A gastric cancer cell derived extracellular compounds suppresses CD161+CD3- lymphocytes and aggravates tumor formation in a syngeneic mouse model
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).
References (47)
Activation and function of natural killer cell responses during viral infections
Curr. Opin. Immunol.
(1997)- et al.
Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo: presented in part at the keystone symposium “Cellular and molecular biology of dendritic cells,” Santa Fe, NM, March 3-9, 1998, and at the annual meeting of the american association for Cancer research, March 28-April 1, 1998
Blood
(1998) - et al.
Immunity, inflammation, and cancer
Cell
(2010) - et al.
In search of the ‘missing self’: MHC molecules and NK cell recognition
Immunol. Today
(1990) - et al.
Tumour immunity: effector response to tumour and role of the microenvironment
Lancet
(2008) - et al.
Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation
Blood
(2006) - et al.
Amplification of tumor-specific regulatory T cells following therapeutic cancer vaccines
Blood
(2006) - et al.
Sunroot mediated synthesis and characterization of silver nanoparticles and evaluation of its antibacterial and rat splenocyte cytotoxic effects
Int. J. Nanomed.
(2015) - et al.
Macrophage infiltration in human non-small-cell lung cancer: the role of CC chemokines
Cancer Immunol. Immunother.
(2000) - et al.
Interleukin-8, a promising predictor for prognosis of pancreatic cancer
World J. Gastroenterol.: WJG
(2012)