on one of the pIGF-IR-positive cell lines (ARO).
A12 inhibits IGF-IR signaling pathways stimulated by IGF-I and IGF-II. To assess the ability of A12 to inhibit IGF-I-stimulated or IGF-II-stimulated IGF-IR signaling, serum-starved ARO cells were treated for 2 hours with various concentrations of A12 and then stimulated with IGF-I or IGF-II in serum-free medium for 15 minutes. Cell lysis and Western blotting for the phosphorylated forms of IGF-IR, IRS-I, Akt, and MAPK revealed that, at a concentration of 100 nmol/L, A12 completely inhibited both IGF-I-induced and IGF-II-induced phosphorylation of IGF-IR. In addition, at concentrations of 1 and 50 nmol/L, A12 inhibited IGF-I-induced and IGF-II-induced phosphorylation of IRS-I, Akt, and MAPK (Fig. 2A ). ARO cells treated for different times with the same dose of A12 (50 nmol/L) also showed pIGF-IRß inhibition (Fig. 2B). However, the inhibitory effects of A12 seemed to be independent of down-regulation of the receptor levels, as we failed to see A12-mediated down-regulation of the receptor when the cells were concomitantly treated with IGF-I. Similar findings have been reported for breast cancer cells (27). It was equally intriguing to note that 10 times more A12 was required to inhibit the IGF-II-mediated than the IGF-I-mediated signaling.
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The MTT assay results strongly supported our hypothesis, because increasing concentrations of A12 with or without IGF-I inhibited the proliferation of ARO, DRO, and C643 cells (Fig. 3A and B ), whereas the other three cell lines either failed to respond or responded minimally to A12 treatment. These results were also confirmed by determining cell counts 72 hours after treatment with A12 (data not shown).
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A12 treatment inhibits xenograft growth and prolongs animal survival in an orthotopic nude mice model of ATC. Preclinical efficacy of A12 as a single agent and in combination with chemotherapy was evaluated in a nude mouse model of ATC. Once ATC tumors were established by orthotopic implantation, animals were randomized into four treatment groups of 10 mice each: placebo (control), A12, irinotecan, and A12 plus irinotecan. Treatment with A12 or irinotecan alone led to a 57% and 80% decrease, respectively, in the tumor volume of ARO ATC xenografts. The differences in the tumor volume compared with the control group were statistically significant (P = 0.041 for A12 and P = 0.004 for irinotecan, respectively). However, the highest growth inhibition was achieved by the coadministration of A12 plus irinotecan. At the end of the 3-week treatment period, the mice treated with A12 plus irinotecan showed a 93% decrease in the estimated tumor volume compared with the control group (P = 0.0002). The decrease in the tumor volume in the mice receiving the combination treatment was also significantly greater than that of the groups receiving A12 or irinotecan alone (P < 0.01; Fig. 4A and B ).
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) it was well tolerated by the animals with no substantial adverse effects. The weights of the animals remained constant throughout the treatment period (data not shown) and none of the animals had to be killed before the end of the study. In the survival study, the survival rate of the mice treated with the combination was significantly greater than that of the mice in the control group (P = 0.0021). The combination group also achieved a greater survival rate than the mice treated with A12 alone (P = 0.004). However, there was no significant improvement in the survival rate between the groups treated with A12 alone or irinotecan alone when compared with the control group (P = 0.578 and 0.0955, respectively). Similarly, treatment with irinotecan alone was not superior to the combination treatment (P = 0.1015) or treatment with A12 alone (P = 0.1710; Fig. 4C).
A12 inhibits IGF-IRß and Akt phosphorylation in vivo. To show in vivo inhibition of IGF-IRß autophosphorylation by A12, immunohistochemical staining was done with antibodies specific to both IGF-IRß and pIGF-IRß. Tumors from all the study groups showed similar levels of total IGF-IRß expression, but the pIGF-IRß levels were decreased only in the tumors of the mice treated with A12 alone or in combination with irinotecan. To determine whether the blockade of IGF-IRß down-regulated its downstream signaling pathway, tumors from the four groups were stained with antibodies specific to Akt and pAkt. Although the level of total Akt expression was similar in all the study groups, pAkt staining was decreased only in the tumors of mice treated with A12 either alone or in combination with irinotecan (Fig. 5A ).
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To assess the degree of intratumoral apoptosis, the tumor sections were stained using the TUNEL method (Fig. 5B). The mean ± SD numbers of apoptotic cells per unit area in the tumors of control mice and the mice treated with irinotecan were 8.5 ± 4.3 and 12.5 ± 6.2, respectively (P = 0.127). However, treatment with A12 alone or A12 plus irinotecan significantly increased the mean numbers of apoptotic cells per unit area to 24.2 ± 9.9 and 25.9 ± 15.4 compared with the control (P < 0.01) and irinotecan-only (P < 0.05) groups.
Microvessel density was determined by staining tumor sections with anti-CD31 antibodies. Treatment with irinotecan did not affect tumor microvessel density compared with the control group. However, treatment with A12 alone or in combination with irinotecan resulted in a statistically significant inhibition of tumor-associated angiogenesis in both mean positive area and mean positive intensity compared with the control group (P < 0.01; Fig. 5B and C).
We also used double-labeled immunohistochemical techniques to stain for CD31 and pIGF-IRß on endothelial cells in the tumor sections. The tumors from the control group or those treated with irinotecan alone showed strong colabeling of fluorescent red anti-CD31 staining specific for endothelial cells and fluorescent green staining of pIGF-IRß. In contrast, the phosphorylation of IGF-IRß in endothelial cells was significantly suppressed in the tumors of mice treated with A12 alone or in combination with irinotecan (Fig. 6 ). This was an important observation. More so, the A12 antibody was raised against human IGF-IR but also seems to act on mouse endothelial IGF-IR, suggesting that it may have more widespread ramifications. The total IGF-IRß level showed no change in the double staining of CD31/total IGF-IRß (data not shown).
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The IGF-I receptor is one of several growth factor receptors implicated in cancer development and progression of various human tumors. The IGFs and IGF-IR signaling thus became an attractive target for new therapeutic strategies in cancer treatment (21, 22). In this study, we assessed IGF-IR expression in both human thyroid cancer specimens and thyroid cancer cell lines. Immunohistochemical analysis showed significantly higher expression of this receptor in human PTC and ATC specimens than in normal thyroid tissue. Using Western blot analysis, we corroborated these findings in thyroid cancer cell lines as well. Our results were similar to those of Belfiore et al. (9) and Vella et al. (10), thus supporting the notion that IGF-IR plays an important role in the progression of ATC.
Interestingly, our Western blot analysis of IGF-I-mediated autophosphorylation of the receptor indicated that although all ATC cell lines expressed detectable levels of IGF-IRß subunit only some of those cell lines autophosphorylated the receptor in response to IGF-I treatment. One possible reason may be that these cell lines harbor or activate specific phosphatases that can continuously dephosphorylate IGF-IR, thereby rendering it nonfunctional and forcing the cells to adapt IGF-independent survival pathways. Our in vitro studies support this hypothesis as in both MTT and cell-counting assays; A12 induced antiproliferative effects only on the ATC cell lines ARO, DRO, and C643 in a dose-dependent manner. In contrast, the Hth74, KAT4, and ATC-A cell lines failed to phosphorylate the receptor as well as failed to respond to A12 treatment.
However, the levels of pIGF-IR may not be the only determinant of A12 responsiveness even within the group of responsive cell lines; C643 cells are 10-fold more sensitive to growth inhibition by A12 compared with ARO and DRO cells. All three of these cell lines express similar levels of IGF-IR and pIGF-IR. Additional cues of A12 responsiveness may thus be obtained by further exploring the biology of C643 cells that makes them more sensitive to A12 treatment.
Our observed inhibitory effects of A12 concur with those reported by others who used small-molecule tyrosine kinase inhibitors of IGF-IR (NVP-AEW541 and NVP-ADW742) that sensitized multiple myeloma and lung cancer cells to chemotherapy drugs, such as doxorubicin and etoposide (28, 29). However, unlike the small-molecule IGF-IR kinase inhibitors studied previously, A12 does not seem to significantly increase ATC cell apoptosis in culture. Our cell cycle analyses showed that ATC cells treated with A12 accumulate in the G0-G1 stage (data not shown), suggesting that, as a mAb, A12 causes decreased cell growth predominantly through a cytostatic effect. Similar results have been found in other studies of IGF-IR antibodies (EM164, h7C10, CP-751, and CP-871; refs. 30, 34, 36).
The principal IGF-IR signaling pathway is through the IGF-IR/IRS-I axis. Both IGF-I and IGF-II can bind to IGF-IR and induce ligand-dependent receptor autophosphorylation. The IGF-IRß then phosphorylates a series of adaptor proteins, including IRS-I, to activate intracellular signaling cascades, including the Akt and MAPK pathways. In our experiments, A12 was found to down-regulate the phosphorylation of IGF-IRß, IRS-I, Akt, and MAPK induced by both IGF-I and IGF-II. The downstream signal inhibition was more extensive in cells stimulated by IGF-I than by IGF-II. This may be attributed to the fact that IGF-II follows a slower binding kinetics to IGF-IR than IGF-I (38). Additionally, IGF-II can signal not only through the IGF-IR but also through insulin and mannose-6-phosphate receptors. A12, on the other hand, is very specific to IGF-IR and has no antagonistic activity against the IR or mannose-6-phosphate receptor. Our results agree with earlier reports where it has been shown that A12 has lower blocking activity for IGF-I with an IC50 equal to 1 nmol/L but significantly higher for IGF-II with an IC50 equal to 6 nmol/L (27).
To precisely determine the effect of A12 on tumor growth in vivo, we used an orthotopic model to study thyroid cancer in nude mice (37, 39). A12 significantly inhibited the growth of ARO mouse tumors with few toxic effects. Treatment with A12 alone produced 50% reduction in tumor volume. In tumor sections, immunohistochemical analyses revealed a decrease in pIGF-IRß, pAkt, and PCNA staining and an increase in apoptosis. However, in the survival study, the survival rate of the group treated with A12 alone was not better than that of the placebo-treated group. This was an unexpected outcome, considering the fact that A12 effectively reduced tumor volumes by 57%. It is conceivable that in an advanced tumor type, such as ATC, the cells have multiple growth receptor pathways or downstream growth-regulatory molecules up-regulated so that interference with a single receptor at the cell membrane does not significantly alter the balance of growth-stimulatory and growth-inhibitory signals to the cytoplasm and nucleus.
However, the most significant tumor growth suppression and longest survival improvement were achieved with the combination of A12 and irinotecan. Therefore, we propose that IGF-IR-targeted therapy should be given in combination with other therapeutic strategies to achieve its maximum antitumor effects (25, 28). Thus, it may provide a further beneficial effects, such as preventing resistance to drugs (e.g., trastuzumab; ref. 40). Another potential benefit of its use as an adjuvant therapy was revealed when immunostaining quantification analyses showed that A12 enhanced the cytotoxic effect of irinotecan by decreasing PCNA staining and inducing the apoptosis of tumor cells. It is likely that part of the antitumor efficacy of A12 may also be due to its ability to induce antibody-dependent cellular cytotoxicity as has been reported for some other therapeutic mAb agents (41). However, this possibility was not specifically explored in this study.
Recent studies have shown that the IGF-IR is abundantly expressed in endothelial cells (42). IGFs have been found to promote the growth, survival, and migration of tumor cells and to induce the syntheses of vascular endothelial growth factors A and C and matrix metalloproteinase-2, which may favor the development of the blood supply essential for the progressive growth of primary malignancies and the development of metastases (43–48). Indeed, in our in vivo study, we found a decrease in microvessel density in the tumors from mice treated with A12 alone or in combination with irinotecan. Although previous studies from our laboratory have shown the antiangiogenic activity of a vascular endothelial growth factor receptor inhibitor (49), we did not detect apoptotic tumor endothelial cells in this study. However, a double-labeled immunohistochemical analysis showed that the phosphorylation of IGF-IR was decreased on tumor-associated endothelium in A12-treated tumors compared with controls, suggesting that IGF-IR signaling may play a role in endothelial cell survival in ATC tumors.
There is certainly precedence for the role of IGF-IR in endothelial cell survival in Ewing's sarcoma where it has been shown that IGF-IR receptor directly affects the levels of vascular endothelial growth factor-A, and there is a strict correlation between the levels of vascular endothelial growth factor-A and human umbilical vascular endothelial cell proliferation and survival (50). It is therefore possible that the antiangiogenic response induced by A12 is the result of reduced levels of proangiogenic factors, such as vascular endothelial growth factor-A, in the tumor endothelium. Further studies have been initiated to test this hypothesis. Similarly, additional studies have been undertaken to determine how and when A12 becomes an effective antiangiogenic agent and if shorter exposures would also elicit a similar response.
In summary, we have shown that a novel IGF-IR antibody, A12, can significantly inhibit the proliferation of ATC cells by down-regulating the IGF-IR signaling pathway in vitro. Treatment with A12 alone or in combination with irinotecan reduced tumor volume and prolonged survival times in an orthotopic ATC nude mouse model by enhancing the cytotoxic effect of irinotecan and inducing antiangiogenesis. These findings suggest that blocking the IGF-IR with A12 seems to be a potential avenue for treating patients with ATC by its direct antitumor effects and its effects on the tumor vasculature.
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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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
4 Personal communication (AACR Abstract 2004; Ludwig et al.). 
Received 12/ 8/05; revised 5/ 9/06; accepted 5/17/06.
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  E. K. Rowinsky, H. Youssoufian, J. R. Tonra, P. Solomon, D. Burtrum, and D. L. Ludwig IMC-A12, a Human IgG1 Monoclonal Antibody to the Insulin-Like Growth Factor I Receptor Clin. Cancer Res., September 15, 2007; 13(18): 5549s - 5555s. [Abstract] [Full Text] [PDF]
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, P = 0.004, compared with the control group; 
, P = 0.0002, compared with the control group; P < 0.01, compared with the A12-alone and irinotecan-alone groups (independent t test). C, combined treatment of A12 + irinotecan significantly prolonged the survival rate in the orthotopic nude mouse model ATC compared with that of the control group (P = 0.0021).
