Anti-Human IGF1 Receptor Recombinant Antibody (Cixutumumab) (CAT#: TAB-078)

Figure 1 Effects of bevacizumab, cixutumumab, and combination bevacizumab and cixutumumab in vitro and in vivo.

A, Western blotting for IGF-1 receptor b, VEGFR-2, and phospho-IGF-1 receptor b in HUVEC cells treated with cixutumumab (20 mg/mL), bevacizumab (75 mg/mL), and the combination of cixutumumab and bevacizumab. GAPDH was used as control. B, Western blotting for IGF-1 receptor b, VEGFR-2, and p-IGF-1 receptor b in SKOV3 cells treated with cixutumumab (20 mg/mL), bevacizumab (75 mg/mL), and the combination. GAPDH was used as control. C, growth curve for SKOV3 xenografts. Results are means AE SE of 6 mice per group; P < 0.05 for overall Kruskal-Wallis test at week 6; cixutumumab was different from the combination therapy. D, growth curve for A2780 xenografts. Results are means AE SE of 5 mice per group, P < 0.05 for overall ANOVA test at day 17. Control was different from all other groups and cixutumumab alone was different from bevacizumab and the combination therapy.

Shao, M., Hollar, S., Chambliss, D., Schmitt, J., Emerson, R., Chelladurai, B.,... & Matei, D. (2012). Targeting the insulin growth factor and the vascular endothelial growth factor pathways in ovarian cancer. Molecular cancer therapeutics, 11(7), 1576-1586.

Figure 2 MVD and apoptosis in SKOV3 xenografts treated with control, bevacizumab, cixutumumab, and combination bevacizumab and cixutumumab.

A, representative immunohistochemical staining for CD31 shows microvessel density in control, cixutumumab, bevacizumab, and combination-treated xenografts (Â100 magnification). B, quantification of microvessel counts was carried out in 5 HPF per specimen in the 4 groups. Results are means of counts in 5 HPF AE SE of 3 specimens per group; results of overall Kruskal-Wallis test were not statistically significant; the 2 bevacizumab groups combined were different from control. C, representative TUNEL immunofluorescent staining in control-, cixutumumab-, bevacizumab-, and combinationtreated xenografts. D, quantification of positive cells in the TUNEL assay.

Shao, M., Hollar, S., Chambliss, D., Schmitt, J., Emerson, R., Chelladurai, B.,... & Matei, D. (2012). Targeting the insulin growth factor and the vascular endothelial growth factor pathways in ovarian cancer. Molecular cancer therapeutics, 11(7), 1576-1586.

Figure 3 Immunohistochemistry for Ki67 and phospho-Ser 473-AKT in control-, cixutumumab-, bevacizumab-, and combinationtreated SKOV3 xenografts.

A, representative immunohistochemical staining for Ki67 in control-, cixutumumab-, bevacizumab-, and combinationtreated xenografts (Â100 magnification). B, quantification of Ki67-positive cells was carried out in 5 HPF per specimen in the 4 groups (n ¼ 3 specimens per group). The overall ANOVA test was significant and all treatment groups were different from control. Representative C, immunohistochemical staining for phosphoSer 473-AKT in control-, cixutumumab-, bevacizumab-, and combination-treated xenografts (Â100 magnification). D, H scores were calculated as described in Materials and Methods. Results are means AE SE of 3 specimens per group; the overall ANOVA test was not statistically significant.

Shao, M., Hollar, S., Chambliss, D., Schmitt, J., Emerson, R., Chelladurai, B.,... & Matei, D. (2012). Targeting the insulin growth factor and the vascular endothelial growth factor pathways in ovarian cancer. Molecular cancer therapeutics, 11(7), 1576-1586.

Figure 4 HNSCC and NSCLC cell lines display differential sensitivities to cixutumumab in the 3D mimic condition.

Indicated HNSCC and NSCLC cells cultured in poly(HEMA)-coated plates (PCP) and in ultralow attached plates (UAP) were treated with hIgG1 (25 μg/mL) or IMC-cixutumumab (25 μg/mL) for 3 (A, C, and D) or 5 days (B) in the presence of FBS or for 6 hours in the absence of FBS and then stimulated with 10% FBS for 30 minutes (A, bottom). A, representative morphologies of LN686 and OSC19 cells (Con, control; Cixu, cixutumumab). A (bottom) and D, Western blot was done for the indicated proteins.

Shin, D. H., Min, H. Y., El-Naggar, A. K., Lippman, S. M., Glisson, B., & Lee, H. Y. (2011). Akt/mTOR Counteract the Antitumor Activities of Cixutumumab, an Anti-Insulin–like Growth Factor I Receptor Monoclonal Antibody. Molecular cancer therapeutics, 10(12), 2437-2448.

Figure 5 Cixutumumab induced the activities and expression levels of EGFR and Akt is through mTOR-mediated protein synthesis.

A, RT-PCR analysis of LN686 and FADU cells grown in PCPs in the presence of vehicle (Con) or cixutumumab (Cixu) for 3 days. B, LN686 cells grown in PCP were treated with cixutumumab in the absence of FBS for indicated time period and then stimulated with 10% FBS for 30 minutes before harvest. The indicated proteins were detected by Western blot analysis. C, LN686 cells grown in PCP in the presence of vehicle (Con) or cixutumumab (Cixu) with or without rapamycin (Rapa) or 3 days were metabolically pulse-labeled with trans 35S-methionine and cysteine and then chased with media containing methionine and cysteine for the indicated time periods.

Shin, D. H., Min, H. Y., El-Naggar, A. K., Lippman, S. M., Glisson, B., & Lee, H. Y. (2011). Akt/mTOR Counteract the Antitumor Activities of Cixutumumab, an Anti-Insulin–like Growth Factor I Receptor Monoclonal Antibody. Molecular cancer therapeutics, 10(12), 2437-2448.

Figure 6 Interaction between cixutumumab-treated cancer cells and stromal cells stimulates tumour angiogenesis.

(a) Representative images of migration and tube formation assay of cixutumumab-treated HUVECs. (b) HUVECs were seeded in the top chamber of the transwell insert and allowed to migrate for 6 h. Left: cixutumumab-treated H1299 cells were seeded in the bottom chambers of the transwell. Right: CM from cixutumumab-treated H1299 cells were filled in the bottom chambers of the transwell. (c) Co-culture of cancer cells and stromal cells.

Lee, J. S., Kang, J. H., Boo, H. J., Hwang, S. J., Hong, S., Lee, S. C.,... & Kim, B. J. (2015). STAT3-mediated IGF-2 secretion in the tumour microenvironment elicits innate resistance to anti-IGF-1R antibody. Nature communications, 6, 8499.

Figure 7 Transcriptionally upregulated IGF-2 mediates the interaction between cancer cells and stromal cells during IGF-1R blockade.

(a) Indicated cancer cells and stromal cells were treated with cixutumumab for 6 days, and expression of ligands and receptors of the IGF axis were determined by real-time PCR. (b) CM from cixutumumab-treated cancer cells were analysed by western blotting to confirm IGF-2 secretion during cixutumumab treatment. Lower blots present Coomassie Blue (CB) staining as a loading control. Graph below shows densitometric analysis of three independent western blot assays. (c) IGF-2 immunofluorescence staining in cixutumumab-treated MDA231 cells (scale bar, 10 μm). (d) IGF-1R expression was stably reduced by shRNA in H1299 cells, and the expression of IGF-2 and IGF-1R was determined by real-time PCR (left) and western blotting (right). (e) Ligand, receptor and IGF-2R expression in stromal cells were determined by RT–PCR and western blotting. (f, left) RT–PCR and western blotting data revealing altered cixutumumab-induced IGF-2 expression. (f, right) Wi38 or THP-1 cells were seeded in the top chamber of the transwell insert. The bottom chambers were filled with CM from cixutumumab-treated H1299 cells transfected with empty vector or shIGF-2.

Lee, J. S., Kang, J. H., Boo, H. J., Hwang, S. J., Hong, S., Lee, S. C.,... & Kim, B. J. (2015). STAT3-mediated IGF-2 secretion in the tumour microenvironment elicits innate resistance to anti-IGF-1R antibody. Nature communications, 6, 8499.

Figure 8 IGF-1R blockade-induced IGF2 transcription via STAT3 activation.

(a) Protein lysates from cixutumumab-treated H1299 cells were incubated with phospho-RTK arrays. Spots are in duplicate, with each pair corresponding to a specific pRTK. The pair spots in the corners are positive controls. STAT3 phosphorylation was determined by western blotting in (b) cixutumumab-treated or (c) IGF-1R-silenced H1299 cells. (d) Luciferase assay for IGF-2 promoter activity. Insert: STAT3 expression was reduced by shRNA in H1299 cells as confirmed by western blotting. Left: P3 promoter. Right: P4 promoter. Each bar represents the mean relative luciferase unit (RLU) ±s.d. of four identical wells of a single representative experiment. (e) The effect of cixutumumab on IGF-2 expression in H1299 cells with reduced STAT3 expression was determined by RT–PCR and western blotting. (f) Wi38 cells were seeded in the top chamber of the transwell insert. Cixutumumab-treated H1299 cells transfected with empty vector or shSTAT3 expression were seeded in the bottom chambers of the transwell. Wi38 cells were allowed to migrate for 16 h. Each bar represents the mean relative unit (RU) ±s.d. of three replicates of a single representative experiment.(i) The excised primary tumours were assessed by immunofluorescence staining using anti-F4/80 (left) and -CD34 (right) antibodies (Scale bar, 50 μm). Data are presented as mean positive cells per field of view (FOV) ±s.d.

Lee, J. S., Kang, J. H., Boo, H. J., Hwang, S. J., Hong, S., Lee, S. C.,... & Kim, B. J. (2015). STAT3-mediated IGF-2 secretion in the tumour microenvironment elicits innate resistance to anti-IGF-1R antibody. Nature communications, 6, 8499.

Figure 9 Increased CXCL8 production from stromal cells through the IGF-2/IGF-2R interaction.

(a) Wi38 or THP-1 cells were co-cultured with cixutumumab-treated cancer cells in the transwell for 24 h and the mRNA level of each target was determined by real-time PCR. (b) Wi38 or THP-1 cells were treated with rhIGF-2 (100 ng ml−1, 24 h) and CXCL8 mRNA levels were examined by real-time PCR. (c) IGF-2R expression was reduced by shRNA (left) or anti-IGF-2R-neutralizing antibody (10 μg ml−1) (right) in Wi38 cells and followed by treatment with CM from cixutumumab-treated cancer cells for 24 h. CXCL8 mRNA levels were determined by real-time PCR. (d) Wi38 cells were incubated with CM from cixutumumab-treated H1299 transfected with empty vector or shIGF-2 (left) or anti-IGF-2-neutralizing antibody (5 μg ml−1) and CM from cixutumumab-treated H1299 cells (right). After 24 h, CXCL8 mRNA levels were examined by real-time PCR. (e, left) Wi38 cells were transfected with negative control siRNA or siCXCL8 and knockdown of CXCL8 was confirmed by real-time PCR. (e, right) Transfected with each siRNA were seeded in the bottom chamber and co-cultured with cixutumumab-treated H1299 cells in the transwell.

Lee, J. S., Kang, J. H., Boo, H. J., Hwang, S. J., Hong, S., Lee, S. C.,... & Kim, B. J. (2015). STAT3-mediated IGF-2 secretion in the tumour microenvironment elicits innate resistance to anti-IGF-1R antibody. Nature communications, 6, 8499.

Figure 10 Clinical relevance of tumour-associated macrophages and fibroblasts in HNSCC patients with cixutumumab treatment.

(a) Representative images of IHC analyses on IGF2 expression, VEGFR+ VE cells, F4/80+ macrophages and FSP-1+ fibroblasts in the tissue samples from patient with HNSCC, either naive or treated with cixutumumab for 3 weeks. Images are from patient 1 of each group. Other images are included in Supplementary 9. (b) Schematic model of events noted in the TME on treatment with IGF-1R-targeted therapy.

Lee, J. S., Kang, J. H., Boo, H. J., Hwang, S. J., Hong, S., Lee, S. C.,... & Kim, B. J. (2015). STAT3-mediated IGF-2 secretion in the tumour microenvironment elicits innate resistance to anti-IGF-1R antibody. Nature communications, 6, 8499.