Elsevier

Molecular Immunology

Volume 120, April 2020, Pages 136-145
Molecular Immunology

A gastric cancer cell derived extracellular compounds suppresses CD161+CD3- lymphocytes and aggravates tumor formation in a syngeneic mouse model

https://doi.org/10.1016/j.molimm.2020.02.016Get rights and content

Highlights

  • This study identified several cytokines/chemokines in the human gastric cancer cell derived extra cellular compound (GC-EC).

  • The GC-EC selectively suppresses NK cell population in splenocytes.

  • The aggravation of tumor formation was prominent in the presence of GC-EC in syngeneic mouse model.

Abstract

Evasion of the immune system is often associated with malignant tumors. The cancer cell microenvironment plays an important role in tumor progression, but its mechanism is largely unknown. Here we show that an extracellular compound derived from gastric cancer (GC-EC) selectively suppresses CD161+CD3 natural killer (NK) cells. Splenocytes treated with GC-EC showed considerable proliferation and the CD161+CD3 NK cell population was time-dependently suppressed. Intracellular staining of IFN-γ was shown to be down-regulated in concert with granzyme B and perforin. A cytotoxicity assay of splenocytes treated with GC-EC against K-562 cells showed a significant reduction in cytolytic activity. Further, the immune-suppressive effect of GC-EC was more evident in a syngeneic tumor model in C57BL/6 mice. Animals treated with B16 F10 and GC-EC exhibited more aggravated tumor formation than animals treated with B16 F10 only. We demonstrated that inhibition of apoptosis while increasing PI3 K/AKT levels may provoke tumor formation by GC-EC. A cytokine array revealed the presence of several cytokines in GC-EC that negatively regulate immune cytolytic activity and could be potential candidates for immune-suppressive effects.

Introduction

Gastric cancer (GC) is the fourth most prevalent cancer in the world (Torre et al., 2015) and the growing scientific evidence indicates that angiogenesis is influenced by various factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and interleukin-8 (IL-8) are major factors involved in angiogenesis in GC (Kitadai, 2010). Cross-talk between cancer cells and cytokines in extracellular regions of the tumor microenvironment (TME) can have a significant impact on the characteristics of GC. Any change in the TME plays a vital role in facilitating tumor progression and activation of cell signaling pathways (Quail and Joyce, 2013). In recent years, immunotherapy has become a standard treatment for various cancers which involves activation of innate and adaptive responses via specific cytokines, monoclonal antibodies, novel immune adjuvants, and anticancer vaccines (Dougan and Dranoff, 2009). However, the success rates of these therapies are limited due to the presence of various immune regulatory pathways (Gajewski et al., 2013; Perwez Hussain and Harris, 2007; Zhou et al., 2006).

The TME has been ascertained to play a crucial role in the initiation and progression of tumors (Chung and Lim, 2014; Kano, 2015). Importantly, extracellular compounds derived from tumors cells are known to influence splenocytes by activating dendritic cells or converting various cell types (Mantovani et al., 2008). In response to the activity immune of cells against tumor cells, the tumor cells undergo a process called “immune editing” and thereby change their phenotypes by down-regulation or loss of tumor antigens capable of recruiting immune cells (Khaled et al., 2013; Ljunggren and Kärre, 1990; Spaggiari et al., 2006). In a recent study, Na et al. reported that gastric cancer cells secrete factors that can cause apoptosis and fibrosis both in vitro and in vivo (Na et al., 2012). However, there is limited knowledge in the area of gastric cancer cell secretion and immune activity.

Natural killer (NK) cells are specialized lymphocytes capable of affecting virus-infected and tumor cells (Biron, 1997; Vivier et al., 2011). They are the primary cells of the innate immune system and also aid in the development of adaptive immune responses (Lotze and Thomson, 2009; A. Moretta et al., 2001). NK cell function is closely regulated by a series of surface receptors that transduce, activate, or inhibit signals. Natural cytotoxicity receptors (NCRs), such as NKp30, NKp44, and NKp46, are crucial receptors for NK cell activation, cytotoxicity, and production of cytokines (L. Moretta and Moretta, 2004; Saunders et al., 2015). IFN-γ is a pleiotropic cytokine primarily produced by NK and T cells, plays an important role in inhibiting tumor formation and metastasis (Farrar and Schreiber, 1993). In a recent study, Kano demonstrated that decreased IFN-γ level is associated with 4T1 tumor cell-conditioned medium, but not with CT26 colon cancer medium. Thus indicating the significance of cancer cell type on specific immune suppression (Kano, 2015). In this background, the aim of the present study is to investigate the immune suppression and aggressive tumor expansion activity of extracellular compounds derived from GC (GC-EC) using various molecular techniques including qRT-PCR, immunoblotting, computed tomography (CT) scan images, histopathology and expression of oncogenic signaling pathway was examined. Furthermore, the possible mechanism that aggravates tumor formation after GC-EC administration in the animal model was investigated.

Section snippets

Preparation of GC-EC

Gastric carcinoma cells (SNU-484) were added (2 × 106 cells) to 75 cm2 dishes containing RPMI medium supplemented with 10 % heat-inactivated FBS and 1% antibiotic-antimycotic solution (Gibco, NY, USA). Following 3 days of incubation at 37 °C with 5% CO2, the supernatant was collected and centrifuged at 3,000×g. To ensure complete removal of cells from the supernatant, the content was filtered using a 0.42 μm filter and transferred to a 3 KDa protein cut-off membrane (Ultracel-3 K; Amicon,

GC-EC induces proliferation without apoptosis

To investigate the effect of GC-EC on the viability and proliferation of splenocytes, different concentrations (150, 300, 400, 500, and 700 μg/mL) were tested. The MTT assay revealed that the addition of GC-EC considerably increased the viability compared to untreated cells (Fig. 1A). Further, the apoptotic effect of GC-EC was analyzed in flow cytometry using Annexin V/PI staining and the results showed that the percentages of annexin V positive cells were relatively similar is revealing that

Discussion

Attenuation of immune cell function, especially NK cells, is regarded as an important mechanism of tumor immune escape in mice and cancer patients (Grivennikov et al., 2010) (Park et al., 2013). The present study particularly addressed the change in CD161+CD3 NK cell activity in the rodent model. The MTT and Annexin V assay revealed that GC-EC does not induce apoptosis even with increasing the concentration up to 700 μg/mL which is equal to half of the media strength present in cancer cells.

CRediT authorship contribution statement

Aravinthan Adithan: Conceptualization, Methodology, Writing - original draft. Judith Sharmila John Peter: Methodology. Amjad Hossain Mohammad: Methodology. Bumseok Kim: Visualization, Supervision. Chang-Won Kang: Visualization, Supervision. Nam Soo Kim: Funding acquisition. Ki-Chul Hwang: Resources. Jong-Hoon Kim: Supervision, Writing - review & editing, Funding acquisition.

Declaration of Competing Interest

The authors declare no competing interests.

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A3B03029075). This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2019R1A6A1A03033084).

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