Abstract C76: Inhibition of galectin-1 expression by OTX008, a novel calyx[4]arene, is associated with antiproliferative and antiangiogenic activity in human tumor xenograft models.
Authors: Astorgues-Xerri Lucile , Tijeras-Raballand Annemilai , Serova Maria , Bieche Ivan , Riveiro Maria E. , Herait Patrice , Cvitkovic Esteban , Noel Kay , Raymond Eric , Faivre Sandrine 
Published: June 2012

Background: Galectin-1 binds neuropilin-1, enhances VEGFR2 signalling, and contributes to membrane anchorage of H-RAS in cancer cells. Those effects may modulate cancer cell proliferation, apoptosis, cell cycle, and tumor angiogenesis. OTX008 is a non-peptide chemical antagonist designed to bind the amphipathic -sheet conformation of galectin-1. We previously showed that OTX008 displays direct antiproliferative effects in cancer cell lines (AACR 2011, Abstract n°685). In this study we focused on the antiproliferative and antiangiogenic effects of OTX008 in xenograft models.

Material and Methods: A2780–1A9 (1A9) ovarian cells and SQ20B head and neck cells, selected for intermediate/high in vitro sensitivity to OTX008, were injected s.c. into female nu/nu athymic mice. When tumors became palpable, treatment was initiated with vehicle (PBS) or 5–10 mg/kg OTX008 i.p. q.d for 3 weeks. At the end of treatment, mice were sacrificed and tumors collected. mRNA expression was evaluated with qRT-PCR. Immunohistochemistry (IHC) was performed on OCT-embedded tumor stained with H&E, MIB1 (Ki67), VEGFR2, CD31, or galectin-1. Image quantification was done with Histolab software (Microvision, France) subtracting the background.

Results: OTX008 inhibited tumor growth in both 1A9 and SQ20B xenografts. OTX008 also decreased the number of secondary tumors (subcutaneous metastases distant from the injection site) in SQ20B xenografts. 1A9 and SQ20B tumors treated with OTX008 expressed significantly lower galectin-1 protein as compared to control tumors (1A9: 11.103 +/− 2.103 μm2 vs 26.103 +/− 6.103 μm2, p&lt;0.05; SQ20B: 3.103 μm2 +/− 0,6.103 vs 28.103 +/− 7.103 μm2, p&lt;0.01). Consistent with antiproliferative effects of OTX008, treated tumors expressed lower MIB1. Tumors treated with OTX008 also showed lower VEGFR2 mRNA and protein levels. Consistent with antiangiogenic effects of OTX008, CD31 immunostaining showed that treated tumors had significantly lower vessels number (p&lt;0.01 for SQ20B) and diameter (1A9: 0,5.103 +/−0,1.103 μm2 vs 6.103 +/− 2.103 μm2, p&lt;0.01; SQ20B: 10.103 +/−1.103 μm2 vs 28.103 +/− 4.103 μm2, p&lt;0.005), suggesting microvessel normalization. In order to confirm that these antiproliferative and antiangiogenic effects are mediated by galectin-1 in vivo, SQ20B cells were transfected with sh-galectin-1 and injected into nude mice. Inhibiting galectin-1 by shRNA reduced tumor growth comparable to the effects seen with OTX008. IHC analysis showed that inhibiting galectin-1 by shRNA recapitulated the effects of OTX008 in SQ20B xenografts, including a decrease of MIB1 (p&lt;0.005) and VEGFR2 (p&lt;0.05) expression in cancer cells, as well as in the number (p&lt;0.05) and the diameter of tumor vessels (p&lt;0.005).

Conclusion: Antiproliferative activity of OTX008 in xenograft models seems to be associated with galectin-1 expression inhibition. In addition to this direct effect, qualitative and quantitative normalization of microvessels may also play a role in the antitumor activity of OTX008. Galectin-1 and VEGFR2 expression as well as quantitative assessment of microvessels are candidate biomarkers for further clinical studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C76.