Introduction

Oncology Reports

1021-335X 1791-2431

D.A. Spandidos

10.3892/or.2018.6809

or-41-01-0608

Articles

CCL9/CCR1 induces myeloid-derived suppressor cell recruitment to the spleen in a murine H22 orthotopic hepatoma model

Baohua

1 2 Zhang

Shu

3 Huang

1 Chen

Haiyan

1 Wang

Peijun

3 Yang

Jun

4 Li

Zongfang

2 3

1Core Research Laboratory, The Second Affiliated Hospital, College of Medicine, Jiaotong University, Xi'an, Shaanxi 710004, P.R. China 2National & Local Joint Engineering Research Center of Biodiagnosis and Biotherapy, The Second Affiliated Hospital, College of Medicine, Jiaotong University, Xi'an, Shaanxi 710004, P.R. China 3Department of General Surgery, The Second Affiliated Hospital, College of Medicine, Jiaotong University, Xi'an, Shaanxi 710004, P.R. China 4Department of Pathology, The Second Affiliated Hospital, College of Medicine, Jiaotong University, Xi'an, Shaanxi 710004, P.R. China

Correspondence to: Professor Zongfang Li, National & Local Joint Engineering Research Center of Biodiagnosis and Biotherapy, The Second Affiliated Hospital, College of Medicine, Jiaotong University, 157 Xi Wu Road, Xi'an, Shaanxi 710004, P.R. China, E-mail: lzf2568@mail.xjtu.edu.cn

012019

18102018

41 1 608 618 07032018 08102018

2019

Myeloid-derived suppressor cells (MDSCs) are the major negative regulators of immune responses and expand in numerous tumor models. They contribute to tumor progression and metastasis, and are involved in limiting the effects of cancer immunotherapy. To selectively target MDSCs, it is required to understand the molecular mechanisms that drive MDSC expansion. The mechanisms of their accumulation in tumor tissue have been extensively studied, while the mechanisms of their expansion in lymphoid organs have been rarely explored. The spleen is the largest lymphoid organ in the human body. A previous study by our group reported that a negative immune status in the spleen facilitated tumor growth, with MDSCs being the major immunosuppressive cells. In the present study, a murine H22 orthotopic hepatoma model was established and the mechanisms of splenic MDSC accumulation were studied, including MDSC proliferation, apoptosis and chemotaxis. The proliferation and apoptosis of splenic MDSCs did not differ between normal and tumor-bearing (TB) mice. Cytokine array and ELISA of splenic tissues indicated elevated chemokine (C-C motif) ligand 9 (CCL9) levels in TB mice. Furthermore, splenic macrophages were able to secrete CCL9. Flow cytometric analysis revealed that splenic MDSCs from TB mice also overexpressed C-C motif chemokine receptor 1 (CCR1), the receptor for CCL9. Taken together, the present results indicate that CCL9 secreted by splenic macrophages induces a CCR1-dependent accumulation of MDSCs in the spleen in a murine H22 hepatoma model.

spleen myeloid-derived suppressor cells hepatoma H22 chemokine (c-c motif) ligand 9 C-C motif chemokine receptor 1

Introduction

Myeloid-derived suppressor cells (MDSCs) form a heterogenous population that consists of myeloid progenitor cells, immature macrophages, immature granulocytes and immature dendritic cells (DCs). The phenotype of MDSCs in mice is granulocyte receptor 1 (Gr-1)⁺CD11b⁺. In the steady state, MDSCs have no inhibitory activity and are mostly present in the bone marrow. The percentage of MDSCs is only 2–4% in the spleen, and they are absent from the lymph nodes in mice. In tumor hosts, MDSCs accumulate in lymphatic organs and tumor tissue, and they produce a large amount of immune-inhibitory factors, including arginase I, inducible nitric oxide synthase and reactive oxygen species, which inhibit T-cell immune responses (1–3), induce regulatory T-cell production, suppress natural killer (NK) cell functions, and affect cytokine production and secretion by macrophages. Furthermore, MDSCs can facilitate epithelial-mesenchymal transition, angiogenesis and metastasis (2,4–6).

The spleen is the largest immune organ, containing nearly 25% of the body's immune cells. It has a vital role in the immune response to tumors, and splenic NK cells and T cells exert important anti-tumor functions (7–10). However, the spleen is also a reservoir of precursor monocytes and granulocytes, which may be mobilized to the tumor tissue and differentiate into tumor-associated macrophages (TAM) 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 immunosuppression in the spleen (12). Similarly, Levy et al (13) reported that the spleen may be a reservoir of MDSCs and that splenectomy may reduce the percentage of MDSCs in peripheral blood and tumor tissue.

To date, numerous studies have reported on the mechanisms of MDSC accumulation in tumor tissue, and the factors involved include chemokines, inflammatory factors, colony-stimulating 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 to the tumor tissue, while blocking of these chemokines or their receptors reduced the number of MDSCs at the tumor sites and inhibited tumor growth (17–23). The accumulation of MDSCs in the tumor may also be regulated by inflammatory environmental factors, including interleukin (IL)-1β, IL-6, prostaglandin E2, S100 calcium-binding protein A8/9 and tumor necrosis factor (24–28).

By contrast, few studies have assessed MDSC accumulation in the spleen under pathological conditions. In the murine MCA203 fibrosarcoma model, nestin-positive splenocytes were able to secrete CCL2 that attracted MDSCs to the spleen in a CCR2-dependent manner (29). The spleen is the primary site of immune responses, and its immune status influences tumor growth. Therefore, it is important to explore the mechanisms of MDSC localization and kinetics in the spleen under pathological conditions. This may facilitate the identification of novel factors and pathways involved in MDSC accumulation in the spleen that may have a role in the contribution of MDSCs to tumor progression. These factors and pathways may be novel targets in immune therapy to block MDSCs in order to suppress tumor growth.

In the present study, the mechanisms of MDSC accumulation in the spleen of tumor-bearing (TB) mice were explored. MDSC proliferation, apoptosis and the expression of cytokines in the spleen were assessed. Chemokines upregulated in TB mice were validated and their cellular origin was identified.

Materials and methods <sec> <title>Cell culture

The H22 murine hepatoma cell line was purchased from the China Center for Type Culture Collection (Wuhan, China). The cells were cultured in RPMI-1640 medium (HyClone; GE Healthcare, Little Chalfont, UK) supplemented with 10% fetal calf serum (HyClone; GE Healthcare) and 1% penicillin/streptomycin (HyClone; GE Healthcare). The cells were grown at 37°C in an atmosphere containing 5% CO₂.

Mice

A total of 96 female BALB/c mice (age, 6–8 weeks; weight, 20–25 g) were purchased from the animal center of Xi'an Jiaotong University (Xi'an, China). The mice were housed under specific pathogen-free conditions at the animal facility under a 12-h light/dark cycle and free access to food and water. All animal procedures complied with the Guide for the Care and Use of Laboratory Animals [National Institutes of Health (NIH); publication from 1996] and were approved by the Xi'an Jiaotong University Animal Care and Use Committee (Xi'an, China).

Orthotopic mouse model of hepatocellular carcinoma

Mice were intraperitoneally injected with 10⁶ H22 cells (at the concentration of 10⁷ cells/ml in saline). After 12 days, ascites fluid containing floating tumor cells was collected from these mice, which was centrifuged at 300 × g for 5 min at 4°C to retrieve the cells, which were washed twice with saline. These cells were subsequently injected under the capsule of the left liver lobe of each mouse (2 ×10⁵ tumor cells at the concentration of 10⁷ cells/ml). Four days later, white tumor nodules had formed on the liver, indicating the successful generation of the H22 hepatoma model.

Generation of single-cell suspensions of splenocytes

Mice were sacrificed and the spleens were collected at one, two and three weeks after tumor inoculation. Spleens were dissociated into single-cell suspensions with the gentleMACS Dissociator (Miltenyi Biotech, Bergisch Gladbach, Germany) in the buffer [0.01 mol/l PBS, 0.5% bovine serum albumin (Amresco, Solon, OH, USA) and 2 mmol/l EDTA], using the C tube and the ‘m_spleen_01’ program according to the manufacturer's protocols. Subsequently, the cell suspensions were centrifuged at 300 × g for 30 sec at room temperature. Next, the splenocytes suspensions were strained through a 70-µm mesh to remove clumps and generate single-cell suspensions.

Flow cytometric analysis

The ammonium-chloride-potassium lysis buffer (0.15 mol/l NH₄Cl, 1 mmol/l KHCO₃ and 0.1 mmol/l EDTA, pH 7.2) was added to the splenocytes to remove red blood cells. Subsequently, the cells were washed twice with PBS. Cells (10⁶) were incubated with anti-CD16/CD32 (cat. no. 101302; 10 µg/ml; BioLegend, San Diego, CA, USA) for 10 min at 4°C to prevent non-specific labeling of surface receptors. Next, cells were incubated with monoclonal antibodies, including anti-mouse granulocyte receptor 1 (Gr-1)-fluorescein isothiocyanate (FITC) (cat. no. 108405), anti-mouse Ly6G-FITC (cat. no. 127605), anti-mouse Ly6C-phycoerythrin (PE) (cat. no. 128007), anti-mouse CD11b-allophycocyanine (APC)-(cyanine)Cy7 (cat. no. 101226), anti-mouse C-C motif chemokine receptor 1 (CCR1)-APC (cat. no. 152503) (all from BioLegend; 2.5 µg/ml dilution), anti-mouse CD11b-PE (cat. no. 12-0112; 0.6 µg/ml; eBioscience, San Diego, CA, USA), anti-mouse Fas-APC (cat. no. 17-0951; 10 µg/ml; eBioscience) and their corresponding isotype antibodies, including FITC rat immunoglobulin (Ig)G2b, κ (cat. no. 400605); FITC rat IgG2a, κ (cat. no. 400505); PE rat IgG2c, κ (cat. no. 400707); APC/Cy7 rat IgG2b, κ (cat. no. 400623); APC rat IgG2b, κ (cat. no. 400611) (all from BioLegend; 2.5 µg/ml dilution); PE rat IgG2b, κ (cat. no. 12-4031; 0.6 µg/ml); APC mouse IgG1, κ (cat. no. 17-4714; 10 µg/ml) (both from eBioscience) as the control for 30 min at 4°C in the dark. Following washing, the cells were analyzed using a FACSCanto instrument with FACSDiva 7.0 software (BD Biosciences, Franklin Lakes, NJ, USA).

To assess splenic MDSC proliferation, splenic cell suspensions were stained with anti-mouse Gr-1-FITC and anti-mouse CD11b-PE antibodies for 30 min at 4°C, followed by incubation with cold 70% ethanol (precooled to −20°C; added in a drop-wise manner to the cells while vortexing) at −20°C for 1 h. Subsequently, the cells were washed three times, stained with anti-mouse Ki-67-APC antibody (cat. no. 652405; 2.5 µg/ml; BioLegend) for 30 min at 4°C in the dark, washed and analysed with a flow cytometer.

To assess splenic MDSC apoptosis, splenic cell suspensions were stained with anti-mouse Gr-1-FITC and anti-mouse CD11b-PE, followed by Annexin V-APC (cat. no. 640920; BioLegend) and 7-aminoactinomycin D (7-AAD; cat. no. 420403; 2.5 µg/ml; BioLegend). In brief, 10⁶ cells were washed twice and re-suspended in Annexin V binding buffer (cat. no. 422201; BioLegend). Next, 5 µl Annexin V-APC and 5 µl 7-AAD were added to the cells. Cells were incubated for 15 min at 4°C in the dark, re-suspended in 400 µl binding buffer and analyzed using a FACSCanto II flow cytometer (BD Biosciences) within 1 h.

Fluorescence-activated sorting of splenic macrophages

To isolate splenic macrophages, single-cell suspensions of splenocytes were first incubated with anti-mouse CD16/32 antibody (cat. no. 101302; 10 µg/ml; BioLegend) for 10 min at 4°C to prevent non-specific labeling of surface receptors, followed by incubation with monoclonal antibodies, including anti-mouse F4/80-PE and (cat. no. 123109; 10 µg/ml; BioLegend) anti-mouse CD11b-APC-Cy7 (cat. no. 101226; 2.5 µg/ml; BioLegend), for 30 min at 4°C in the dark. Cells were washed twice and F4/80⁺CD11b⁺ double-positive cells were isolated with a FACSAria II instrument (BD Biosciences). The purity of the sorted macrophages was verified by flow cytometry (FACSAria II; BD Biosciences).

Cytokine array

The concentrations of cytokines in the spleens of normal and TB mice were quantified using the Mouse Cytokine Array G3 (cat. no. AAM-CYT-G3-4; RayBiotech, Norcross, GA, USA) according to the manufacturer's protocols. After all of the procedure steps, the chip was scanned with an Axon GenePix scanner (GenePix 4000B; Axon Instruments, Inc., San Jose, CA, USA). Protein array data were annotated and processed with GenePix Pro 6.0 software (Molecular Devices, LLC, Sunnyvale, CA, USA). The expression levels (fluorescent signal intensities) of each cytokine were calculated by subtracting the mean absorbance of the blank sample, with normalization to a positive control.

ELISA

The levels of murine CCL9 and CXCL2 in the spleens of normal and TB mice were measured using ELISA kits (cat. nos. F111602 and F1117; Westang, Shanghai, China). Assays were performed in duplicate according to the manufacturer's protocols.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

For analysis of CCL9 gene expression, total RNA was extracted from the isolated splenic macrophages using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Complementary (c)DNA was synthesized using the PrimeScript™ RT reagent kit with a genomic DNA eraser (cat. no. RR047A, Takara Bio, Inc., Otsu, Japan). Real-time PCR was performed using SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus) kit (cat. no. RR820A, Takara Bio, Inc.) in a 20-µl reaction system, including 10 µl Premix Ex Taq II (Takara Bio, Inc.), 0.4 µl 5-carboxy rhodamine X reference dye II (Takara Bio, Inc.), 0.8 µl of 0.4 µM forward primer and 0.8 µl of 0.4 µM reverse primer, 2 µl cDNA (resembling the transcription product of 50 ng RNA) and 6 µl distilled water. The following primers were used: CCL9 FP, 5′-CCCTCTCCTTCCTCATTCTTACA-3′ and RP, 5′-AGTCTTGAAAGCCCATGTGAAA-3′; GAPDH FP, 5′-AGGTCGGTGTGAACGGATTTG-3′ and RP, 5′-TGTAGACCATGTAGTTGAGGTCA-3′. Amplifications of CCL9 and GAPDH transcripts were performed during 40 PCR cycles using the ABI 7500 fast real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The initial denaturation step was at 95°C for 30 sec. Each PCR amplification cycle included a denaturation step at 95°C for 5 sec, and a primer annealing and elongation step at 60°C for 30 sec. The expression levels were calculated as the relative cDNA content normalized to GAPDH expression (2^−ΔΔCq method) (30). Three independent replicates were performed.

Statistical analysis

Values are expressed as the mean ± standard deviation. Data were analyzed with Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Student's t-test was used to compare the difference between the two groups. For more than two groups, one-way ANOVA followed by Dunnett's test was performed. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>H22 tumor induces accumulation of MDSCs in the spleen

The murine H22 orthotopic HCC model was established and it was determined whether the development of H22 tumors was associated with an increased number of MDSCs in the spleen. The percentage of splenic MDSCs increased significantly in TB mice compared with that in normal mice at week 1 (6.82±2.92 vs. 3.44±0.60%, P<0.05), week 2 (43.20±11.03 vs. 4.90±0.90%, P<0.001) and week 3 (13.28±4.66 vs. 3.42±0.48%, P<0.001) post-tumor inoculation, with the greatest difference observed at week 2 (Fig. 1). These results suggest that a H22 tumor may induce the accumulation of CD11b⁺Gr-1⁺ cells in the spleen in vivo.

Accumulation of MDSCs in the spleen is not associated with their proliferation and apoptosis

It was investigated whether the accumulation of splenic MDSCs in TB mice resulted from their increased proliferation and/or reduced apoptosis. First the proliferation status of splenic MDSCs was assessed by staining for Ki-67, a marker of cell proliferation. The percentage of Ki-67⁺ splenic MDSCs was not significantly different between TB mice and normal mice at any of the time-points. The mean fluorescent intensity (MFI) of Ki-67 in splenic MDSCs was also not significantly different between TB mice and normal mice (Fig. 2).

Next, the apoptosis of splenic MDSCs was assessed. No difference in the amount of total (AnnexinV⁺) apoptotic MDSCs was identified between TB and normal mice at weeks 1, 2 and 3 post-tumor inoculation (Fig. 3). Fas protein is an apoptotic protein expressed on the cell surface, and therefore, Fas expression on the splenic MDSCs was also assessed. Neither the percentage of Fas⁺ splenic MDSCs nor the MFI of Fas in splenic MDSCs was different between TB mice and normal mice at any of the time-points assessed (Fig. 4).

These results indicate that the accumulation of MDSCs in the spleen was not associated with their proliferation or apoptosis.

Accumulation of splenic MDSCs in TB mice is associated with increases in the level of splenic CCL9

The accumulation of splenic MDSCs in TB mice may be caused by their chemotaxis to the spleen (29). To identify the factors involved, a protein chip assay was then performed for the simultaneous detection of 62 cytokines in the spleen of TB and untreated mice at weeks 1, 2 and 3 post-tumor inoculation (Fig. 5A). At certain time-points, the fluorescent signals of 11 cytokines were higher and those of 8 cytokines were lower in TB mice compared with those in untreated mice. Among the upregulated cytokines, there were 7 chemokines: Chemokine (C-X-C motif) ligand 13 (CXCL13), CXCL16, chemokine (C-C motif) ligand 5 (CCL5), CCL12, CCL9, CXCL2 and CXCL4 (Fig. 5B). However, two of them, CXCL16 and CCL12, were only expressed at low levels and one of them, CXCL13, also known as B lymphocyte chemoattractant, is a chemotactic factor for B lymphocytes. Another chemokine, CCL5, is a chemotactic factor for T cells, eosinophils and basophils. CXCL4 has been reported to negatively control the amount of MDSCs (31), as well as to inhibit angiogenesis and tumor growth (32,33). Therefore, its elevated expression was not correlated with the accumulation of MDSCs (Table I). Subsequently, the role of CCL9 and CXCL2 in MDSC accumulation in the spleen of TB mice was further investigated.

The expression of CCL9 was significantly higher in the spleen of TB mice compared with that in untreated mice at week 2 post-tumor inoculation (Fig. 6A), while there was no significant difference in the expression of CXCL2 at any of the time-points assessed (Fig. 6B). Next, it was determined whether splenic MDSCs expressed CCR1, the receptor for CCL9. It was revealed that splenic MDSCs isolated from TB mice and normal mice expressed CCR1. Furthermore, granulocyte-like MDSCs (G-MDSCs, CD11b⁺ly6G⁺ly6C^low) and monocyte-like MDSCs (MO-MDSCs, CD11b⁺ly6G⁻ly6C^hi) expressed CCR1 (Fig. 7A), suggesting that the increased expression of CCL9 in the spleen of TB mice may attract MDSCs in a CCR1-dependent manner. Of note, the expression of CCR1 on MO-MDSCs in TB mice was higher compared with that in normal mice, while such a difference was not observed for G-MDSCs (Fig. 7B).

Splenic macrophages produce elevated levels of CCL9 in tumor-bearing mice

Next, it was determined which splenic cells secrete CCL9. CCL9 may be secreted by mononuclear phagocytic cells (34). It was examined whether macrophages were the source of CCL9 in spleen of TB mice. Splenic macrophages (CD11b⁺/F4/80⁺) from untreated and TB mice were isolated by fluorescence-assisted cell sorting (Fig. 8A) and the expression levels of CCL9 were detected by RT-qPCR. Splenic macrophages from TB mice produced higher levels of CCL9 than control macrophages at weeks 1, 2 and 3 (Fig. 8B). These results suggest that macrophages may be the source of CCL9 the in the spleen of H22 hepatoma mice.

Discussion

Previous studies have reported that the frequencies of MDSCs increased not only in tumor tissue but also in the spleen of TB mice (12,24,35). The accumulation of splenic MDSCs in TB mice may be caused by their increased proliferation, decreased apoptosis or enhanced chemotaxis to the spleen. In the present study, all three aspects were investigated.

It remains elusive whether the proliferation of MDSCs occurs mainly in the bone marrow or in the spleen. The spleen is the major site of MDSC proliferation in the 4T1 mammary cancer model, and the expansion of splenic MDSCs was caused by their increased survival and decreased apoptosis (36). By contrast, in murine 3LL lung cancer, B16 melanomas and Meth A fibrosarcomas, the proliferation of MDSCs primarily occurs in the bone marrow and not in the peripheral blood, the spleen or the tumor tissue (37). Therefore, in the murine H22 orthotopic hepatoma model of the present study, changes in splenic MDSC proliferation and apoptosis were first assessed. It was identified that the expansion of splenic MDSCs was not associated with their proliferation or apoptosis.

Next, the mechanisms of MDSCs recruitment to the spleen were assessed. In the murine MCA203 fibrosarcoma model, nestin-positive splenocytes are able to secrete CCL2, which caused an elevated level of CCL2 in the spleen and induced MDSC accumulation in the spleen via CCR2 (29). In the present H22 hepatoma model, it was investigated which factor is able to attract MDSCs to the spleen by screening for 62 cytokines. The cytokine protein array and ELISA results revealed that the CCL9 chemokine was highly expressed in the spleen of TB mice.

The overexpressed CCL9 exerts its chemotactic function through binding to its receptor CCR1. Therefore, it was next determined whether MDSCs expressed CCR1. The splenic MDSCs (including G-MDSCs and MO-MDSCs) from normal and TB mice were identified to express CCR1. This indicates that the recruitment of MDSCs to the spleen of TB mice was driven by increases of CCL9 as well as its receptor CCR1, leading to the accumulation of MDSCs in the spleen.

It was then investigated which cell type in the spleen secretes CCL9 in TB mice. CCL9 may be secreted by macrophages, DCs, Langerhans cells, gliocytes, osteoclasts and certain types of tumor cell, including intestinal cancer cells. MDSCs may also be a source of CCL9 (38–40). As the percentage of macrophages in the spleen is higher than that of DCs (35,41,42), the expression of CCL9 in macrophages was examined and it was revealed that splenic macrophages from TB mice had significantly elevated levels of CCL9 compared with those in normal mice. Therefore, it was concluded that the elevated CCL9 secreted by splenic macrophages attracted MDSCs to the spleen of TB mice.

In the H22 orthotopic hepatoma model, a reduction in spleen weight from week 2 to week 3 was previously observed (12). The total number of splenocytes, including macrophages, may decrease at week 3. Macrophages viability and activity may decrease, resulting in reduced cytokine secretion (43). This may explain for the lower expression of CCL9 in the spleen and reduced recruitment of MDSCs to the spleen at week 3 compared with that at week 2.

In conclusion, the results of the present study suggest that in the H22 hepatoma model, splenic macrophages secreted CCL9 that induced MDSC accumulation in the spleen in a CCR1-dependent manner. Further studies should assess which other splenocytes, including DCs or MDSCs, secrete CCL9, whether the mobilization of MDSCs to the spleen may be inhibited by targeting CCL9 or CCR1, and how such an inhibition may affect tumor growth.

Acknowledgements

The authors would like to thank Professor Malgorzata A. Garstka (Core Research Lab of the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China) for critical reading of the manuscript.

Funding

This work was supported by the Program for Changjiang Scholars and Innovative Research Teams at University (grant no. 1171), the National Natural Science Foundation of China (grant no. 81001309) and the Key Research and Development Program of Shaanxi Province of China (grant no. 2017ZDCXL-SF-02-05, 2018SF-197).

Availability of data and materials

All data generated and analyzed during this study are included in this manuscript.

Authors' contributions

BL performed the experiments and wrote the first draft of the manuscript. NH, HC and PW assisted in the establishment of the tumor model and the sample preparation of flow cytometry. SZ assisted in the statistical analysis and data interpretation. JY and ZL designed the study and supervised the experimental work. All authors reviewed the manuscript prior to its submission and approved the final manuscript.

Ethics approval and consent to participate

All animal procedures complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication, 1996) and were approved by the Animal Care and Use Committee of Xi'an Jiaotong University (Xi'an, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figure 1.

MDSCs accumulate in the spleen during tumor growth. Single-cell suspensions of splenocytes were prepared and stained with anti-mouse-Gr-1 monoclonal antibody conjugated with FITC and anti-mouse CD11b monoclonal antibody conjugated with PE, and analyzed by flow cytometry. (A) Representative fluorescence-assisted cell sorting plots of splenocyte preparations. A live gate P1 was set in the FSC/SSC plot. Subsequently, the populations of CD11b-PE and Gr-1-FITC cells were displayed in dot plots. (B) The percentage of splenic MDSCs (Gr-1⁺CD11b⁺, gate P2 within live gate) in H22 hepatoma mice (▲) and normal mice (■) at weeks 1, 2 and 3 post-tumor inoculation (n=6); Values are expressed as the mean ± standard deviation. *P<0.05 and ***P<0.001 compared with normal mice. TB, tumor-bearing; MDSCs, myeloid-derived suppressor cells; FITC, fluorescein isothiocyanate; PE, phycoerythrin; SSC, side scatter; FSC, forward scatter; Gr-1, granulocyte receptor 1.

Figure 2.

Accumulation of splenic MDSCs in H22 hepatoma mice is not associated with their proliferative status. Single-cell suspensions of splenocytes were stained with anti-mouse Gr-1 monoclonal antibody conjugated with FITC and anti-mouse CD11b monoclonal antibody conjugated with PE, followed by permeabilization with 70% ethanol and staining with anti-mouse Ki-67 antibody conjugated with APC. (A) Representative flow cytometry plots and gating strategy. A live gate P1 was set in the FSC/SSC plot and CD11b⁺/Gr-1⁺ were included in the further analysis with gate P2. Subsequently, the selected cells were displayed in a histogram for Ki-67-APC. (B) The percentage of Ki-67⁺ MDSCs (left panel) and the MFI of Ki-67 in MDSCs (right panel) in H22 hepatoma and normal mice (n=6). Values are expressed as the mean ± standard deviation. TB, tumor-bearing; APC, allophycocyanine; MDSCs, myeloid-derived suppressor cells; FITC, fluorescein isothiocyanate; PE, phycoerythrin; SSC, side scatter; FSC, forward scatter; Gr-1, granulocyte receptor 1; MFI, mean fluorescent intensity.

Figure 3.

Accumulation of splenic MDSCs in H22 hepatoma mice is not caused by alterations in their apoptotic rate. Single-cell suspensions of splenocytes were stained with anti-mouse Gr-1 monoclonal antibody conjugated with FITC and anti-mouse CD11b monoclonal antibody conjugated with PE, followed by staining with anti-mouse AnnexinV antibody conjugated with APC and viability dye 7-AAD. (A) Representative flow cytometry plots and gating strategy. A live gate P1 was set in the FSC/SSC plot and CD11b⁺/Gr-1⁺ cells were included in the further analysis with gate P2. Subsequently, the selected cells were displayed in histograms for Annexin-APC or 7-AAD. Annexin V⁺/7-AAD⁻ indicates cells in early apoptosis and Annexin V⁺/7-AAD⁺ indicates cells in late apoptosis. (B) Total apoptotic MDSC populations (AnnexinV⁺) in TB and normal mice are presented. Values are expressed as the mean ± standard deviation (n=6). TB, tumor-bearing; APC, allophycocyanine; MDSCs, myeloid-derived suppressor cells; FITC, fluorescein isothiocyanate; PE, phycoerythrin; SSC, side scatter; FSC, forward scatter; Gr-1, granulocyte receptor 1; 7-AAD, 7-amino actinomycin D; PerCP, peridinin chlorphyll protein; Cy, cyanine.

Figure 4.

Mice with hepatoma and normal mice do not exhibit any difference in the expression of apoptotic protein Fas in splenic MDSCs. Single-cell suspensions of splenocytes were stained with anti-mouse Gr-1 antibody conjugated with FITC, anti-mouse CD11b antibody conjugated with PE and anti-mouse Fas antibody conjugated with APC. (A) Representative flow cytometry plots and gating strategy. A live gate P1 was set in the FSC/SSC plot and CD11b⁺/Gr-1⁺ cells were included in a further analysis with gate P2. Subsequently, the selected cells were displayed in a histogram for Fas-APC. (B) The percentage of Fas⁺ MDSCs (left panel) and the MFI of Fas in MDSCs (right panel) in H22 hepatoma and normal mice. Values are expressed as the mean ± standard deviation (n=6). TB, tumor-bearing; APC, allophycocyanine; MDSCs, myeloid-derived suppressor cells; FITC, fluorescein isothiocyanate; PE, phycoerythrin; SSC, side scatter; FSC, forward scatter; Gr-1, granulocyte receptor 1; MFI, mean fluorescent intensity.

Figure 5.

Differential expression of 62 cytokines in the spleen of H22 hepatoma and normal mice. A RayBiotech mouse cytokine antibody array was used to compare the expression of 62 cytokines in the splenic tissues of untreated and TB mice. (A) Mouse cytokine antibody array membranes. (B) Quantification of A. The upregulated (upper part) and downregulated (lower part) cytokines in the spleen of TB mice compared with untreated mice. Among the upregulated cytokines, seven chemokines are indicated by rectangles. TB, tumor-bearing; 1w, 1 week; G-CSF, granulocyte-colony stimulating factor; IGFBP3, insulin-like growth factor-binding protein 3; CCL12, chemokine (C-C motif) ligand 12; CXCL2, chemokine (C-X-C motif) ligand 2; sTNFR, soluble tumor necrosis factor receptors; TIMP1, tissue inhibitors of metalloproteinase 1; IFN, interferon; IL, interleukin; XCL1, chemokine (C motif) ligand.

Figure 6.

Accumulation of splenic MDSCs is caused by increases of CCL9 levels in the spleens of H22 hepatoma mice. The expression of (A) CCL9 and (B) CXCL2 in the spleen tissues of untreated and TB mice was determined using ELISA. The spleen tissues were harvested at week 1, week 2 and week 3 post-tumor inoculation and lysed. Lysates were clarified and the protein concentration was measured using ELISAs. Values are expressed as the mean ± standard deviation (pg/mg of chemokine/splenic tissue, n=5). *P<0.05 compared with untreated mice. TB, tumor-bearing; 1w, 1 week; CCL9, chemokine (C-C motif) ligand 9; CXCL2, chemokine (C-X-C motif) ligand 2.

Figure 7.

Splenic MDSCs express CCR1, the receptor for chemokine (C-C motif) ligand 9. Splenic single-cell suspensions were stained with anti-mouse ly6G monoclonal antibody conjugated with FITC, anti-mouse ly6C monoclonal antibody conjugated with PE, anti-mouse CD11b monoclonal antibody conjugated with APC-Cy7 and anti-mouse CCR1 monoclonal antibody conjugated with APC. (A) Representative flow cytometry plots and gating strategy. Left panel: A live gate P1 was set in the FSC/SSC plot and CD11b⁺ cells were included in further analysis with gate P2 (APC/Cy7 rat immunoglobulin G2b, κ isotype antibody was used as the negative control for the separation of negative and positive cell populations). Subsequently, the selected cells were displayed in a histogram for ly6C-PE and ly6G-FITC. The population of CD11b⁺ly6G⁺ly6C^low cells refers to G-MDSCs, and the population of CD11b⁺ly6G⁻ly6C^high cells refers to MO-MDSCs. Right panel: Representative plots for the expression of CCR1 on G-MDSCs and MO-MDSCs from the spleen of normal and TB mice. (B) The MFI of CCR1 on G-MDSCs (left panel) and MO-MDSCs (right panel) from H22 hepatoma and normal mice (n=6). Values are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01. TB, tumor-bearing; G-MDSCs, granulocytic myeloid-derived suppressor cells; MO-MDSCs, monocytic MDSCs; TB, tumor-bearing; APC, allophycocyanine; FITC, fluorescein isothiocyanate; PE, phycoerythrin; SSC, side scatter; FSC, forward scatter; MFI, mean fluorescent intensity; CCR1, C-C motif chemokine receptor 1; Cy, cyanine.

Figure 8.

Splenic macrophages produce elevated levels of CCL9 in H22 hepatoma mice. (A) Single-cell suspensions of splenocytes were stained with anti-mouse F4/80 monoclonal antibody conjugated with PE and anti-mouse CD11b monoclonal antibody conjugated with APC-Cy7. The double-positive cells refer to splenic macrophages. Splenic macrophages from untreated and TB mice were isolated by fluorescence-assisted cell sorting. (B) Total RNA was extracted from the sorted splenic macrophages, and the expression of CCL9 relative to GAPDH was examined by reverse transcription-quantitative polymerase chain reaction analysis. ***P<0.001 compared with splenic macrophages from untreated mice. TB, tumor-bearing; 1w, 1 week; APC, allophycocyanine; Cy, cyanine; PE, phycoerythrin; Q, quadrant; CCL9, chemokine (C-C motif) ligand 9.

Table I.

Upregulated chemokines in spleen of H22 hepatoma mice. A total of 62 cytokines were detected in spleen tissue of untreated and TB mice at weeks 1, 2 and 3 post-tumor inoculation using a mouse cytokine antibody array.

	Fluorescent signal intensity (normalized to positive control)

Abbreviation	Untreated	TB-1w	TB-2w	TB-3w	Chemotactic cells
CXCL13	374	282	270	563	B
CXCL16	155	184	171	206	T, NKT
CCL5	916	1132	729	91	T, eosinophils, basophils
CCL12	42	53	18	184	Eosinophils, monocytes, lymphocytes
CCL9	3298	4770	5644	7677	DC
CXCL2	955	502	1343	926	Polymorphonuclear leukocytes
CXCL4	3907	256	9609	4638	Neutrophils, monocytes, fibroblasts

DC, dendritic cells, NKT, natural killer T-cells; TB, tumor-bearing; 1w, 1 week; CXCL13, chemokine (C-X-C motif) ligand 13; CCL5, chemokine (C-C motif) ligand 5.