Znati, Sami; (2022) A Translational Study of Vandetanib Treatment for Hepatocellular Carcinoma. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background: Transarterial chemoembolisation (TACE) occludes tumour vascular supply and is administered to advanced hepatocellular carcinomas (HCC) with a 26 month median overall survival. The effect of vandetanib, which targets VEGFR-II to disrupt new vasculature, when loaded in TACE beads and delivered with and without radiotherapy was studied in preclinical models. Methods: Various 2D culture assays and 3D spheroids grown in Matrigel and RAFT systems were employed. Vandetanib titration measured dose-dependent lethality. Drug loading on TACE beads was quantified and elution was measured with fluorescence. Radiosensitisation was measured by 2-D clonogenic survival assay. Cell cycle arrest after radiation was studied with FACS. VEGFR-II expression of HCC cell lines was measured via western blot and the role of autophagy in vandetanib radiosensitisation was studied. Gapfilling invasion was measured following vandetanib and radiation. Serum samples from patients receiving vandetanib TACE were analysed with cross-correlation and PCA. Combined treatment outcomes were tested in vitro and in vivo. 3-D spheroids in matrigel were treated with vandetanib alone or with radiation. Tumour growth was measured in an immunocompetent mouse model under vandetanib, radiation and combination treatment. Results: In 2-D culture, an IC50 range of 2.7-83 μM was measured. Drug loading was found to be 0.92-1.66 μg per bead with complete elution in matrigel in <4 hours. Exposure-time dependent radiosensitisation by vandetanib was significant (p<0.05). Spheroids treated with radiation showed a cell cycle delay in G2/M. VEGFR-II expression was null in all HCC lines tested. Autophagic exhaustion was found in cells treated with both vandetanib and radiation. 2-D migration assays showed a dose-dependent reduction in migration after vandetanib treatment (p<0.05) and additive reduction in cells receiving combined treatment (p<0.05). Patient serum revealed inflammatory and vascular markers respond to TACE treatment. Significant (p<0.05) growth reduction was observed in 3-D spheroids treated with both 5 μM vandetanib and 4 Gy radiation compared to spheroids treated with 4 Gy radiation alone and 5 μM vandetanib alone. In 3-D spheroid co-culture with HCC and fibroblasts, combination treatment significantly reduced invasion over radiation (p<0.05) and vandetanib (p<0.0005). In animal models, HCC tumours experienced a growth delay with significant (3x2 Gy + VAN vs. 3x2 Gy p<0.005, 3x2 Gy vs. VAN p<0.0001) size reduction compared with control. Conclusion: The advantages of 3-D over 2-D models were demonstrated via direct comparisons. Markers of response to vandetanib TACE were identified in patient serum. Vandetanib inhibited spheroid growth and 3-D co-cultures of HCC and stromal cells were significantly inhibited by combined vandetanib and radiation. A reduction in tumour size was observed in vandetanib and radiation treated animals compared to controls, supporting vandetanib treatment of HCC.

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