Poster Presentation Australasian Cytometry Society 43rd Annual Conference and Workshop

Development of an 18-colour/ 21-parameter flow cytometry panel for immunophenotypic characterisation of human peripheral blood leukocytes obtained from people with bladder cancer (#117)

Patrick B Thomas 1 2 3 4 , Kate Zimmermann 4 5 , Nathan Mackenzie 1 2 4 , Clarissa Nicholls 1 2 4 , Ian Vela 1 2 3 4 6 , Tony J Kenna 2 4 5 , Elizabeth D Williams 1 2 3 4
  1. Queensland Bladder Cancer Initiative (QBCI) , School of Biomedical Sciences at the Translational Research Institute, Brisbane, Queensland, Australia
  2. Centre for Personalised Analysis of Cancers (CPAC), Brisbane, Queensland, Australia
  3. Australian Prostate Cancer Research Centre – Queensland (APCRC-Q), Brisbane, Queensland, Australia
  4. School of Biomedical Sciences, Queensland University of Technology (QUT), Brisbane, Queensland, Australia
  5. School of Biomedical Sciences, Experimental Rheumatology Lab, Brisbane, Queensland, Australia
  6. Department of Urology, Princess Alexandra Hospital, Brisbane, Queensland, Australia

Background: Bladder cancer (BC) is a biologically diverse and lethal urological malignancy with variable clinical outcomes and high rates of recurrence. Given its significant prevalence and high tumour recurrence, BC forms a substantial part of cancer care in Australia. Following a diagnosis of BC, and longitudinally during monitoring and treatment, we aim to employ a multiparametric immunophenotyping panel to observe changes in immune cell subsets, immunological markers, and immune checkpoint receptors (ICRs) on peripheral blood mononuclear cells (PBMCs) and tissue infiltrating lymphocytes (TILs). Here, we present a 18-colour/21-parameter panel for enumeration and characterisation of leukocyte cell subsets and its utility within a cohort of BC patients that represent diverse and clinically relevant histopathology’s.

 

Methodology: This panel is designed to run on the LSRFortessa™ X-20 Cell Analyzer on fresh and cryopreserved PBMCs and examines the frequency of CD45+ cells, including B-cells, CD4+/ CD8+ T-cells, regulatory T-cells, γδ T-cells, NKT, NK, and dendritic cells, as well as memory, activation markers and expression of immune checkpoints (PD-1, CTLA-4, TIM-3). All antibodies were titrated on healthy donor PBMCs followed by full-panel assessment for spreading error and marker resolution. Following immunophenotyping of PBMCs from healthy donors (n=5) and patients with BC (n=8), we show alterations in circulating CD4+ and CD8+ T-cells, and B-cells, as well as expression of ICRs. Our protocol is currently being adapted to enable immunophenotyping of paired PBMC/ TIL populations from individuals with BC.

 

Conclusion: Our study aims to simultaneously define the immunological changes in the peripheral blood and tumour microenvironment of patients with BC. This will provide important context to parallel ex vivo immune-oncology and chemotherapeutic efficacy assays performed using patient matched organoid cultures and tissue explant technologies. These analyses will generate novel insights into the identification of effective personalised treatments in a range of advanced BCs with unclear prognostic outcomes.