608 ONCOLOGY REPORTS 41: 608-618, 2019 CCL9/CCR1 induces myeloid‑derived suppressor cell recruitment to the spleen in a murine H22 orthotopic hepatoma model BAOHUA LI1,2, SHU ZHANG3, NA HUANG1, HAIYAN CHEN1, PEIJUN WANG3, JUN YANG4 and ZONGFANG LI2,3 1Core Research Laboratory; 2National & Local Joint Engineering Research Center of Biodiagnosis and Biotherapy; Departments of 3General Surgery and 4Pathology, The Second Affiliated Hospital, College of Medicine, Jiaotong University, Xi'an, Shaanxi 710004, P.R. China Received March 7, 2018; Accepted October 8, 2018 DOI: 10.3892/or.2018.6809 Abstract. Myeloid-derived suppressor cells (MDSCs) are the Introduction major negative regulators of immune responses and expand in numerous tumor models. They contribute to tumor progres- Myeloid-derived suppressor cells (MDSCs) form a heterog- sion and metastasis, and are involved in limiting the effects enous population that consists of myeloid progenitor cells, of cancer immunotherapy. To selectively target MDSCs, it is immature macrophages, immature granulocytes and imma- required to understand the molecular mechanisms that drive ture dendritic cells (DCs). The phenotype of MDSCs in mice MDSC expansion. The mechanisms of their accumulation in is granulocyte receptor 1 (Gr-1)+CD11b+. In the steady state, tumor tissue have been extensively studied, while the mecha- MDSCs have no inhibitory activity and are mostly present nisms of their expansion in lymphoid organs have been rarely in the bone marrow. The percentage of MDSCs is only 2-4% explored. The spleen is the largest lymphoid organ in the in the spleen, and they are absent from the lymph nodes in human body. A previous study by our group reported that a mice. In tumor hosts, MDSCs accumulate in lymphatic negative immune status in the spleen facilitated tumor growth, organs and tumor tissue, and they produce a large amount with MDSCs being the major immunosuppressive cells. In the of immune-inhibitory factors, including arginase I, induc- present study, a murine H22 orthotopic hepatoma model was ible nitric oxide synthase and reactive oxygen species, which established and the mechanisms of splenic MDSC accumula- inhibit T-cell immune responses (1-3), induce regulatory T-cell tion were studied, including MDSC proliferation, apoptosis production, suppress natural killer (NK) cell functions, and and chemotaxis. The proliferation and apoptosis of splenic affect cytokine production and secretion by macrophages. MDSCs did not differ between normal and tumor-bearing (TB) Furthermore, MDSCs can facilitate epithelial-mesenchymal mice. Cytokine array and ELISA of splenic tissues indicated transition, angiogenesis and metastasis (2,4-6). elevated chemokine (C-C motif) ligand 9 (CCL9) levels in TB The spleen is the largest immune organ, containing mice. Furthermore, splenic macrophages were able to secrete nearly 25% of the body's immune cells. It has a vital role in CCL9. Flow cytometric analysis revealed that splenic MDSCs the immune response to tumors, and splenic NK cells and T from TB mice also overexpressed C-C motif chemokine cells exert important anti-tumor functions (7-10). However, receptor 1 (CCR1), the receptor for CCL9. Taken together, the the spleen is also a reservoir of precursor monocytes and present results indicate that CCL9 secreted by splenic macro- granulocytes, which may be mobilized to the tumor tissue phages induces a CCR1-dependent accumulation of MDSCs in and differentiate into tumor-associated macrophages (TAM) the spleen in a murine H22 hepatoma model. and neutrophils (TAN) that promote tumor progression. Splenectomy has been indicated to markedly reduce TAM and TAN responses, thus retarding tumor growth (11). A previous study by our group identified an immune‑suppressive status in the spleen of a H22 orthotopic hepatoma mouse model. MDSCs were the major inhibitory cells causing the immunosuppres- sion in the spleen (12). Similarly, Levy et al (13) reported that Correspondence to: Professor Zongfang Li, National & Local the spleen may be a reservoir of MDSCs and that splenectomy Joint Engineering Research Center of Biodiagnosis and Biotherapy, The Second Affiliated Hospital, College of Medicine, Jiaotong may reduce the percentage of MDSCs in peripheral blood and University, 157 Xi Wu Road, Xi'an, Shaanxi 710004, P.R. China tumor tissue. E-mail: [email protected] To date, numerous studies have reported on the mechanisms of MDSC accumulation in tumor tissue, and the factors involved Key words: spleen, myeloid-derived suppressor cells, hepatoma, H22, include chemokines, inflammatory factors, colony‑stimulating chemokine (c-c motif) ligand 9, C-C motif chemokine receptor 1 factor and complements (14-16). Tumor-derived chemokines, including C-C motif chemokine ligand (CCL)2, CCL5, CCL21 and C-X-C motif ligand (CXCL)8 were able to recruit MDSCs LI et al: CCL9/CCR1 INDUCES SPLENIC MDSC RECRUITMENT IN HEPATOMA 609 to the tumor tissue, while blocking of these chemokines or Generation of single‑cell suspensions of splenocytes. Mice their receptors reduced the number of MDSCs at the tumor were sacrificed and the spleens were collected at one, two sites and inhibited tumor growth (17-23). The accumulation of and three weeks after tumor inoculation. Spleens were disso- MDSCs in the tumor may also be regulated by inflammatory ciated into single-cell suspensions with the gentleMACS environmental factors, including interleukin (IL)-1β, IL-6, Dissociator (Miltenyi Biotech, Bergisch Gladbach, prostaglandin E2, S100 calcium-binding protein A8/9 and Germany) in the buffer [0.01 mol/l PBS, 0.5% bovine serum tumor necrosis factor (24-28). albumin (Amresco, Solon, OH, USA) and 2 mmol/l EDTA], By contrast, few studies have assessed MDSC accumula- using the C tube and the ‘m_spleen_01’ program according tion in the spleen under pathological conditions. In the murine to the manufacturer's protocols. Subsequently, the cell MCA203 fibrosarcoma model, nestin-positive splenocytes suspensions were centrifuged at 300 x g for 30 sec at room were able to secrete CCL2 that attracted MDSCs to the spleen temperature. Next, the splenocytes suspensions were strained in a CCR2-dependent manner (29). The spleen is the primary through a 70-µm mesh to remove clumps and generate site of immune responses, and its immune status influences single-cell suspensions. tumor growth. Therefore, it is important to explore the mecha- nisms of MDSC localization and kinetics in the spleen under Flow cytometric analysis. The ammonium-chloride-potas- pathological conditions. This may facilitate the identification sium lysis buffer (0.15 mol/l NH4Cl, 1 mmol/l KHCO3 and of novel factors and pathways involved in MDSC accumula- 0.1 mmol/l EDTA, pH 7.2) was added to the splenocytes tion in the spleen that may have a role in the contribution of to remove red blood cells. Subsequently, the cells were MDSCs to tumor progression. These factors and pathways washed twice with PBS. Cells (106) were incubated with may be novel targets in immune therapy to block MDSCs in anti-CD16/CD32 (cat. no. 101302; 10 µg/ml; BioLegend, order to suppress tumor growth. San Diego, CA, USA) for 10 min at 4˚C to prevent In the present study, the mechanisms of MDSC accumula- non-specific labeling of surface receptors. Next, cells tion in the spleen of tumor-bearing (TB) mice were explored. were incubated with monoclonal antibodies, including MDSC proliferation, apoptosis and the expression of cytokines anti-mouse granulocyte receptor 1 (Gr-1)-fluorescein in the spleen were assessed. Chemokines upregulated in TB isothiocyanate (FITC) (cat. no. 108405), anti-mouse mice were validated and their cellular origin was identified. Ly6G-FITC (cat. no. 127605), anti-mouse Ly6C-phyco erythrin (PE) (cat. no. 128007), anti-mouse CD11b-all Materials and methods ophycocyanine (APC)-(cyanine)Cy7 (cat. no. 101226), anti-mouse C-C motif chemokine receptor 1 (CC Cell culture. The H22 murine hepatoma cell line was R1)-APC (cat. no. 152503) (all from BioLegend; purchased from the China Center for Type Culture Collection 2.5 µg/ml dilution), anti-mouse CD11b-PE (cat. no. 12-0112; (Wuhan, China). The cells were cultured in RPMI-1640 0.6 µg/ml; eBioscience, San Diego, CA, USA), anti-mouse medium (HyClone; GE Healthcare, Little Chalfont, UK) Fas-APC (cat. no. 17-0951; 10 µg/ml; eBioscience) and supplemented with 10% fetal calf serum (HyClone; their corresponding isotype antibodies, including FITC GE Healthcare) and 1% penicillin/streptomycin (HyClone; rat immunoglobulin (Ig)G2b, κ (cat. no. 400605); FITC rat GE Healthcare). The cells were grown at 37˚C in an atmo- IgG2a, κ (cat. no. 400505); PE rat IgG2c, κ (cat. no. 400707); sphere containing 5% CO2. APC/Cy7 rat IgG2b, κ (cat. no. 400623); APC rat IgG2b, κ (cat. no. 400611) (all from BioLegend; 2.5 µg/ml Mice. A total of 96 female BALB/c mice (age, 6-8 weeks; dilution); PE rat IgG2b, κ (cat. no. 12-4031; 0.6 µg/ml); APC weight, 20-25 g) were purchased from the animal center of mouse IgG1, κ (cat. no. 17-4714; 10 µg/ml) (both from eBiosci- Xi'an Jiaotong University (Xi'an, China). The mice were ence) as the control for 30 min at 4˚C in the dark. Following housed under specific pathogen‑free conditions at the animal washing, the cells were analyzed using a FACSCanto instru- facility under a 12-h light/dark cycle and free access to food ment with FACSDiva 7.0 software (BD Biosciences, Franklin and water. All animal procedures complied with the Guide for Lakes, NJ, USA). the Care and Use of Laboratory Animals [National Institutes To assess splenic MDSC proliferation, splenic cell suspen- of Health (NIH); publication from 1996] and were approved sions were stained with anti-mouse Gr-1-FITC and anti-mouse by the Xi'an Jiaotong University Animal Care and Use CD11b‑PE antibodies for 30 min at 4˚C, followed by incuba- Committee (Xi'an, China).