Novel Adiponectin Receptor Agonist Inhibits Cholangiocarcinoma via Adenosine Monophosphate-activated Protein Kinase.

BACKGROUND: Cholangiocarcinoma (CCA) has a poor prognosis and only limited palliative treatment options. The deficiency of adiponectin and adenosine monophosphate-activated protein kinase (AMPK) signaling was reported in several malignancies, but the alteration of these proteins in CCA is still unclear. OBJECTIVES: This study aimed to assess the role of adiponectin and AMPK signaling in CCA. Furthermore, AdipoRon, a novel adiponectin receptor (AdipoR) agonist, was evaluated in vitro and in vivo as a new anti-tumor therapy for CCA. METHODS: The expression of AdipoR1 and p-AMPKα in human tissue microarrays (TMAs) was evaluated by immunohistochemistry staining (IHC). The effect of 2-(4-Benzoylphenoxy)-N-[1-(phenylmethyl)- 4-piperidinyl]-acetamide (AdipoRon) was investigated in vitro with proliferation, crystal violet, migration, invasion, colony formation, senescence, cell cycle and apoptosis assays and in vivo using a CCA engineered mouse model (AlbCre/LSL-KRASG12D/p53L/L). RT-qPCR and western blot methods were applied to study molecular alterations in murine tissues. RESULTS: AdipoR1 and p-AMPKα were impaired in human CCA tissues, compared to adjacent non-tumor tissue. There was a positive correlation between the AdipoR1 and p-AMPKα levels in CCA tissues. Treatment with AdipoRon inhibited proliferation, migration, invasion and colony formation and induced apoptosis in a time- and dose-dependent manner in vitro(p<0.05). In addition, AdipoRon reduced the number of CCA and tumor volume, prolonged survival, and decreased metastasis and ascites in the treated group compared to the control group (p<0.05). CONCLUSIONS: AdipoR1 and p-AMPKα are impaired in CCA tissues, and AdipoRon effectively inhibits CCA in vitro and in vivo. Thus, AdipoRon may be considered as a potential anti-tumor therapy in CCA.

The safety of CAR-T cells and PD-1 antibody combination on an experimental model.

Chimeric antigen receptor (CAR) T cells and PD-1 antibodies (PD-1 Ab) are emergent immunotherapies with unprecedented efficacy. The presence of PD-1 on T cells contributes to hypofunction of CAR-T therapy and inhibition of PD-1 enhances anti-cancer effect of CAR-T cells. Therefore, the combination of CAR-T cells and PD-1 antibody is a promissing strategy for cancer treatment. This study aims to establish our in-house CAR-T cells and evaluate the safety of CAR-T cells in combination with PD-1 antibody in animals. The toxicity of CD19-CAR-T cells was examined using Swiss Webster mice. Four mouse groups were treated with control, CAR-T, PD-1 antibody or CAR-T + PD-1 antibody. Mice's overall status was monitored and recorded. At the end-point, hematological and biochemical indices were quantified, histopathology of liver and kidney was evaluated by pathologists. The relative abnormal ratio and absolute values were compared between groups. We generated our in-house CAR-T cells and used them for safety evaluation in mice. The increase in mouse weight was observed in all groups after treatment and the weight was comparable between groups. The hematological, biochemical and histopathological parameters were equivalent between groups, except for liver grain degeneration occurred in treatment groups. Thus, CAR-T cells, PD-1 Ab and their combination were safe in mice. We successfully produced our in-house CAR-T cells and the combination of our CAR-T cells and PD-1 antibody was safe in mice with comparable values of hematopoietic indices, liver and kidney functions. Longer follow-up might be necessary to evaluate their effect on the liver.

X-ray-irradiated K562 feeder cells for expansion of functional CAR-T cells.

Immunotherapy, particularly CAR-T therapy has recently emerged as an innovator for cancer treatment. Gamma-irradiated K562 cells is a common and effective method to stimulated CAR-T cells prior to treatment. However, high cost and limited equipment of gamma-irradiation is drawback of this method. This requires the establishment of CAR-T-expanding alternatives, such as X-ray-irradiated K562 cells. X-ray irradiation was used to deactivate K562 cells. The post-irradiative cell survival was investigated by counting of the number of cells, staining with Trypan Blue and PI. FACS analysis was applied to detect the expression of cell surface markers. The production of CD19-CAR-T cells were executed from fresh blood donor by CD19-CAR-plasmid transfection, followed by the stimulation with X-ray-irradiated K562 feeder cells. The function of produced CAR-T cells was checked by their ability to kill Daudi cells. X-ray-irradiation inhibited the propagation and viability of K562 cells in a dose- and time-dependent manner. Interestingly, CAR-T-stimulating effectors were remained on the surface of X-ray-irradiated K562 cells. CD-19-CAR-T cells were produced successfully, suggested by number of CAR-positive cells in transfected and stimulated population, compared to un-transfected group. Lastly, our data showed that engineered CAR-T cells effectively killed Daudi cells. Our data demonstrated the efficacy of X-ray on deactivation K562 feeder cells which subsequently stimulated and expanded functional CAR-T cells. Thus, X-ray can be used as an alternative to inactivate K562 cells prior to using as a feeder of CAR-T cells.