The increase in circulating myeloid-derived suppressor cells correlated with clinical stage of cervical carcinoma

Abstract

The myeloid-derived suppressor cells (MDSCs) are important tumor-induced immune suppression cells which have gained great attention in recent years. In this study, the levels of MDSCs in peripheral blood of cervical carcinoma (CC) patients, cervical intraepithelial neoplastic (CIN) patients, and healthy donors were analyzed by flow cytometry. It was shown that the proportion of circulating MDSCs was elevated in CC and CIN patients compared to that in healthy donor (HD). Further analyses revealed that the percentage of MDSCs is increased along with the stage of CC, but has no difference among other clinicopathological factors, including age, tumor grade, lymph node invasion, and histopathology. Taken together, our results suggested that MDSC levels are indicative of suppressive functions within the tumor environment of CC. Therefore, large validation studies could help to develop new immunotherapeutic strategies for CC patients by inhibiting or eliminating MDSC functions.

Introduction

Invasive cervical carcinoma (CC) ranks fourth in incidence of common female cancers. In general, surgical resection, radiography, and chemotherapy are relatively effective treatments for cervical cancer patients. However, the cancer patients suffer greatly from side effects of the present therapeutic methods and there is no curative treatment for advanced cancer. Thus, we need to investigate novel therapy such as immunotherapeutic methods to treat cervical cancer and improve survival rate and living quality.

Myeloid-derived suppressor cells (MDSCs) have emerged as important elements of cancer-induced immune dysfunction. These cells were first described in tumor-bearing murine in the late 1970s. ¹ In healthy individuals, hematopoietic stem cells differentiate into immature myeloid cells (IMCs) which then migrate to different peripheral organs and differentiate into mature myeloid cells such as macrophages, dendritic cells, and granulocytes. However, if the body suffers from inflammation, infection, or malignant tumors, IMCs will not differentiate into normal mature myeloid cells, but MDSCs. ² MDSCs do not belong to any subpopulation of bone marrow cells, but have immunosuppressive function. Phenotypically, MDSCs in humans usually express CD11b and myeloid molecular marker CD33, but lack Human Leukocyte Antigen–antigen D Related (HLA-DR).^3,4

MDSCs from tumor-bearing hosts can suppress natural killer (NK) cells and antigen-specific T-cell-mediated immunity mainly by producing highly concentrated arginase-1 (ARG-1), inducible nitric oxide synthase (iNOS), nitric oxide (NO), and reactive oxygen species (ROS) group. Furthermore, MDSCs themselves can also produce matrix metalloproteinase (MMPS), vascular endothelial growth factor (VEGF), interleukin-1 (IL-1), interleukin-1 (IL-10), transforming growth factor (TGF), interleukin-6 (IL-6), and a variety of other cytokines, which can promote development and progression of inflammation and tumor.^5–8 In this study, we focused on investigating the frequency of MDSCs in the peripheral blood of CC patients and explored their clinical implications.

Materials and methods

Patients

A total of 80 CC patients (range 32–68 years, mean 48.5 years), 30 patients with cervical intraepithelial neoplastic (CIN; range = 30–63 years, mean = 46 years), and 30 healthy donors (HDs; range = 24–80 years, mean = 37 years) were enrolled from the First Affiliated Hospital of Soochow University from April 2014 to June 2015. All patients had been histologically diagnosed by preoperative endoscopy biopsy and postoperative biopsy and had not received any treatment prior to the study. The clinical stages were determined according to FIGO standard. The characteristic features of all patients are shown in Table 1. Written informed consent was obtained from each subject or a legally authorized representative before blood sampling. The study was approved by the local ethics committee.

Table 1.

Analysis of MDSC in CC subjects.

	Number	p
Age (years)
<50	42	0.656
≥50	38
FIGO stage
I	58	0.026
II	22
Grade
Well/moderate	31	0.802
Poor	49
Lymph node
Positive	9	0.074
Negative	71
Histopathology
Squamous	73	0.576
Adenocarcinoma	7

FIGO: International Federation of Gynecology and Obstetrics; MDSC: myeloid-derived suppressor cell; CC: cervical carcinoma.

Reagents

The following antibodies (mAb) were used to identify MDSC subsets by means of flow cytometer: fluorescein isothiocyanate (FITC)–conjugated CD33 (catalog number 11-0339), phycoerythrin (PE)-conjugated CD11b (catalog number 12-0118), and allophycocyanin (APC)-conjugated HLA-DR (catalog number 17-9956). All mAbs were purchased from eBiosciences (San Diego, CA, USA). An isotype control was employed in each test to adjust for background fluorescence. We performed fluorescence minus one (FMO) staining controls to establish the parameters for gating.

Staining and flow cytometry

In brief, 30 μL of ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood was stained with 5 μL of one of the antibodies as previously indicated. The samples were incubated for 30 min at 4°C in the dark. Afterward, the cells were lysed with the use of Optilyse C Lysis Solution (from Immotech SAS, MC, France) and incubated in a water bath at 37°C for 10 min. Subsequently, the tubes were washed with phosphate-buffered saline (PBS) solution twice and centrifuged at 1500 r/min for 5 min. The supernatant was discarded and the cell pellets were suspended in 500 μL of fluorescence-activated cell sorting (FACS) buffer and then analyzed in a Beckman Coulter flow cytometer (FC500; Fullerton, CA, USA). The prevalence of MDSCs was expressed by a ratio of CD33⁺CD11b⁺HLA-DR^– cells among CD33⁺CD11b⁺ cells. Analysis of the flow cytometric data was performed by the Beckman FACSuite software.

Statistical analysis

All statistical analyses were conducted with GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). Unpaired Student’s t-test (Mann–Whitney U test) was used to assess the differences between circulating MDSCs and clinical characteristics. The data are shown as mean ± standard deviation (SD). In all the analyses, the threshold for significance was p < 0.05.

Results

Frequency of circulating MDSCs is significantly elevated in cervical cancer patients

The proportion of circulating MDSCs was analyzed in peripheral blood among 80 CC patients, 30 CIN patients, and 30 HD (Figure 1). Neither patients nor healthy volunteers revealed any statistical significance with regard to age (p > 0.05). The frequency of CD33⁺CD11b⁺HLA-DR^-MDSCs was significantly higher in CC patients (12.34% ± 1.08%) than that in CIN (3.92% ± 0.49%), and HD (2.18% ± 0.27%). Furthermore, the proportion of MDSCs was also elevated in CIN patients compared to HD (Figure 2).

Figure 1.

Flow cytometric evaluation of CD33, CD11b, and HLA-DR in patients with CC (a), CIN (b), and HD (c). An example of representative dot plots is shown for each subgroup. Gates were set based on negative controls. Numbers represent the percentages from the original populations gated.

Figure 2.

Scatter plot of the percentage of CD33⁺CD11b⁺ HLA-DR⁻ cell among CD33⁺CD11b⁺ cell in the blood of CC, CIN patients, and HD. Bar represents median in each group (*p = 0.0017, **p = 0.0062, ***p = 0.0029).

Increase in circulating MDSCs correlated positively with FIGO stage in CC patients

Afterwards, we set to investigate the correlations between the proportion of MDSCs and the clinical characteristics of CC patients. The frequency of MDSCs was elevated as stage increased from FIGO I to II (p = 0.026). No difference in frequency of MDSCs was observed in context of age, lymph node metastasis, grade of differentiation, or histopathologic type (Table 1).

Discussion

Progress in immunology has highlighted the importance of the immune system in cancer prevention and prognosis. Tumor cells can escape from attack by the immune system through various mechanisms, such as immune evasion, immune suppression, or both. Over the past several years, a large number of reviews have described mechanisms of immune evasion in cancer.^9,10 Although specific information on immune escape mechanisms is rare, recent findings indicate that tumor-induced mechanisms of immune suppression are the main reasons for bad prognosis and the base of immunotherapy.^11–13 Immune suppression is mediated by both soluble factors, such as prostaglandins, TGF-β, and IL-10, and suppressor cells including MDSCs.

MDSCs are important tumor-induced immune suppression cells which have gained great attention in recent years. MDSCs represent a group of immature cells that are morphologically, phenotypically, and functionally heterogeneous and play an important role in cancer immune suppression. Expansion of MDSCs has been described both in patients with pancreatic cancer, ¹⁴ gastric cancer, ⁹ and in mice bearing carcinomas such as Lewis lung carcinoma, ¹⁵ Ehrlich carcinoma, mammary adenocarcinoma, ¹⁶ chemically induced fibrosarcoma, ¹⁷ and colon carcinoma.^18–20 In our study, we reached the similar conclusion that circulating MDSCs were significantly elevated in cervical cancer. It was suggested that MDSCs expansion may be a feature indicative of suppressive functions and a representation of functional state within the tumor environment and can be further exploited as a diagnostic biomarker.

MDSCs were also reported in chronic inflammation. These conditions can lead to the production and accumulation of MDSCs in lymph nodes, spleen, blood, and bone marrow. Cervical cancer is frequently associated with human papillomavirus (HPV) infection and chronic inflammation. Although our data indicate that MDSCs expansion is increased in CIN, we found a clear difference in number of MDSCs among different stages of CC, which suggested that it may act as an indicator of disease progression in cervical cancer instead of HPV infection. Notably, this also supported the role of MDSCs in the development of CC and the fact that increase in MDSCs might be a risk of HPV-associated CC.

Several limitations should be noted here. We defined MDSCs as CD33⁺CD11b⁺HLA-DR⁻ without further characterization of granulocytic or monocytic subsets. Additionally, all blood samples were from patients who were in the stage eligible for surgery. Thus, we require larger and more accurate studies in the future. Taken together, MDSCs may become potent biomarkers of cervical cancer, especially early stages of this disease, which may be very useful for further clinical trials of MDSC-targeting drugs and vaccines.