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 Jan;376(1):21-28.
doi: 10.1124/jpet.120.000284. Epub 2020 Nov 6.

Downregulation of Interferon- γ Receptor Expression Endows Resistance to Anti-Programmed Death Protein 1 Therapy in Colorectal Cancer

Chunxiao Lv  1 , Dongfen Yuan  1 , Yanguang Cao  2
Affiliations

Downregulation of Interferon- γ Receptor Expression Endows Resistance to Anti-Programmed Death Protein 1 Therapy in Colorectal Cancer

Chunxiao Lv et al. J Pharmacol Exp Ther. 2021 Jan.

Abstract

Immune checkpoint inhibitors have emerged as a frontline treatment of a variety of malignancies. However, only a subset of patients respond to these therapies, and many initial responders eventually develop resistance, leading to tumor relapse. Programmed death protein 1 is one of the checkpoint inhibitors that is expressed on activated T cells and suppresses the antitumor immune response when binding to its ligand, programmed death ligand 1, on tumor cells. Previous studies indicated that loss-of-function mutations in the IFN-γ pathway could result in acquired resistance to immune checkpoint inhibitors in human patients with cancer. Here, we investigated the effects of the IFN-γ receptor downexpression on the response to an anti-PD-1 antibody (αPD1) in a murine colorectal cancer model and the underlying mechanisms of resistance. IFN-γ receptor (IFNGR) 1 was knocked down in MC38 cells, a murine colon adenocarcinoma cell line using IFNGR1 short hairpin RNA (shRNA) lentiviral particles. Then, MC38 IFNGR1 knockdown (KD) cells and negative control (SC) cells were used in this study. In the C57BL/6 xenograft model, the KD tumor demonstrated resistance to αPD1 in comparison with SC cells. The observed treatment resistance might be associated with reduced tumor-infiltrating immune cells (TILs). When mixed, the resistant (KD) and control cells (SC) grew in spatially separated tumor areas, and αPD1 did not impact this pattern of spatial distribution. Our findings have proved that downregulation of the IFNGR1 endowed resistance to αPD1 and provided the potential mechanisms involving the TILs. SIGNIFICANCE STATEMENT: Immunological checkpoint blockades have achieved substantial efficacy in a variety of tumors. However, only a subset of patients respond to these therapies, and innate and acquired resistance is widely present. Our study found that the downregulation of the IFN-γ receptor caused resistance to an anti-PD-1 antibody in a murine colorectal cancer model associated with the reduced tumor-infiltrating lymphocytes. Our findings have substantial implications for improving the efficacy of checkpoint blockades.

PubMed Disclaimer

Conflict of interest statement

No author has an actual or perceived conflict of interest with the contents of this article.

Figures

Fig. 1.
Fig. 1.
Knockdown of IFN-γ receptor in MC38 colorectal cells. (A) The workflow for the development of the SC-GFP and KD-RFP cell lines. (B) The flow cytometry results for sorting SC-GFP and KD-RFP cell lines after 1 month of culture. SSC-A is for side-scatter area. (C) Western blot confirmed knockdown of IFN-γR1 protein expression in KD cells and KD-RFP cells and not in SC cells and SC-GFP cells in comparison with MC38 WT cells. (D) The schematic and research questions that will be addressed in this study.
Fig. 2.
Fig. 2.
Tumor growth trajectories for SC-GFP, KD-RFP, and SC-GFP/KD-RFP 1:1 xenografts in C57BL/6 mice treated with control IgG or aPD1. (A) Animal study plan. (B) Individual tumor growth trajectory group by tumor type and treatment. The number of mice with tumor volume above 1000 mm3 at day 25 was labeled. SC, KD, and KD-SC are mice inoculated with SC-GFP cells, KD-RFP cells, or a 1:1 mixture of SC-GFP and KD-RFP cells, respectively.
Fig. 3.
Fig. 3.
The knockdown of IFN-γR1 conferred resistance to αPD1 therapy. (A) Mean tumor growth curves of C57BL/6 mice bearing different MC38 tumors and treated with control IgG or αPD1. Student’s t test was used to compare the tumor volume at the end of the study between the two treatments. (B) Kaplan-Meier survival analysis of the same mice. SC, KD, and KD-SC are mice inoculated with SC-GFP cells, KD-RFP cells, or a 1:1 mixture of SC-GFP and KD-RFP cells, respectively.
Fig. 4.
Fig. 4.
Analysis of TILs and tumor cells by flow cytometry. Percentage of CD45+ cells in TILs, pecentage of CD8+ T cells in CD45+ cells, pecentage of CD4+ T cells in CD45+ cells, pecentage of CD8+ and CD4+ T cells in CD45 + cells, percentage of PD-1+ cells in CD45+ cells, and PD-L1+ cells in tumor cells. Statistical significance was determined by t test. SC, KD, and KD-SC are mice inoculated with SC-GFP cells, KD-RFP cells, or a 1:1 mixture of SC-GFP and KD-RFP cells, respectively.
Fig. 5.
Fig. 5.
The spatial distribution of the tumor cells and immune cells (CD8+, CD4+, FoxP3+) in KD-SC xenografts by immunofluorescence staining. (A) Distribution of the SC-GFP and KD-RFP tumor cells. (B) Staining of CD4+ regulatory cells (Treg) and CD8+ cytotoxic T cells. DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that binds strongly to DNA. (C) Staining of Foxp3 regulatory T cells and Ki67+ proliferating cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ayers M, Lunceford J, Nebozhyn M, Murphy E, Loboda A, Kaufman DR, Albright A, Cheng JD, Kang SP, Shankaran V, et al. (2017) IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade. J Clin Invest 127:2930–2940. - PMC - PubMed
    1. Bertrand F, Montfort A, Marcheteau E, Imbert C, Gilhodes J, Filleron T, Rochaix P, Andrieu-Abadie N, Levade T, Meyer N, et al. (2017) TNFα blockade overcomes resistance to anti-PD-1 in experimental melanoma. Nat Commun 8:2256. - PMC - PubMed
    1. Callahan MK, Postow MA, Wolchok JD. (2016) Targeting T cell co-receptors for cancer therapy. Immunity 44:1069–1078. - PubMed
    1. Champiat S, Dercle L, Ammari S, Massard C, Hollebecque A, Postel-Vinay S, Chaput N, Eggermont A, Marabelle A, Soria JC, et al. (2017) Hyperprogressive disease is a new pattern of progression in cancer patients treated by anti-PD-1/PD-L1. Clin Cancer Res 23:1920–1928. - PubMed
    1. Chen DS, Mellman I. (2013) Oncology meets immunology: the cancer-immunity cycle. Immunity 39:1–10. - PubMed

Publication types

MeSH terms

Substances