Accumulating evidence suggests that the immunological landscape plays a key role in the progression of neuroendocrine liver metastases (LM-NENs), which is often characterised by immune cell infiltration. Anti-tumour functions of infiltrating lymphocytes are often silenced through hyper-expression of inhibitory checkpoint receptors (CRs) such as PD-1, but favourable outcomes with anti-PD-1 therapy have been low. Recently, functional soluble (cell-free) CRs beyond PD-1 have been described but their role in LM-NENs is unexplored. Our aim was to characterise soluble CR landscape of LM-NEN patients and examine their expression from the tumour microenvironment using a novel human immunocompetent organotypic tissue slice model for LM-NENs.
We measured plasma levels of stimulatory/inhibitory soluble CRs including sBTLA; sCD27; sCD28; sCD40; sCD80; sCD137; sCTLA-4; sGITR; sHVEM; sIDO-1; sPD-1; sPD-L1; sPD-L2 by multiplex luminex assay in LM-NEN (n=27), primary liver cancer (n=11), healthy controls (n=15). Precision cut tumour slices (PCTS) were prepared from resected LM-NEN tumours (n=6) and cultured for up to 15 days. PCTS viability was assessed by measuring ATP, apoptosis (CK18) and lactate dehydrogenase (LDH) release. Proliferative capacity (Ki67) and neuroendocrine differentiation (Chromogranin A) were assessed by immunofluorescence. Immune genes were quantified by microarrays in tumour and peri-tumour tissue. Soluble CR expression was assessed in PCTS supernatants.
A novel and distinct plasma soluble CR signature was observed in LM-NEN patients. We identified ten soluble CRs that were significantly higher compared to healthy controls which interestingly were similar to primary liver cancer. LM-NEN-PCTS were viable for up to 15 days in culture and maintained tissue histoarchitecture, patient specific cellular heterogeneity, neuroendocrine differentiation and mitotic grade compared to the original histopathology of the resected tumour. Immune-specific gene signatures were readily detectable in LM-NEN PCTS, confirming immune infiltration in the tumour microenvironment. LM-NEN-PCTS produced the same soluble CRs signature observed in the plasma.
Soluble checkpoint receptors beyond PD1 are actively involved in LM-NENs offering new insights into the pathogenesis and the development of immunomodulatory therapeutic agents for this cancer. Soluble CRs are actively produced by LM-NET tumour tissue and the PCTS model provides a hitherto unattainable platform for drug development and pre-clinical testing.
*Ewald Doornebal and Nicola Harris are joint first authors
*Rajaventhan Srirajaskanthan and Shilpa Chokshi are joint last authors