Hedgehog signaling induces PDL‐1 expression and subsequently inactivates effector T cell function and promotes gastric cancer cell proliferation
Authors: Chakrabarti Jayati , Syu LiJyun , Holokai Loryn , Steel Nina , Chang Julie , Dlugosz Andrzej , Zavros Yana 
Published: June 2021

BackgroundHedgehog (Hh) signaling plays a crucial role in tumor growth and chemoresistance in a variety of tumors including gastric cancer. Tumor cells often express elevated programmed cell death 1 (PD‐L1) that interacts with PD‐1 on CD8+ cytotoxic T lymphocytes (CTLs). This interaction inhibits CTL effector function, leading to immune evasion and cancer cell proliferation. Regulation of PD‐L1 may be a promising therapeutic strategy, but the regulatory mechanism remains unclear in gastric cancer.HypothesisHh signaling induces PDL‐1 expression and subsequently inactivates effector T cell function and promotes gastric cancer cell proliferation.MethodsMouse‐derived gastric cancer organoids were generated from a triple‐transgenic model engineered to express an activated GLI2 allele, GLI2A, in Lgr5‐expressing stem cells when treated with doxycycline (iLgr5;GLI2A mTGOs). Organoids were also derived from normal mouse gastric tissue (mGOs). Conditioned media was collected from mTGOs (mTGOCM) and mGOs (mGOCM). Bone marrow‐derived dendritic cells (DCs) were pulsed with either mGOCM or mTGOCM for 24 hrs and co‐cultured with CTLs for 48 hrs. Pulsed DCs and CTLs were then co‐cultured with either mGOs or mTGOs for 24 hrs in presence or absence of PD‐L1 inhibitor (PDL‐1I). Apoptosis and cell proliferation were measured using TUNEL or Edu Imaging Assay Kits respectively. mTGOs were treated with GANT61. PD‐L1 expression and cell proliferation were measured after 48 hrs. iLgr5;GLI2A mice were injected with either Hh inhibitor GANT61 or phosphate buffered saline for 20 days and stomach tissues were immunostained for PD‐L1 and metaplastic markers CD44v9 and lectin Griffonia simplicifolia (GSII). Human‐derived gastric cancer organoids (huTGO) were derived from resected tumors and analyzed for Gli2 and PD‐L1 expression. HuTGOs were treated for 48 hours with gastric cancer chemotherapeutic agents (Epirubicin, Oxaliplatin, and 5‐FU) in presence or absence of GANT61 and organoid proliferation measured by MTS proliferation assay.Results

Increased Gli‐2 correlates with the elevated PD‐L1 expression: ILgr5;GLI2A mice developed rapid gastric adenocarcinoma within the antrum. Organoids derived from these tumors (mTGOs) expressed Gli2 that correlated with elevated PD‐L1 expression when compared to mGOs.
Inhibition of PD‐L1 in mTGO/immune cell co‐cultures induces organoid death: CTLs co‐cultured with DCs pulsed with mTGOCM had increased expression of PD‐1, IFNg, and IL‐2, compared to co‐cultures using unpulsed DCs or DCs pulsed with mGOCM. Live time‐lapse microscopy revealed migration of DCs and CTLs toward mTGOs in co‐culture. Pulsed DC/CTL/mTGO co‐cultures revealed no apoptosis or organoid death. Interestingly, when pulsed DC/CTL/mTGO co‐cultures were treated with PDL‐1I, there was induced organoid apoptosis and death.
Hedgehog signaling regulates PD‐L1 expression and organoid proliferation: Hh inhibitor, GANT‐61 significantly reduced PD‐L1 expression and tumor cell proliferation both in vitro in mTGOs and in vivo in ILgr5;GLI2A mice. In vivo, GANT61 treatment resulted in decreased PD‐L1 expression that correlated with loss of tumor growth and decreased metaplasia.
Inhibition of Hedgehog signaling sensitizes organoids to chemotherapy: Chemotherapy‐induced cell death in Gli2/PD‐L1+ve huTGOs pretreated with GANT61.
ConclusionHh signaling plays a role as a regulator of PDL‐1 expression in gastric cancer cells to promote tumor growth.Support or Funding Information1RO1DK083402 (Zavros RO1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.