Abstract

Introduction

Liquid biopsies are important samples for providing
biomarkers in clinical studies in a non-invasive manner. They are particularly
relevant for immuno-oncology trials, where regulation of circulating immune
cells may reflect immune changes in tumours in response to immune targeting
therapies. For example, RXC004, a potent and selective inhibitor of the Wnt
pathway regulator porcupine is hypothesised to have immunomodulatory
anti-cancer functions. Therefore, as part of an RXC004 safety and tolerability
study in cancer patients with solid tumours (NCT03447470), we aim develop
methods to analyse immune cell changes in liquid biopsies. This includes
analysis of whole blood by flow cytometry to quantify a range of immune cell
subsets and functional markers.

Methods

Flow cytometry analysis is carried out in house using 7
multi-colour panels, analysed on the ACEA Biosciences Novocyte 3000 flow
cytometer. Antibodies included in these panels have been validated using
healthy donor peripheral blood mononuclear cells (PBMCs), healthy donor whole
blood and Biolegend VeriCells^TM. PBMCs were used to titrate all
antibodies with basal expression. Various in
vitro culture conditions were used to induce expression of other functional
markers. Healthy donor whole blood samples were used to carry out sample
stability testing and staining protocol optimisation. VeriCells^TM
were implemented for staining panel quality control.

Results

The immune cell subsets that can be identified in whole
blood samples include monocytes, granulocytes, B and T lymphocytes and natural
killer cells. Naïve and memory T cell subsets can also be identified and these
cell types been validated using naïve T cell enrichment from PBMCs. Dendritic
cells (DCs) are a very small proportion of immune cells in whole blood samples,
but this panel has been validated using dendritic cell enrichment from PBMCs.
Activation markers CD69, CD25, Ki67 and CD154 have been validated using T cells
purified from PBMCs and cultured in vitro
with various stimuli. A regulatory T cell (T_reg) panel has been validated using naïve CD4 T cells
and in vitro culture conditions to
induce T_regs. A
dataset of the key immune cell subsets’ identified using these staining panels
has been generated, and demonstrates the stability and natural variance in
healthy individuals over a 6 week period. VeriCells^TM have been used
to show reproducibility of the staining protocol over time and to help set QC
criteria for ongoing sample screening.

Conclusion

We have established a protocol for the analysis of
immune cell subsets by flow cytometry, suitable to assess potential therapy-induced
changes in circulating immune cells as an exploratory end-point in patients
with solid tumours enrolled in the RXC004 clinical study.