Immune profiling of the tumour microenvironment in prostate cancer

Ntala, Chara (2020) Immune profiling of the tumour microenvironment in prostate cancer. PhD thesis, University of Glasgow.

Abstract

Prostate cancer is the most common cancer among men in the UK and is characterised by large biological and clinical heterogeneity. There is an urgent need for better-personalised patient stratification, for example in accurately identifying patients with regional lymph node metastasis. Nodal involvement negatively impacts on patient survival outcomes and the current pre-operative staging tools to determine the need for extended pelvic lymph node dissection at time of radical prostatectomy are far from precise. The primary tumour immune microenvironment influences tumour immune editing and therefore disease progression. The primary aim of this research was to investigate the in situ phenotype of prostate cancer tumour infiltrating immune cells and determine their potential as biomarkers for regional lymph node invovlement and further explore possible underlying mechanisms for their distribution.
The discovery tissue microarray comprised of index lesions from 94 patients undergoing radical prostatectomy and pelvic node dissection (50 with and 44 without histologic evidence of pelvic nodal disease respectively, referred to as LN+ and LN- thereafter). Two multiplex immunofluorescence panels were optimised to comprehensively characterise the immune microenvironment: (1) The macrophage and B cell panel includes CD68, CD163, CD20, AE1/3 (PanCK) and DAPI and (2) The T lymphocytic panel assays for CD4, CD8, FoxP3, PD-1, AE1/3 and DAPI. The macrophage (CD68, CD163+), T (CD8+, CD4+) and B (CD20+) cell immune cell subpopulations within the malignant epithelium and associated stroma were measured and correlated to the nodal status. Stromal infiltration by M1-like macrophages (CD68+CD163-) (p=0.047), CD8 effector (CD8+FoxP3-PD-1-) (p=0.008) and CD4 effector (CD4+FoxP3-PD-1-) T cells (p=0.0003, Mann Whitney test) were lower in LN+ patients. Stromal CD4 effector immune cell density remained a statistically significant independent predictor of lymph node spread in multivariate regression analysis (OR= 0.15, p=0.004). Additionally, in an independent validation cohort of 184 radical prostatectomy specimens, stromal CD4 effector immune cell density predicted the presence of nodal metastasis (OR=0.26, p=0.0004). Addition of stromal CD4 effector T cell density to currently used clinicopathological factors, namely T stage, PSA level, Gleason score and percentage of tumour positive cores, improved the predictive accuracy of current nomograms (from 63.5% to 76.8%, p<0.0001). Tumour infiltrating immune cells did not however correlate with common molecular alterations of prostate cancer such as ERG overexpression and PTEN deletion.
Transcriptomic analysis (by HTG EdgeSeq) of the tumour microenvironment was performed to assay 1,041 host immune response related genes. Surprisingly, I did not observe significant differences in the expression levels of adhesion molecules or chemokines (common regulators of immune cell migration) between LN+ and LN- cases. Instead, there was significant upregulation (FC>1.5, adj p value <0.05) of extracellular matrix components (collagen I, collagen III, fibronectin 1) in LN+ tumours, suggesting increased extracellular matrix fibrosis to be associated with reduced T lymphocytic infiltration and tumour immune evasion. Increased collagen III and fibronectin 1 protein expression were confirmed in LN+ patients. Collagen I had increased density score (by second generation harmonic), but not overall abundance, in LN+ patients, eluding to a disorganised stroma with increased cross-linking and elongated fibres.
B7-H3 is a newly discovered member of the B7 family of immune checkpoint molecules with both immune and non-immune functions. I investigated the relationship of B7-H3 to the tumour microenvironment as well as its non-immune functions in prostate cancer. Contrast to PD-1, high B7-H3 expression correlated with worse clinicopathological patient features: higher T stage (p<0.0001), perineural invasion (p=0.01) and lymph node spread (p=0.0006). Furthermore, there was significant decrease in migration and invasion in vitro following suppressed B7-H3 expression in multiple human prostate cancer cell lines. RNA sequencing identified extracellular space chemotactic cytokines and their receptors to be highly downregulated genes in PC3M cells with B7-H3 knocked out. Future experiments will investigate the mechanistic downstream pathways of this phenotype and further evaluate the role of B7-H3 in metastasis in vivo.
Data presented in this thesis reveal differences in the immune infiltrates, particularly CD4 effector (CD4+FoxP3-PD-1-) T cells between LN+ and LN- patients. Prospective clinical studies are needed to test the predictive value of stromal CD4 effector T cell density in diagnostic prostatic biopsies for regional nodal disease. The role of increased extracellular matrix components in determining tumour immune infiltrates also warrants additional research.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Prostate cancer, tumour microenvironment, multipex immunofluorescence, tumour infiltrating immune cells.
• Subjects: R Medicine > RB Pathology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Funder's Name: Cancer Research UK (CRUK)
• Supervisor's Name: Leung, Professor Hing
• Date of Award: 2020
• Depositing User: Dr Chara Ntala
• Unique ID: glathesis:2020-81660
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 18 Sep 2020 15:14
• Last Modified: 18 Sep 2020 15:14
• URI: https://theses.gla.ac.uk/id/eprint/81660