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Investigating the role of high endothelial venules in cancer immunity

Milutinovic, Stefan ORCID: 2020. Investigating the role of high endothelial venules in cancer immunity. PhD Thesis, Cardiff University.
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The extent of T-cell infiltration is a key parameter that dictates cancer immunity and response to immunotherapy. The depletion of Foxp3+ regulatory T cells (Tregs) in a 3-methylcholanthrene (MCA) carcinogenesis model was previously shown to lead to the formation of high endothelial venules (HEVs) in approximately 50% of treated mice. Importantly, HEV formation was found to correlate both with the extent of T cell infiltration and control of tumour growth. To examine how intra-tumoral HEVs are globally distributed and whether they can be augmented to improve control of tumour growth, a 3-dimensional (3D) imaging technique known as light sheet fluorescence microscopy (LSFM) was used. In-vivo labelling, auto-fluorescence reducing, and organic solvent clearing protocols were successfully optimized for imaging blood vessel and HEV networks by LSFM. In-silico vessel quantification tools were further optimized to process LSFM acquired datasets of discrete HEV networks. These tools were validated by mapping the topology of naïve popliteal and inguinal lymph node (LN) HEV networks and were used to show that LN HEVs expand following Treg depletion and following the establishment of a fibrosarcoma tumour. LSFM imaging of a non-treated fibrosarcoma revealed the presence of HEVs in a small subset of vessels suggesting Treg depletion may not be an essential pre-requisite for intra-tumoral HEV formation. Furthermore, LSFM imaging of a regressor fibrosarcoma revealed a heterogenous global distribution and structural organization of HEVs. Lastly, LSFM imaging was used to show that combinatory Treg depletion with LTR agonist or anti-IL-27 neutralizing antibody treatment augments intratumoural HEVs. Such changes were not accompanied by an increase in immune cell infiltrate or improved control of tumour growth. Whilst LSFM imaging has demonstrated the plasticity of HEVs, the relationship between their presence, T cell infiltrate and tumour control is complex and requires further investigation before they can be harnessed for immunotherapy.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Medicine
Funders: Medical Research Council
Date of First Compliant Deposit: 26 March 2021
Last Modified: 09 Nov 2022 10:37

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