Abstract 1681: The antitumor effect of a soluble recombinant human thrombomodulin as growth suppression against gastrointestinal tumor in murine peritonitis model
Authors: En Amada, Kazumasa Fukuda, Koshi Kumagai, Koichi Suda, Hiroya Takeuchi, Yuko Kitagawa
Journal: Cancer Research (2017)

Abstract Introduction: Severe postoperative inflammatory response (PIR) with CARS increases the risk of tumor recurrence after cancer surgery by Tregs suppressing antitumor immunity. Also, cancer recruits Tregs into its tissue by secreting CCL22 and TGF-beta that strengthen its survival. We established a model to represent tumor dynamics undergo PIR by mice that are performed cecal ligation and puncture (CLP) followed by subcutaneous injection of cancer cells into the dorsum. In this model, we previously revealed significant tumor growth and increase number of Tregs both in tumor tissue and blood compared to the normal mice. A soluble recombinant human thrombomodulin (rhTM) is developed as a treatment drug against DIC. It has anti-inflammatory effect and several reports showed that it prevents cancer metastasis. We hypothesized that rhTM normalizes PIR and prevents cancer growth caused by inflammation. Method: Ten-week-old C57BL/6 mice were divided in CLP/rhTM, CLP/normal saline (NS), and control (simple laparotomy plus NS) groups. rhTM (3mg/kg/12hrs) was injected subcutaneously for 7 consecutive days from the day before CLP. CT26 cells (1×104) were implanted 4 hours after CLP. Mice were sacrificed 28 days after CLP. Then, the tumor tissue and blood were collected. To analyze dynamics of Treg, peripheral blood leukocytes the tumor cells were isolated. CD4, CD25 and Foxp3 were stained by fluorescent antibody and stained cells underwent flow cytometric analysis. The quantity of Tregs is measured by the rate of CD25+/Foxp3+ population in CD4+ T cells. The level of CCL22 and TGF-beta were measured both in serum and tumor tissue. The ELISA was used for analyzing serum samples. And for tumor tissue, immunostaing by DAB was performed and positively stained areas were calculated by image-editing software. Results: CLP/NS group mice exhibited significant enhanced tumor growth compared to controls (6.0±1.9 g vs. 3.5±1.6 g, p=0.03), while CLP/rhTM group produced significantly smaller tumors (2.2±1.1 g) than CLP/NS (p=0.01). The Tregs in the blood stream was smaller in CLP/rhTM group compared to CLP/NS group. Intratumoral Tregs showed significant difference between CLP/rhTM group and CLP/NS group (8.89±3.5 % vs. 24.1±15.0 %, p=0.02). We found significant elevation of serum TGF-beta level by CLP (4.7±1.2 pg/ml vs. 15.4±7.0 pg/ml, p=0.02) and relative reduction to 15.4±7.0 pg/ml by administration of rhTM (p=0.05). However, we did not recognize any change of serum CCL22 level in this study. Intratumoral rate of CCL22 positive areas increased by CLP (41% vs. 50%), while rhTM made it decreased to 43%. We found little difference of TGF-beta positive areas among three groups in this study. Conclusion: Acute inflammation induced by CLP enhances the growth of implanted tumors, while administration of rhTM suppresses tumor growth by possibly affecting dynamics Tregs via TGF-beta and CCL22. Citation Format: En Amada, Kazumasa Fukuda, Koshi Kumagai, Koichi Suda, Hiroya Takeuchi, Yuko Kitagawa. The antitumor effect of a soluble recombinant human thrombomodulin as growth suppression against gastrointestinal tumor in murine peritonitis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1681. doi:10.1158/1538-7445.AM2017-1681