Abstract
Colorectal cancer (CRC) has a severe mortality rate in New Zealand, with over 1200 annual disease-related deaths. The Consensus Immunoscore®, developed by Galon et al. is a prognostic measure of CD3+ and CD8+ T cells in the invasive margin (IM) and centre of the tumour (CT) that is superior to the current staging. High Immunoscore® patients show longer disease-free survival (DFS) than low Immunoscore® patients. To improve accessibility to this proprietary platform for low and middle-income countries, the Kemp Lab is developing its own open-source Immunoscore. Cancer-associated fibroblasts (CAFs) are heterogenous cells abundant in the tumour microenvironment (TME) that are associated with inhibition of T cell function in many cancer types and may affect Immunoscore status. CAFs have been associated with an immunosuppressive role influencing T cell function; however, whether CAFs directly influence T cells in CRC is unknown. The overall goal of the research in this thesis was to examine heterogenous CAF populations using high-parameter imaging and examine whether the spatial distribution of specific CAF types affect T cell frequencies, localisation, and phenotype and, hence, may affect Immunoscore status.
The Immunoscore was validated in a small cohort (n=61) and large cohort (n=389) of CRC patients. The Immunoscore was predictive of 5-year disease-free survival (DFS) in a small cohort, while the large cohort was predictive of DFS during a maximal 234-month follow-up period.
A multiplex IHC panel was optimised to identify CAFs in CRC, and CAFs were quantified in the IM, CT, and stroma. The density of vimentin+αSMA+FXIIIa+ CAFs was higher in the IM of high Immunoscore tumours compared to that of low Immunoscore tumours, and CAF density correlated with CD3+ T cell density in the CT.
CAFs were imaged with T cells using a 24-marker imaging mass cytometry™ (IMC™) panel to determine a relationship between the cell types in CRC tumours. Higher frequencies of Podoplanin+αSMA+FXIIIa+ CAFs were associated with higher CD3+ and CD8+ T cell density, and pairwise testing revealed that Podoplanin+αSMA+ CAFs significantly localised with CD8+ T cells in the CT, relative to an empirical null distribution.
To determine whether these cells aggregated in specific locations, a novel whole slide IMC™ was optimised using a 42-marker IMC™ panel to obtain whole slide imaging (WSI) images of the tumour sections. Quantification of heterogenous cell types with WSI using Tissue Mode was equivalent to quantification at a cellular level. Inflammatory, tumour-like, matrix, and vascular CAF populations were identified. Analyses of CAF spatial distributions with T cells showed that both matrix CAFs and αSMA- inflammatory CAFs were in close proximity to CD8 T cells in situ. CD8 T cells that resided with αSMA- inflammatory CAFs expressed Granzyme B. αSMA- inflammatory CAFs and Granzyme B+ CD8 T cells were abundant in high Immunoscore tumours.
Overall, this study showed a positive relationship between CAFs and T cells, although CAFs are usually characterised as immunosuppressive cells in the TME. This unique finding highlights that CAFs are highly diverse, and some types may be beneficial in the T cell response to the tumour. Granzyme B+ CD8 T cells associated with αSMA- inflammatory CAFs, and both were abundant in high Immunoscore tumours, validated in this thesis to represent good prognosis. Further functional studies are warranted to determine if there is a functional link between inflammatory CAFs and CD8 T cells to uncover their role in the T cell response.