Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 May 19;10(10):2191.
doi: 10.3390/jcm10102191.

Prognostic Role of Immune Checkpoint Regulators in Cholangiocarcinoma: A Pilot Study

Lu Cao  1   2 , Prashanth Prithviraj  3   4 , Ritu Shrestha  1   2 , Revati Sharma  3   4 , Matthew Anaka  5 , Kim R Bridle  1   2 , George Kannourakis  3   4 , Darrell H G Crawford  1   2 , Aparna Jayachandran  1   2   3
Affiliations

Prognostic Role of Immune Checkpoint Regulators in Cholangiocarcinoma: A Pilot Study

Lu Cao et al. J Clin Med. .

Abstract

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy associated with steadily increasing incidence and poor prognosis. Ongoing clinical trials are assessing the effectiveness and safety of a few immune checkpoint inhibitors (ICIs) in CCA patients. However, these ICI treatments as monotherapies may be effective for a proportion of patients with CCA. The prevalence and distribution of other immune checkpoints (ICs) in CCA remain unclear. In this pilot study, we screened databases of CCA patients for the expression of 19 ICs and assessed the prognostic significance of these ICs in CCA patients. Notably, expression of immune modulator IDO1 and PD-L1 were linked with poor overall survival, while FASLG and NT5E were related to both worse overall survival and progression-free survival. We also identified immune modulators IDO1, FASLG, CD80, HAVCR2, NT5E, CTLA-4, LGALS9, VTCN1 and TNFRSF14 that synergized with PD-L1 and correlated with worse patient outcomes. In vitro studies revealed that the expression of ICs was closely linked with aggressive CCA subpopulations, such as cancer stem cells and cells undergoing TGF-β and TNF-α-mediated epithelial-to-mesenchymal transition. These findings suggest that the aforementioned IC molecules may serve as potential prognostic biomarkers and drug targets in CCA patients, leading to lasting and durable treatment outcomes.

Keywords: CD73; EMT; Galectin-9; IDO1; PD-L1; cancer stem cells; cholangiocarcinoma; immune checkpoints.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The OncoPrint analyses of CCA patients reveal changes in expression of ICs. Rows and columns represent genes and CCA patients, respectively. Genetic alterations, such as copy number alterations (homozygous deletions and amplifications), mutations and gene expression changes, are represented by glyphs and color codes. The patient order is presented as per alterations.
Figure 2
Figure 2
Association between immune modulators and survival in CCA patients. The Kaplan–Meier survival curves in SurvExpress CCA patients for the gene expression of (A) CD274 (PD-L1), (B) PDCD1 (PD-1), (C) CTLA4 and (D) IDO1. Low-risk groups are indicated in green and high-risk groups are indicated in red. The x-axis presents the study time in months. HR, CI and p-values are shown in the insert.
Figure 3
Figure 3
Relationship of immune modulators in combination with CD274 (PD-L1) and survival in CCA patients. The Kaplan–Meier survival curves in SurvExpress CCA patients for the gene expression of (A) CD274/IDO1, (B) CD274/NT5E, (C) CD274/CD80, (D) CD274/FASLG, (E) CD274/HAVCR2, (F) CD274/LGALS9, (G) CD274/TNFRSF14 and (H) CD274/VTCN1. Low-risk groups are indicated in green and high-risk groups are indicated in red. The x-axis presents the study time in months. HR, CI and p-values are shown in the insert.
Figure 4
Figure 4
Anchorage-independent three-dimensional spheroid culture enriches HuCCT-1 CSCs. (A) Monolayer culture and 3-D culture of HuCCT-1 cells (scale bar = 200 µm). (B–J) qRT-PCR analysis demonstrated increased expression of embryonic stemness and surface CSC markers in HuCCT-1 spheres compared with HuCCT-1 adherent monolayer culture. Values are mean ± SD of three experiments in triplicate (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, ns: not significant). ActB: β-Actin.
Figure 5
Figure 5
Anchorage-independent three-dimensional spheroid culture of CCLP-1 cells enriches CSCs. (A) Monolayer culture and 3-D culture of CCLP-1 cells (scale bar = 200 µm). (B–I) Enhanced expression of embryonic stemness and cell surface CSC markers were observed in CCLP-1 spheres compared with CCLP-1 adherent monolayer culture by qRT-PCR. Values are mean ± SD of three experiments in triplicate (*** p < 0.005, **** p < 0.001, ns: not significant). ActB: β-Actin.
Figure 6
Figure 6
HuCCT-1 and CCLP-1 CSCs have elevated expression of immune modulators. (A–F) qRT-PCR analysis showed upregulation of immune modulators NT5E, LGALS9, FASLG, TNFRSF14 and VTCN1 in HuCCT-1 spheres compared with the parental adherent HuCCT-1 cells. (G–J) Increased expression of immune modulators NT5E, LGALS9, FASLG and TNFRSF14 was detected in mRNA from CCLP-1 spheres versus the parental adherent cells. (K) Western blots demonstrated the upregulation of NT5E and LGALS9 in both HuCCT-1 and CCLP-1 spheres versus the adherent parental cells. (L) Graphs represent quantification of Western blot analyses. Values are mean ± SD of three experiments in triplicate (* p < 0.05, *** p < 0.005, **** p < 0.001, ns: not significant). ActB: β-Actin.
Figure 7
Figure 7
TGF-β1-mediated EMT induced upregulation of immune modulators in HuCCT-1 cells. qRT-PCR analysis revealed the upregulation in the expression of (A) TGF-β and (B) TNF-α in HuCCT-1 spheres compared with parent adherent cells. (C–H) qRT-PCR showed epithelial markers E-Cad, Occludin and KRT19 were decreased, and mesenchymal markers N-cad, Fibronectin and Slug were increased after 72 h of TGF-β1 treatment in HuCCT-1 cells. (I–J) qRT-PCR demonstrated elevated expression of immune modulators NT5E and PD-L1 upon 72 h of TGF-β1 treatment in HuCCT-1 cells. Values are mean ± SD of three experiments in triplicate (* p < 0.05, *** p < 0.005, **** p < 0.001, ns: not significant). ActB: β-Actin.
Figure 8
Figure 8
TNF-α-mediated EMT induced upregulation of immune modulators in CCLP-1 cells. (A–F) qRT-PCR showed reduction in the expression of epithelial markers E-Cad, ZO-1 and KRT19, and elevation in the expression of mesenchymal markers N-cad, Fibronectin and ZEB1 after 72 h of TNF-α treatment in CCLP-1 cells. (G–I) qRT-PCR demonstrated elevated expression of immune modulators NT5E and PD-L1 and decreased level of LGALS9 upon 72 h of TNF-α treatment in CCLP-1 cells. Values are mean ± SD of three experiments in triplicate (** p < 0.01, *** p < 0.005, **** p < 0.001, ns: not significant). ActB: β-Actin.
Figure 9
Figure 9
Association between immune modulators, EMT markers, CSC markers and survival in CCA patients. The Kaplan–Meier survival curves in SurvExpress CCA patients for the gene expression of (A) CD274/E-Cad/Vimentin, (B) CD274/E-Cad/Fibronectin, (C) ALDH1A1/NT5E, (D) ALDH1A1/LGALS9, (E) ALDH1A1/CD274 in CCA patients. Low-risk groups are indicated in green and high-risk groups are indicated in red. The x-axis presents the study time in months. HR, CI and p-values are shown in the insert.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Rizvi S., Borad M.J., Patel T., Gores G.J. Cholangiocarcinoma: Molecular pathways and therapeutic opportunities. Semin. Liver Dis. 2014;34:456–464. doi: 10.1055/s-0034-1394144. - DOI - PMC - PubMed
    1. Bergquist A., von Seth E. Epidemiology of cholangiocarcinoma. Best Pract. Res. Clin. Gastroenterol. 2015;29:221–232. doi: 10.1016/j.bpg.2015.02.003. - DOI - PubMed
    1. Bridgewater J., Galle P.R., Khan S.A., Llovet J.M., Park J.W., Patel T., Pawlik T.M., Gores G.J. Guidelines for the diagnosis and management of intrahepatic cholangiocarcinoma. J. Hepatol. 2014;60:1268–1289. doi: 10.1016/j.jhep.2014.01.021. - DOI - PubMed
    1. Rizvi S., Gores G.J. Pathogenesis, diagnosis, and management of cholangiocarcinoma. Gastroenterology. 2013;145:1215–1229. doi: 10.1053/j.gastro.2013.10.013. - DOI - PMC - PubMed
    1. Valle J., Wasan H., Palmer D.H., Cunningham D., Anthoney A., Maraveyas A., Madhusudan S., Iveson T., Hughes S., Pereira S.P., et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N. Engl. J. Med. 2010;362:1273–1281. doi: 10.1056/NEJMoa0908721. - DOI - PubMed