Cholinergic signalling influences the expression of immune checkpoint inhibitors, PD-L1 and PD-L2, in human colorectal cancer tissues and cell lines

Abstract Background Cancer cells express immunosuppressive molecules, such as programmed death ligands (PD-L)1 and PD-L2, enabling evasion from the host's immune system. Cancer cells synthesize and secrete acetylcholine (ACh), acting as an autocrine or paracrine hormone to promote their proliferation, differentiation, and migration. Methods We correlated the expression PD-L1, PD-L2, cholinergic muscarinic receptor 3 (M3R), alpha 7 nicotinic receptor (α7nAChR), and choline acetyltransferase (ChAT) in colorectal cancer (CRC) tissues with the stage of disease, gender, age, risk, and patient survival. The effects of a muscarinic receptor blocker, atropine, and a selective M3R blocker, 4-DAMP, on the expression of immunosuppressive and cholinergic markers were evaluated in human CRC (LIM-2405, HT-29) and intestinal epithelial (T4056) cells. Results Increased expression of PD-L1, M3R and ChAT at stages III-IV was associated with a high risk of CRC and poor survival outcomes independent of patients' gender and age. α7nAChR and PD-L2 were not changed at any CRC stages. Atropine and 4-DAMP suppressed proliferation and migration of human CRC and T4056 cells, induced apoptosis, decreased PD-L1, PD-L2 and M3R expression in CRC cells via inhibition of EGFR and phosphorylation of ERK and STAT3. Conclusions The expression of immunosuppressive and cholinergic markers may increase the risk of recurrence of CRC. These markers might be used in determining prognosis and treatment regimens for CRC patients. Blocking cholinergic signalling may be a potential therapeutic for CRC through anti-proliferation and anti-migration via inhibition of EGFR and phosphorylation of ERK and STAT3. These effects allow the immune system to recognize and eliminate cancer cells.