(M1530-02-14) β-catenin Inhibition Using XAV939 Nanoparticle Promotes Immunogenic Cell Death and Suppresses Tumor Progression

Purpose: The evolutionarily conserved Wnt-/b-catenin pathway is critical for regulating embryonic development and adult tissue homeostasis(1). However, tumors induce aberrant activation of Wnt/b-catenin pathway to promote stem-cell like phenotype, tumorigenesis, immunosuppression and resistance to targeted cancer therapies(2-4). Therefore, targeting this pathway is a promising therapeutic approach to suppress tumor progression and elicit anti-tumor immune response. This study aims to provide a nanoparticle formulation for XAV939 (XAV-Np) to promote β-catenin degradation and modulate immune response in conjunctival melanoma (CM).

Methods: The study prepared XAV939 micelles using a film-dispersion method and quantified their loading concentrations through UV-VIS absorbance at 330nm. The morphology of the TPGS-XAV939 micelle was determined using Scanning electron microscopy (SEM) and atomic force microscopy (AFM). The size of micelles was determined with dynamic laser scattering (DLS). The anti-cancer effects of the micelles were evaluated in 2D and 3D cultured cell lines using Cell Counting Kit (CCK-8), Calcein AM-PI staining, and CellTiter-Blue assays(4). Wound healing assay and Transwell migration and invasion assay, and detection kits were applied to measure the ability of the micelles to prevent metastasis and induce immunogenic cell death. 1 x 10⁴ B16F10 cells were injected into the temporal subconjunctival space of the right eye to establish mice CM model and the treated group received XAV-Np on days 5, 8, and 11 pi at 0.4 mg/kg (5).

Results: XAV-Np with a uniform and spherical morphology had a drug loading efficiency (4.71± 0.17)%. The CMC value is 15 mg/L. XAV-Np showed a rapid 28 ± 3.79 % release of XAV939 within first 6 h and sustained release up to 120 h in vitro. The IC50 of XAV-Np was determined as 1.38 uM whereas there was no significant death in Con-Np-treated group.It showed sustained release of the drug over a period of 120 hours.(Fig1) The in-vitro cell-based studies indicated that AVA939 NP was effective in killing tumor cells and inhibiting their migration and invasion(Fig2). XAV-Np also promoted immunogenic cell death, with increased expression of cell surface calreticulin (CRT) and extracellular release of HMGB1 and ATP. In a mouse model of conjunctival melanoma, XAV-Np significantly reduced tumor growth compared to control animals. (Fig3).

Conclusion: The study developed a nano-formulation of the drug XAV939 for the treatment of melanoma. The nanoparticles exhibit decent drug loading and uniform morphology. The NPs also exert slow-release profile and stability in serum. Cell growth assays both in 2D and 3D levels indicate the efficacy of XAV939 NP. The in-vitro metastasis-associated studies show the anti-metastasis potential of XAV939 NP. The ICD effects induced by XAV939 NP suggests the potency of XAV939 NP in immune modulation. Based to the in-vitro and in- vivo assays, it can conclude that XAV-Np may be a promising therapeutic approach for the treatment of melanoma.

References: 1. Clevers, H. and R. Nusse, Wnt/beta-catenin signaling and disease. Cell, 2012. 149(6): p. 1192-205.
2. Jamieson, C.H.M., et al., The role of Wnt signaling in myeloid leukemogenesis. Blood, 2002. 100(11): p. 26a-26a.
3. Nusse, R. and H. Clevers, Wnt/beta-Catenin Signaling, Disease, and Emerging Therapeutic Modalities. Cell, 2017. 169(6): p. 985-999.
4. Kang, X., Cai, Y., Wang, Q., Wang, C., Chen, W., Yang, W., Suryawanshi, A., Zhou, G., Chen, P. and Li, F., 2021. Near-infrared light triggered activation of pro-drug combination cancer therapy and induction of immunogenic cell death. International Journal of Pharmaceutics, 607, p.120972.
5. Schlereth, S.L., et al., A Novel Model of Metastatic Conjunctival Melanoma in Immune-Competent Mice. Invest Ophthalmol Vis Sci, 2015. 56(10): p. 5965-73.

Acknowledgements: This study was supported by research funding from the Intramural Grants Program, Auburn University Research Initiative in Cancer (AURIC), and start-up funds from the Department of Pathobiology, College of Veterinary Medicine, Auburn University. PC’s work is funded by NIH MIRA R35GM133795.We thank Chunhui Huang for critical feedback during manuscript preparation. We thank Jiayi Ren for proofreading the manuscript.


Fig2. β-catenin inhibition using XAV-Np reduces tumor cell wound-healing (A), migration (B) and invasion (C).

Fig3. (A-D)In vitro. β-catenin inhibition induces immunogenic cell death in tumor cells indicated by CRT,ATP,HMGB1.(E-H) in vivo, XAV-Np suppresses conjunctival melanoma progression.