IRF3 Knockout Results in Partial or Complete Rejection of Murine Mesothelioma

Our plain-English summary of IRF3 Knockout Results in Partial or Complete Rejection of Murine Mesothelioma, by M. Aoki, L. Wu, J. Murakami, et al (Journal of Clinical Medicine, 2021).

The study

• Authors: M. Aoki, L. Wu, J. Murakami, Y. Zhao, H. Yun, M. de Perrot
• Journal: Journal of Clinical Medicine
• Published: 2021;10(21)
• Cited by: 4 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Malignant pleural mesothelioma (MESO) has a poor prognosis despite aggressive treatment with surgery, radiation and chemotherapy, and novel therapeutic approaches are needed. IRF3 is a downstream molecule of the cGAS/STING signaling pathway, but its roles have not been investigated in MESO.

Methods: Various murine mesothelioma cell lines were inoculated into wild type (WT) and IRF3 knockout (IRF3KO) mice to compare tumor growth. AE17-bearing mice were treated with local radiotherapy (LRT) to evaluate the effect on tumor growth, and immune cell infiltration was analyzed by flow cytometry 20 days after tumor inoculation. TCGA data were used to examine the relationship between mRNA expression of IRF3 and genes of the cGAS/STING signaling cascade on prognosis in MESO. Correlations between gene expression of IRF3, cGAS/STING signaling pathway, and immune checkpoints were analyzed in TCGA MESO and our scRNA-Seq data from MESO patients.

Results: In mouse mesothelioma models, AK7, RN5 and ZiP3 were completely rejected in IRF3KO mice 20 days after the tumor challenge. AE17tumor volume was slightly larger than WT mice around day 10 before shrinking and becoming significantly smaller than WT mice on day 20. LRT accelerated tumor shrinkage of AE17 tumors in IRF3KO mice. Compared with WT mice, the number of macrophages infiltrating the tumor of IRF3KO mice was significantly reduced, and CD4+ T cells and CD8+IFNγ+ T cells were significantly increased. TCGA data showed that IRF3 expression was an unfavorable prognostic factor in MESO patients. IRF3 expression, the cGAS/STING signaling pathway, and immune checkpoints were positively correlated.

Conclusion: IRF3 could play a critical role in the tumor immune microenvironment of MESO.

In plain English

This was a laboratory study in mice, combined with an analysis of gene data from people with malignant pleural mesothelioma. The authors grew four mouse mesothelioma cell lines (AK7, RN5, ZiP3 and AE17) under the skin of normal (wild-type) mice and mice lacking the gene IRF3. Two of the lines, RN5 and ZiP3, had originally been made from mice injected in the abdomen with asbestos fibers. In IRF3-lacking mice, AK7, RN5 and ZiP3 tumors grew, then shrank and disappeared around day 20. AE17 tumors shrank but did not disappear. On day 20, mean AE17 tumor volume was 214.0 cubic millimetres in IRF3-lacking mice versus 1145.0 in normal mice. In both kinds of mice, tumors given local radiation (15 Gy over three days) were significantly smaller on day 20 than untreated tumors. Tumors in IRF3-lacking mice had fewer macrophages and more of certain T cells. In a public cancer database, mesothelioma patients with high IRF3 messenger RNA levels had significantly poorer overall survival. The authors concluded that IRF3 may play an important role in mesothelioma and could be a potential treatment target. They stated that the underlying immune mechanisms remain to be worked out.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Animal experiment (mouse tumor models, with and without local radiotherapy), plus analysis of public TCGA gene-expression and survival data and single-cell RNA sequencing of patient samples
• Who was studied: Wild-type C57BL/6 mice and IRF3 knockout mice (radiotherapy experiment: four groups of 5 mice each); the TCGA mesothelioma cohort (size not stated in the text); and tumor biopsies from five treatment-naive patients with malignant pleural mesothelioma recruited 5 September 2019 to 16 September 2020, with pleural effusion from 3 patients, in Toronto
• Exposure: Lack of the IRF3 gene (knockout mice), with or without local radiotherapy of 15 Gy in three daily 5 Gy fractions on days 10 to 12. The RN5 and ZiP3 mouse cell lines were derived from mice given intraperitoneal injection of asbestos fibers; the fiber type is not stated.
• Main results:
  • In IRF3 knockout mice, AK7, RN5 and ZiP3 tumors grew at first, began shrinking around day 10 and completely disappeared around day 20
  • AE17 tumors in IRF3 knockout mice shrank but did not disappear
  • On day 20, untreated AE17 tumors in wild-type mice measured (1145.0 ± 321.7) mm3 (mean ± SEM)
  • On day 20, untreated AE17 tumors in IRF3 knockout mice measured (214.0 ± 55.7) mm3 and were significantly smaller than in wild-type mice (p = 0.004)
  • With local radiotherapy, day-20 AE17 tumors were (79.3 ± 31.1) mm3 in IRF3 knockout mice versus 370.5 ± 83.1 mm3 in wild-type mice, a significant difference (p less than 0.001)
  • Tumor-infiltrating macrophages were significantly fewer in the IRF3 knockout and IRF3 knockout plus radiotherapy groups than in the untreated wild-type group (p = 0.031 and 0.002)
  • CD4+ T cells were significantly increased in the wild-type plus radiotherapy group and the IRF3 knockout group compared with the untreated wild-type group (p = 0.042 and 0.014)
  • Total CD8+ T cells did not differ significantly between groups, but CD8+IFNγ+ T cells were significantly increased in the IRF3 knockout group compared with the wild-type group (p = 0.029)
  • In TCGA mesothelioma data, patients with high IRF3 mRNA expression had significantly poorer overall survival than those with low expression (p = 0.0058); expression of MB21D1, TMEM173 (STING) and TBK1 was not associated with prognosis
  • In TCGA data, expression of IRF3 and cGAS/STING pathway genes was significantly higher in the non-epithelioid subtype than in the epithelioid subtype
  • In single-cell RNA sequencing data, IRF3 and LGALS1 (Galectin-1) were highly expressed in both mouse and human mesothelioma cells
• The authors' conclusion: The authors concluded that IRF3 may play an important role in mesothelioma and could be a potential therapeutic target for designing new immunotherapy
• Limitations the authors state:
  • The immune cell populations infiltrating the tumor and contributing to mesothelioma development in knockout versus wild-type mice remain to be delineated
  • It is unclear what effects IRF3 knockout has on immune cells and cytokines in mice whose tumors were rejected
  • The four cell lines were not characterized for major genetic mutations or for expression of IRF3-related and immune-related factors
  • In the IRF3 knockout plus radiotherapy group, tumors from two mice were too small for flow cytometry, so only three mice were analyzed

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma, Pleural Mesothelioma, Prognosis, Chemotherapy, Macrophage.

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