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

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 of 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) 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 the proliferation and migration of human CRC cells, induced apoptosis, and decreased PD-L1, PD-L2, and M3R expression in CRC cells via inhibition of EGFR and phosphorylation of ERK.

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 signaling may be a potential therapeutic for CRC through anti-proliferation and anti-migration via inhibition of EGFR and phosphorylation of ERK. These effects allow the immune system to recognize and eliminate cancer cells.