title: Investigation into the roles of neutrophil infiltration in bladder cancer using murine models
Creators: name: Ismail, Nur Faezah
Publisher: name: University of Glasgow
Year: 2020
Subjects: subject: Bladder cancer, CXCR2, neutrophils.

description: Current literature suggested that the role of tumour infiltrating neutrophils could be pro- or anti-tumour and therefore double-edged. The heterogeneous nature of this population is dictated by environmental signals coming from the host and the tumour immune microenvironment. In bladder cancer, a high level of neutrophils is associated with poor prognosis. However, the functional role of neutrophil infiltration along the courses of bladder tumorigenesis is yet to be understood.
This study aimed to elucidate the roles of neutrophils in regulating bladder tumorigenesis using FGFR3-mutated and Cxcr2-deleted mouse models with wildtype (wt) mice as a control. FGFR3 is one of the most commonly mutated genes in bladder cancer. Cxcr2 is an essential receptor protein that mediates chemotaxis of neutrophils. 
Bladder tumours were induced by a tobacco carcinogen N-butyl-N-(4- hydroxybutyl) nitrosamine (OH-BBN) for 10 weeks and tumour pathogenesis was analysed at the timepoint of 2, 12, 16 and 20 weeks from the start of treatment. A combined qualitative and quantitative approaches, namely haematoxylin and eosin (H&amp;E) staining, immunohistochemistry (IHC), real-time PCR-based microarray were used to evaluate the changes in the urothelial and tumour histopathology, immune cell influx and gene expression in the tissue context.
Carcinogen-dependent bladder tumorigenesis was increased in Tg(UroII-hFGFR3IIIbS249C) (FGFR3S249C) and in mice with Cxcr2 deletion in myeloid lineage, LysMCre Cxcr2fl/fl (Cxcr2 flox) compared to wt. The tumour phenotype in FGFR3S249C and Cxcr2 flox was more advanced and invasive. The acute inflammatory response was suppressed at the initial time of carcinogen treatment in both FGFR3S249C and Cxcr2 flox with a significant reduction in neutrophils recruitment. 
Further mechanistic evaluation of the immune cell recruitment in Cxcr2 flox showed changes in the levels of infiltrations of neutrophils, macrophages and T cells along the stages of tumour initiation and progression. Unexpectedly, Cxcr2 flox tumours were highly infiltrated with neutrophils as examined by H&amp;E for their morphological appearance. However, many of them lacked expression of well-established markers for neutrophils. The increased level of of neutrophils was in concomitant with that of tumour-infiltrating CD3+ T-cells. 
Evaluation of the transcriptional changes showed that genes associated with immunity and inflammation were differently expressed in the bladder at the acute inflammation stage compared to bladder tumours in Cxcr2 flox compared to wt. Differences were also observed in the gene expression between neutrophil-infiltrated and non-neutrophil infiltrated Cxcr2 flox tumour samples. 
Depletion of neutrophils using Ly6G monoclonal antibody (1A8) in the first 10 weeks of carcinogen treatment in wt mice reduced the recruitment of neutrophil at acute inflammation stage. The suppression of neutrophil recruitment at acute inflammation stage resulted in an increase of inflammation at a later stage of bladder tumorigenesis. Depletion of neutrophils in tumour-bearing mice was also attempted, however the effectiveness of depletion remained inconclusive. 
The results in this study suggested that, firstly, bladder tumorigenesis was induced by the suppression of acute immune responses. Secondly, an increased level of neutrophil infiltrations resulted in an enhanced tumour progression. Thirdly, neutrophils influenced the levels of macrophages and T cells infiltrations, and this is likely to be caused by their regulation of gene expression associated with inflammation and immune signalling during early and late stages of bladder tumorigenesis. The results support further investigations towards clinical translation of FGFR3, CXCR2 and neutrophils, as therapeutic targets in bladder cancer.