Insulin-Like Growth Factor 1 Receptor and Response to Anti-IGF1R Antibody Therapy in Osteosarcoma

<div><p>Background</p><p>Survival outcomes for patients with osteosarcoma (OS) have remained stagnant over the past three decades. Insulin-like growth factor 1 receptor (IGF1R) is over-expressed in a number of malignancies, and anti-IGF1R antibodies have and are currently being studied in clinical trials. Understanding the molecular aberrations which result in increased tumor response to anti-IGF1R therapy could allow for the selection of patients most likely to benefit from IGF1R targeted therapy.</p><p>Methods</p><p><i>IGF1R</i> mRNA expression was assessed by RT PCR in OS patient primary tumors, cell lines, and xenograft tumors. <i>IGF1R</i> copy number was assessed by 3 approaches: PCR, FISH, and dot blot analysis. Exons 1–20 of <i>IGF1R</i> were sequenced in xenograft tumors and 87 primary OS tumors, and surface expression of <i>IGF1R</i> was assessed by flow cytometry. Levels of mRNA and protein expression, copy number, and mutation status were compared with tumor response to anti-<i>IGF1R</i> antibody therapy in 4 OS xenograft models.</p><p>Results</p><p><i>IGF1R</i> mRNA is expressed in OS. Primary patient samples and xenograft samples had higher mRNA expression and copy number compared with corresponding cell lines. <i>IGF1R</i> mRNA expression, cell surface expression, copy number, and mutation status were not associated with tumor responsiveness to anti-IGF1R antibody therapy.</p><p>Conclusions</p><p><i>IGF1R</i> is expressed in OS, however, no clear molecular markers predict response to IGF1R antibody-mediated therapy. Additional pre-clinical studies assessing potential predictive biomarkers and investigating targetable molecular pathways critical to the proliferation of OS cells are needed.</p></div

Relative IGF1-R mRNA Gene Expression in OS Primary Samples and Corresponding Cell Lines.

<p>Quantitative Real Time PCR detecting IGF1R mRNA expression was performed in OS primary samples (A) and OS cell lines (B). Grey bars represent relative quantification of IGF1-R expression using the ΔΔCT method using GAPDH as an endogenous control and MSC as a calibrator. Scatter plot comparing IGF1-R expression fold change in primary OS samples and their corresponding cell lines (C).</p

Summary of IGF1R mRNA Expression and Copy Number in OS Xenograft Models.

<p>Summary of IGF1R mRNA Expression and Copy Number in OS Xenograft Models.</p

OS Xenograft Model Response to anti-IGF1R Antibody SCH 717454 Treatment.

<p>Three OS xenograft models were treated with an anti-IGF1R antibody SCH 717454 (A) (open square) or vehicle (closed circle), small molecule IGF1R inhibitor BMS 754807 (B) (open square) or vehicle (closed circle) or IMC A12 (C) (open square) or vehicle (closed circle). Mice were treated with 0.5 mg SCH 717454 administered twice weekly via intraperitoneal injection for 4 weeks, BMS 754807, 25 mg/kg administered orally BID for 6 days, repeated for 6 weeks, or IMC A12, 1 mg/mouse administered intraperitoneally twice weekly for 6 weeks. These studies were previously reported; however, the figures for M17 and M31 were not published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106249#pone.0106249-Cao1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106249#pone.0106249-Kolb2" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106249#pone.0106249-Yuen1" target="_blank">[20]</a>. Ten mice were included in each arm of the experiments. Tumor size was monitored weekly for a maximum of 6 weeks of treatment or until a relative tumor volume increased to 4× baseline.</p

IGF1R Cell Surface Expression in OS Xenograft Models.

<p>FACS analysis of IGF1R surface expression in 4 OS xenograft models (M1: high responder, M2: intermediate responder, M17: intermediate responder, M31: high responder) and a positive control (MCF7) (A). The isotype control is represented by a red line and cells stained with IGF1R antibody are represented by a blue line.</p

LY5 inhibits pSTAT3 in sarcoma cell lines.

<p>(A) The tumor cell lines RH30, EW8, and RD were treated with increasing concentrations of LY5 for 30 minutes. Cell lysates were collected for Western blotting of p-STAT3, total STAT3, and GAPDH. GAPDH was used as loading control. (B-C) The SJSA cell line was serum-starved overnight and then left untreated or treated with LY5 for two hours followed by stimulation with 50 ng/mL of OSM, IL-6, IFN-γ, IFN-α, or IL-4 for 30 minutes prior to collection of cell lysates for Western blotting of p-STAT3, total STAT3 p-STAT1, total STAT1, p-STAT2, p-STAT4, or p-STAT6. GAPDH was used as loading control.</p

Effects of LY5 are not mediated via STAT3 inhibition.

<p>(A) Antitumor activity of LY5 against OS-1 PDX model of OS. Left panel, growth of individual control (untreated OS-1 xenografts); Center panel, growth of individual OS-1 xenografts in mice receiving LY5 (40 mg/kg twice daily for 5 days/week for 4 consecutive weeks); Right panel, median relative tumor growth (RTV) from control and treated groups. (B) Sarcoma cell lines (RMS cell line RH30 and ES cell lines ES-3 and ES-7) were transfected with either negative control siRNA (NC siRNA) or STAT3-targeting siRNA #7. After 72 hours of transfection, cells were harvested and analyzed for total (T) STAT3 and GAPDH protein expression by immunoblotting. (C) RH30, ES-7, and EW-8 cell lines were transfected with either NC siRNA or STAT3-targeting siRNA. After 48 hrs, cells were incubated with LY5 (1μM) for an additional 48 hrs. Cell viability was then determined using Alamar Blue staining. Data represent the mean ± standard deviation, n = 3. * denotes p < 0.05 by ANOVA.</p

LY5 treatment did not inhibit RMS or OS xenograft tumor growth.

<p>LY5 treatment did not inhibit RMS or OS xenograft tumor growth.</p

Biologic activity of LY5 in sarcoma cell lines.

<p>Dose response curves for LY5: RMS cell lines (RH30, RD, JR-1), ES cell lines (ES-3, ES-6, ES-7, ES-8), and an OS cell line (OS-17) were treated with LY5 (concentrations ranging from 10 nM to 10 μM) for 96 hours. Cell viability was determined using Alamar Blue staining. IC₅₀ values are indicated for each cell line. Analyses were performed in triplicate and data are plotted as means ± standard deviation.</p

LY5 inhibits STAT3 phosphorylation in lung metastases but does not inhibit lung metastasis formation.

<p>(A-B) IHC staining of lung metastasis from mice treated with LY5. The metastatic tumor sections were stained with pSTAT3 or hematoxylin and eosin. Western blotting was performed to confirm inhibition of STAT3 phosphorylation in lung tumor sections. (C) Mice were administered OS-17 cells stably expressing luciferase by tail vein injection and subsequently treated with LY5. After 30 days, imaging was performed to evaluate the effects of LY5 on the development of pulmonary metastasis.</p