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Potential involvement of IL-32 in cell-to-cell communication between macrophages and hepatoblastoma

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Abstract

Purpose

This study investigated the expression of interleukin 32 (IL-32) in hepatoblastoma, the most common primary pediatric liver tumor, and its possible roles in tumorigenesis.

Methods

IL-32 expression was investigated in two hepatoblastoma cell lines (Hep G2 and HuH 6) in the steady state and after co-culture with macrophages by RNA-seq analysis and RT-qPCR, and after stimulation with chemotherapy. Cultured macrophages were stimulated by IL-32 isoforms followed by RT-qPCR and western blot analysis. IL-32 immunohistochemical staining (IHC) was performed using specimens from 21 hepatoblastoma patients. Clustering analysis was also performed using scRNA-seq data downloaded from Gene Expression Omnibus.

Results

The IL-32 gene is expressed by hepatoblastoma cell lines; expression is upregulated by paracrine cell–cell communication with macrophages, also by carboplatin and etoposide. IL-32 causes protumor activation of macrophages with upregulation of PD-L1, IDO-1, IL-6, and IL-10. In the patient pool, IHC was positive only in 48% of cases. However, in the downloaded dataset, IL-32 gene expression was negative.

Conclusion

IL-32 was detected in hepatoblastoma cell lines, but not in all hepatoblastoma patients. We hypothesized that stimulation such as chemotherapy might induce expression of IL-32, which might be a critical mediator of chemoresistance in hepatoblastoma through inducing protumor activation in macrophages.

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Data availability

The datasets generated and/or analyzed in the current study are available from the corresponding author on reasonable request.

Abbreviations

Bcl-2:

B-cell lymphoma 2

CD163:

Cluster of differentiation 163

EGF:

Endothelial growth factor

HB:

Hepatoblastoma

HMDMs:

Human monocyte derived macrophages

Iba1:

Ionized calcium-binding adapter molecule 1

IC50:

Half maximal inhibitory concentration

IDO-1/2:

Indoleamine dioxygenase-1/2

IHC:

Immunohistochemical staining

IL-1β/6/10/32/34:

Interleukin-1 beta/6/10/32/34

Kyn pathway:

Kynurenine pathway

LPS:

Lipopolysaccharide

M-CSFR:

Macrophage colony stimulating factor receptor

MMP-2,7,9:

Matrix metalloproteinases-2,7,9

MTB:

Mycobacterium tuberculosis

NF-κB:

Nuclear factor kappa-light-chain-enhancer of activated B cells

NKs:

Natural killer cells

PCNSL:

Primary central nervous system lymphoma

PDGF:

Platelet-derived growth factor

PD-L1/2:

Programmed death ligand 1/2

PLAA:

Phospholipase A2-activating protein

RNA-seq:

Ribonucleic acid sequencing

RT-qPCR:

Real time quantitative polymerase chain reaction

SCC:

Squamous cell carcinoma

STAT3:

Signal transducer and activator of transcription 3

TAMs:

Tumor-associated macrophages

Tet-On/Off:

Tetracycline-On/Off

TGF-β:

Tumor growth factor beta

TME:

Tumor microenvironment

TNF-α:

Tumor necrosis factor alpha

VEGF:

Vascular endothelial growth factor

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Acknowledgements

We thank Mr. Takenobu Nakagawa and Mrs. Yuka Watanabe for their technical assistance.

Funding

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 20H03459 to Y.K., and No. 21K08622 to D.Y). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Authors and Affiliations

  1. Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Chuouku, Kumamoto, 860-8556, Japan

    Ahmad Adawy, Lianbo Li, Hiroki Hirao, Tomoaki Irie, Daiki Yoshii, Hiromu Yano, Yukio Fujiwara & Yoshihiro Komohara

  2. Department of Pediatric Surgery and Transplantation, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan

    Ahmad Adawy, Lianbo Li, Hiroki Hirao, Tomoaki Irie, Masaki Honda & Taizo Hibi

  3. Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan

    Yoshihiro Komohara

  4. Department of Anatomy and Neurobiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

    Shigeyuki Esumi

  5. Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, Japan

    Shinya Suzu

  6. Department of Pediatric Surgery, Faculty of Medicine, Mansoura University, Mansoura, Egypt

    Ahmad Adawy

  7. Chengdu Women’s and Children’s Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 611731, China

    Lianbo Li

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  1. Ahmad Adawy
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Contributions

Conceptualization and study design: AA, YK and TH; data collection: AA, LL, HH, TI, DY, HY, YF, SE, MH and SS; data analysis and interpretation: AA and YK; writing the manuscript (original draft preparation, review & editing): AA and YK; critical revision: TH.

Corresponding author

Correspondence to Yoshihiro Komohara.

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Conflict of interest

All of the authors declare that they have no conflicts of interest.

Ethical approval

This study was performed in line with the principles of the Declaration of Helsinki. Informed consent to be included in the study was obtained from the patients’ caregivers. The study design and proposal were approved by the Institutional Review Board (IRB) at Kumamoto University (IRB No. 2224).

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Supplementary Information

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383_2023_5557_MOESM1_ESM.tif

Supplementary file1 Image showing results of western blot analysis. Chemiluminescence (left) and chemiluminescence with molecular weight markers (right) for PD-L1 (a) and IDO-1 (b) are shown (TIF 310 KB)

383_2023_5557_MOESM2_ESM.tif

Supplementary file2 IC50 for carboplatin and etoposide. HepG2 (a) and HuH-6 (b) cell lines were cultured with various concentrations of carboplatin and etoposide for 48 hours and then the cell viability assay was performed (TIF 106 KB)

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Adawy, A., Li, L., Hirao, H. et al. Potential involvement of IL-32 in cell-to-cell communication between macrophages and hepatoblastoma. Pediatr Surg Int 39, 275 (2023). https://doi.org/10.1007/s00383-023-05557-0

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