Abstract
Worldwide, colorectal cancer (CRC) is the third most diagnosed cancer and is the second leading cause of death due to cancer. Similarly, in New Zealand, is the third most diagnosed cancer and is the second leading cause of death due to cancer. Overall, CRC patient survival is highly variable, even in CRC classified into the same histological cancer stage. The variability in the patient survival rate might be due to the complex nature of the CRC tumour microenvironment (TME).
The CRC TME is characterised by high WNT signalling activation and WNT agonists. WNT signalling drives CRC progression and influences different cellular components of the CRC TME. An important immune cell population within the CRC TME is the T cells. A high infiltrate of T cells within the CRC TME is correlated with better overall patient survival. However, T cells within the CRC TME are heterogeneous. It is currently poorly understood how WNT signalling activation affects T cell function. Further, the role of T cell heterogeneity within the CRC TME on the overall survival rate of CRC patients is not fully understood.
A subset of regulatory T cells (Tregs) within the CRC TME are the effector Tregs (eTregs). Our laboratory has previously shown that CRC patients with higher infiltrates of eTregs at the invasive tumour margin had better disease outcome compared to patients with lower infiltrates. Because of this, I studied the effect of WNT signalling on T cells using an eTreg polarisation media in vitro. WNT activation lowered the frequency of Tregs (CD4+CD25+CD127-FOXP3+) in healthy control and CRC peripheral blood mononuclear cells (PBMCs). Furthermore, WNT activation led to a decreased expression of inhibitory receptors (iRs) CTLA4 (in CRC PBMCs and tumours), PD1 (in CRC tumours), and TIM3 (in healthy control PBMCs and CRC tumours), and LAG3 (in healthy control PBMCs) on T cells. In healthy control PBMCs, the frequency of IL-2+ T cells was decreased. However, in CRC PBMCs WNT activation resulted in an increased expression of IFN-γ, TNF, and granzyme B on T cells. WNT activation in CRC tumours resulted in a decrease in the frequency of IFN-γ+ T cells. Taken together, WNT signalling activation negatively regulated FOXP3 and iRs expression on activated T cells while the effect on cytokine expression in T cells was dependent on the local microenvironment the T cells were isolated from.
Archived patient samples (e.g. formalin-fixed paraffin embedded (FFPE) tissue blocks, cryoembedded tissue blocks, and frozen whole tissue samples) are a source of immune information with corresponding patient data. I optimised a technique to immunophenotype T cells from archived patient samples through spectral flow cytometry. Frozen whole tissue samples were feasible to immunophenotype using surface markers through spectral flow cytometry. Similar to previous findings in our laboratory, there was a higher frequency of iRs CTLA4, PD1, LAG3, and TIM3 in frozen whole CRC tumours compared to adjacent non-tumour bowel. Developing the technique further would allow us to better understand how T cell heterogeneity affect CRC patient survival and prognosis.
The original plan of the PhD was to use the findings of the effects of WNT signalling on T cells to inform what T cell populations to look for on FFPE CRC tissue samples. Because of the challenges encountered working with cells isolated from FFPE tissue blocks on spectral flow cytometry (e.g. autofluorescence and lack of optimal antibody binding), I decided to investigate which archived sample type (e.g. FFPE tissue blocks, cryoembedded tissue blocks, and frozen whole tissue samples) would be usable on spectral flow cytometry.