Bottomley, M. | Search bioRxiv

bioRxiv2025

Machine perfusion and single-cell spatial transcriptome mapping identifies novel immune escape mechanisms in colorectal cancer liver metastasis

Liver metastasis is the terminal stage of colorectal cancer. Immune checkpoint blockade (ICB) has heralded remarkable clinical success across a range of cancer types, but with limited efficacy in replacement-type colorectal liver metastasis (CRLM), the most common and lethal histological subtype. Using a novel human normothermic perfusion model, we demonstrate for the first time, that T-cells preferentially extravasate within the peri-tumoural liver rather than the CRLM. We use single-cell spatial transcriptomics and multiplexed immunofluorescence to validate CRLM T-cell exclusion and show that CRLM endothelia are anergic, lacking key receptors required for T-cell extravasation. CD4 T-cells that extravasate within the peri-tumoural liver are TCR-reactive, yet exhausted, whilst CD4 T-cells within the CRLM demonstrate a stress response, impaired cytokine expression and lack of TCR reactivity. We identify the spatial cellular and molecular interactions underlying these observations, providing novel targets for future attempts to sensitise to immunotherapeutics and a justification for failed ICB efficacy in CRLM.

cancer biology↗

bioRxiv2022

Structural and Computational Design of a SARS-CoV-2 Spike Antigen with Increased Receptor Binding Domain Exposure and Improved Immunogenicity

Emerging SARS-CoV-2 variants of concern challenge the efficacy of approved vaccines and emphasize the need for improved antigens. Using an evolutionary-based design approach starting from the widely used engineered Spike antigen, S-2P, we sought to increase antigen production levels and the exposure of highly conserved and neutralization sensitive receptor-binding domain (RBD) epitopes. Thirty-six prototypes were generated in silico, of which fifteen were produced and tested in biochemical assays. Design S2D14, which contains 20 mutations within the Spike S2 domain, showed a 6-fold increase in expression while preserving similar thermal stability and antigenicity as S-2P. Cryo-EM structures indicate that the dominant populations of S2D14 particles have RBDs in exposed states, and analysis of these structures revealed how modifications within the S2 domain balance trimer stability and RBD accessibility through formation and removal of hydrogen bonds and surface charge alterations. Importantly, vaccination of mice with adjuvanted S2D14 resulted in higher levels of neutralizing antibodies than adjuvanted S-2P against SARS-CoV-2 Wuhan strain and four variants of concern. These results can guide the design of next generation vaccines to combat current, and future coronaviruses and the approaches used may be broadly applicable to streamline the successful design of vaccine antigens.

immunology↗