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Soluble recombinant human thrombomodulin suppresses inflammation-induced gastrointestinal tumor growth in a murine peritonitis model

Molecular and Cellular Biochemistry volume 475, pages 195–203 (2020)Cite this article

Abstract

Regulatory T cells (Tregs) and transforming growth factor β (TGF-β) are believed to play key roles in both postoperative pro-inflammatory and anti-inflammatory responses of malignancies. Recombinant human thrombomodulin (rTM) is implied to inhibit the interaction between TGF-β and Tregs. The aim of this study is to evaluate the antitumor effects of rTM against gastrointestinal tumors under systemic inflammation. Mice were subjected to cecal ligation and puncture and percutaneous allogeneic tumor implantation. rTM were introduced by percutaneous injection into the abdominal cavity. The effects of rTM were evaluated by weight of implanted tumor, proportion of Tregs in peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL) and temporal evaluation of serum cytokines. The effect of rTM was also evaluated on the in vitro differentiation of naïve T cells into induced Tregs induced by TGF-β and interleukin (IL) -2. rTM significantly inhibited the proliferation of the implanted tumor cells in an inflammation-dependent manner. rTM also reduced the fractions of regulatory T cells and induced regulatory T cells among both PBL and TIL. Temporal evaluation of serum cytokine levels in the model mice showed that rTM significantly suppressed the increases in the serum levels of IL-2 and TGF-β. An in vitro differentiation assay revealed that rTM inhibited the differentiation of naïve T cells into Tregs triggered by IL-2- and TGF-β. rTM has suppressive effects on inflammation-induced gastrointestinal tumor growth by suggestively affecting differentiation of Tregs.

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Data availability

Cell line: CT26.CL25 cell line, ATCC (RRID: CVCL_7255). Mouse: BALB/cCrSlc mice (RRID: MGI:2161077). Software: FACSDiva v8.0.1 (RRID: SCR_001456). SPSS (RRID:SCR_002865). Antibody: CD4 (D702Z) rabbit mAb (RRID: AB_2798898). Foxp3 (D608R) rabbit mAb (RRID: AB_2797979). CD8α (D4W2Z) XP rabbit mAb (AB_2756376). Anti-Mouse CD4 FITC (RM4-5)) (RRID: AB_464902). Anti-Mouse CD25 APC (PC61.5) (RRID: AB_469366). Anti-Mouse/Rat Foxp3 PE (FJK-16s) (RRID: AB_469978). Anti-Mouse CD125-APC-Vio770 (RRID: AB_2654795). Anti-mouse CD62L PE-Cy™7 (RRID: AB_394182). Murine anti-TGF-beta 1 antibody (RRID: AB_10562492).

All data generated or analyzed during this study are included in this published article.

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Funding

En Amada, Kazumasa Fukuda, Koshi Kumagai and Hirofumi Kawakubo received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Yuko Kitagawa was funded by AsahiKASEI Co., Ltd., TAIHO PHARMACEUTICAL CO., LTD, CHUGAI PHARMACEUTICAL CO., LTD., DAIICHI SANKYO COMPANY, LIMITED, Merck Serono Co., Ltd., EA Pharma Co., Ltd., Yakult Honsha Co. Ltd., Otsuka Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Factory Inc., SHIONOGI & CO., LTD., KAKEN PHARMACEUTICAL CO.,LTD., Kowa Pharmaceutical Co., Ltd., Astellas Pharma Inc., MEDICON INC., DAINIPPON SUMITOMO PHARMA Co., Ltd., Taisho Toyama Pharmaceutical Co., Ltd., Kyouwa Hakkou Kirin Co., Ltd., Pfizer Japan Inc., ONO PHARMACEUTICAL CO., LTD., NIHON PHARMACEUTICAL CO., LTD., Japan Blood Products Organization, Medtronic Japan Co., Ltd., Sanofi K.K., grants from Eisai Co., Ltd., TSUMURA & CO., KCI Licensing, Inc., ABBOTT JAPAN CO., LTD., and FUJIFILM Toyama Chemical Co., Ltd.

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Affiliations

  1. Department of Surgery, School of Medicine, Keio University, 35, Shinanomachi, Shinjuku, Tokyo, Japan

    En AMADA, Kazumasa Fukuda, Hirofumi Kawakubo & Yuko Kitagawa

  2. Department of Gastroenterological Surgery, The Cancer Institute Hospital of JFCR, 3-8-31, Ariake, Koto, Tokyo, Japan

    Koshi Kumagai

Authors
  1. En AMADA
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  2. Kazumasa Fukuda
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  3. Koshi Kumagai
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  4. Hirofumi Kawakubo
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  5. Yuko Kitagawa
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Contributions

EA designed the experiments, performed the experiments, analyzed the data and drafted the manuscript. KF and KK participated in the coordination of study and designing of the experiments. HK and YK participated in interpretation and collection of data. All authors have approved the final version of the manuscript and are accountable for all aspects of this study.

Corresponding author

Correspondence to Koshi Kumagai.

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Conflict of interest

En Amada, Kazumasa Fukuda, Koshi Kumagai and Hirofumi Kawakubo declared no potential conflicts of interest with respect to the research, authorship, or publication of this article. Yuko Kitagawa has received compound of recombinant thrombomodulin (rTM) from Asahi Kasei Pharma, Tokyo, Japan.

Ethical approval

All animal procedures were conducted in accordance with Institutional Guidelines on Animal Experimentation of Keio University and were approved by the ethics committee (approval number: 18077-(0)). All staff that participated in the study received special training in animal care and handling from the Laboratory Animal Center of Keio University of Medicine.

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AMADA, E., Fukuda, K., Kumagai, K. et al. Soluble recombinant human thrombomodulin suppresses inflammation-induced gastrointestinal tumor growth in a murine peritonitis model. Mol Cell Biochem 475, 195–203 (2020). https://doi.org/10.1007/s11010-020-03872-x

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  • DOI: https://doi.org/10.1007/s11010-020-03872-x

Keywords

  • Thrombomodulin
  • Anti-tumor immunity
  • Regulatory T cell
  • Transforming growth factor beta