Phenotypic Analysis of Circulating Myeloid Derived Suppressor Cells and Their Subpopulations in Egyptian Females with Breast Cancer: A Single-Centre Case-Control Study

Background: Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with suppressive function that has been thoroughly documented in the setting of cancer. Our purpose was to evaluate levels of MDSC and their subsets in a cohort of Egyptian patients with breast cancer. Methods: Evaluation of peripheral blood total MDSC and its subset was done using multicolor flowcytometry in 30 malignant, 10 benign breast tumor patients and 10 healthy control females. Results: BC patients had higher total MDSC levels compared to controls (p= 0.01) particularly the Monocytic MDSC (M-MDSC) and abnormal MDSC subsets (p = 0.001 and p <0.001, respectively). A tumor size > 2 cm exhibited significantly higher granulocytic MDSCs (G-MDSCs) compared to tumor size < 2 cm (p= 0.02) whereas abnormal MDSCs were significantly higher in patients with a tumor size < 2 cm (p = 0.037). Conclusion: MDSC and its subsets can be used as a prognostic marker of tumor size as well as a potential targets for treatment in breast cancer patients.


Introduction
Breast cancer (BC) is the most frequently diagnosed malignancy worldwide and is the second leading cause of cancer-related deaths in females [1].Remarkably, more than half of BC diagnoses and two-thirds of BC related deaths were reported in low-and middle-income countries in 2020 [2].In Egypt, BC accounts for 38.8% of cancers in females, with the estimated number of BC cases nearly 22,700 in 2020 and is forecasted to rise exponentially over time [3].Several factors contribute to BC heterogenicity including variations in genomic, epigenomic, transcriptomic, and proteomic characteristics RESEARCH ARTICLE Phenotypic Analysis of Circulating Myeloid Derived Suppressor Cells and Their Subpopulations in Egyptian Females with Breast Cancer: A Single-Centre Case-Control Study immunoediting by innate and adaptive immune cells that together form the BC Immune Microenvironment (BCIM) is a vital determinant of tumor progression [6].Surprisingly, immunosuppressive cells, especially myeloid-derived suppressor cells (MDSC), hinder the anticancer immune response generated by immunostimulating cells like macrophages, lymphocytes, natural killer cells, and innate lymphoid cells [7].
Despite the fact that treatment alternatives have improved BC patients' outcomes, many still develop metastatic disease, which is still challenging to cure [10,11].One study provided scientific evidence about the benefits of beetroot extract when given with neoadjuvant regimen in cases of breast cancer which improved the pathological response of cancer cells by increasing the immune response in the tumor environment, especially increasing CD8 T cells and decreasing MDSC [12].
Several studies suggested that MDSC play a detrimental role in BC progression [5].Research reveals the association between raised levels of MDSC in peripheral blood of BC patients and the disease prognosis, since it's associated with advanced stages, higher tumor burden, and lower progression-free survival and overall survival, and lesser response to chemotherapy, radiotherapy, immunotherapy, and targeted therapies [13].Interestingly, clinical trials combining immunotherapy with other types of therapies in BC to target MDSC are currently underway [14].
To the best of our knowledge, there is a paucity of information concerning the relationship of MDSC in Egyptian patients with BC [15,16], hence, we aimed to determine the frequency of different MDSC subsets in a cohort of female patients with BC compared to subjects with benign breast lesions as well as healthy controls.We also aimed to investigate the potential association of MDSC and their subsets with tumor size, distinct tumor histopathology, hormonal markers, and intake of neoadjuvant therapy.

Ethical approval
The collection of blood for PBMC isolation and MDSC analysis was approved by the medical ethics committee of Faculty of Medicine, Cairo University (approval number: N-143-2018).All procedures were carried out in accordance with the Helsinki Declaration's ethical principles.The study purpose was clarified to all participants prior to study enrollment.Prior to participating in the study, each subject provided informed consent.

Study population
We enrolled 30 patients that had histologically confirmed BC that was treated in the Department of General Surgery, Cairo University, between March 2019 and November 2019.All patients were staged in accordance with National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology: Breast Cancer Screening and Diagnosis [17].Fifty percent of patients received preoperative anticancer treatment.Ten patients with benign breast lesions were enrolled.Exclusion criteria for participation were presence of active infection(s), coexistent malignancies or inflammatory or autoimmune disease(s) at the time of sampling.The control group included 10 healthy volunteers without chronic disorders.All the study participants were females.

Blood sampling and isolation of peripheral blood mononuclear cells (PBMNs)
Six mL of blood were collected into K2 ethylene diamine tetra acetic acid Vacutainers™ for the immediate isolation of PBMCs using the Ficoll density gradient centrifugation method (Ficoll-Hypaque; GE Healthcare Life Sciences, Chalfont, UK).After two washes in phosphate buffer saline, cells were assessed for viability by trypan blue exclusion.The number of viable cells present in the cell suspension ranged between 1.68 x 106 and 26 x 106 per ml.For freezing, Roswell Park Memorial Institute medium-1640 (RPMI; ThermoFisher Scientific) supplemented with 40% fetal bovine serum (FBS; Biowest) and 10% dimethyl sulfoxide was used, and cells were stored in liquid nitrogen immediately at −80 °C for analysis later.After collection of all samples which took around one-year, frozen PBMCs were thawed at 37°C in a water bath.Cells were washed twice in 3−5 ml pre-warmed RPMI and resuspended in 1 ml PBS.Cell viability was determined by trypan blue exclusion and all samples showed viability of more than 90%.
Data acquisition and analysis were performed on a FACSCanto 10 flow cytometer (BD Biosciences, San Jose, CA, USA) using BD FACS DIVATM software.The labelled cells were first gated based on their lack of expression of HLA-DR; was composed of HLA-DR− cells.The fraction of cells in this population that expressed the myeloid marker CD33 was then determined.Then within this population, the fraction of cells expressing CD15 and CD14 were evaluated.In the present study, MDSC were defined as DR-/low/CD33+.Moreover, MDSC subsets were determined as G-MDSC: CD15+/ CD14-, M-MDSC: CD14+/CD15-, e-MDSC: CD14-/ CD15-and abnormal MDSC: CD15+/CD14+ MDSC. Figure 1 presents flowcytometry scatter plot results, based on the gating strategy for one of the study participants.

Statistical studies
Analysis of data was done by IBM computer using statistical program for social science version 21.Description of quantitative variables as mean, standard deviation (SD), median and interquartile range (IQR) for quantitative variables.Description of qualitative variables as number and percent.Mann Whitney test was used instead of independent t-test to compare quantitative variables between two groups in non-parametric data (SD>30%mean).Comparison between quantitative variables between more than two categories done using one-way Anova test in noramally distributed data and cancer within 3 generations.3/30 (10%) patients had a previous history of BC.At the time of blood draw, fifteen patients (50%) were untreated, and 15 (50%) patients received neoadjuvant therapy prior to tumor resection in the form of chemotherapy (30%), hormonal therapy (13.3%), combined hormonal and chemotherapy (6.7%).Ten female patients with biopsy-proved breast benign tumors were also recruited.Their mean ages were 34.8 ± 9.8 (range, 28-60).The pattern of benign breast disease, fibroadenoma was the most common lesion constituting 6/10 (60%) cases followed by hamartoma in 2/10 (20%) cases, fibrocystic disease in 1/10 (10%) patient and phylloid in 1/10 (10%) patient.2/10 (20%) patients with benign tumors had a positive family history of BC.All ten patients had no previous history breast tumors.Control group was 10 age-and gender-matched healthy participants with no chronic disorders, randomly selected from the out-patient clinic with a mean age 48.1 ± 7.65 (range, 35-60) years.
The percentages of circulating MDSC total cells were significantly increased in patients with BC compared to controls (median= 1%, IQR= 0.7-1.3 vs. median= 0.3%, Kruskal wallis test in not normal distributed test.In both conditions pairwise comparison checked.Chi square test used to compare between qualitative variables and exact correction when cell contain count less than 5. P ≤0.05 was considered significant while p <0.01 was considered highly significant.
Notably, no differences between patients were observed regarding total MDSC and its subsets in relation to BC histopathology, lymph node involvement, distant metastasis, the presence of estrogen receptors, progesterone receptors or human epidermal growth factor receptor (HER2/neu).Patients who received neoadjuvant therapy compared to those who did not get neoadjuvant therapy prior to surgery did not differ in terms of levels of MDSC and its subsets (The data are summarized in electronic supplementary Tables S1-S8).

Discussion
MDSC have been implicated in sustaining progression of numerous malignancies including BC [18].We demonstrated that circulating total MDSC were significantly increased in patients with BC, but not in subjects with benign breast lesions, compared with the control group (p = 0.01).Notably, alteration in MDSC levels in BC was reported in several studies, but the results are inconsistent, with some researchers demonstrating elevated MDSC in peripheral blood of patients with BC [19,20], whereas another report demonstrated that tumor-infiltrating MDSC expansion was not reflected in the peripheral blood of BC patients [21].The lack of consistency of markers to identify MDSC and the clinical parameters of study groups may explain the discrepancies in the results among different studies.
As a draw back in the current study, the usage of thewed MDSCs may affect quality of the results [22] but as a compensation for that draw back viability testing done directly before using MDSCs in flowcytometry demonstrated that every sample used showed viability more than 90% as mentioned in the methodology section.
No significant differences in MDSC levels were observed among our patients with BC with regards to lymph nodes involvement, lymph vascular emboli and distant metastasis.Intriguingly, MDSC play a fundamental role in orchestrating immunosuppression within the TME via multiple mechanisms that dampen antitumor immunity and promote tumor progression including induction of a highly oxidative microenvironment [23], production of cytokines and immunosuppressive mediators [24], metabolite depletion, and expression of immune checkpoint molecules [25].Tumor cells recruit MDSC, T-regulatory cells, and M2 macrophages, to shape a pro-tumorigenic microenvironment [26].
Even though immunotherapies improved the outcome of many BC patients, nevertheless, it has been demonstrated that the immune context of the TME plays a key role in the development of resistance mechanisms to immunotherapy in BC [27,24].Currently, there are various immunotherapeutic strategies in BC to target MDSC, either decreasing their number or inhibiting their immunosuppressive functions [28].Interestingly, recent research revealed that MDSC depletion combined with HER2-targeted passive immunotherapy using monoclonal antibodies (mAbs) dramatically reduced tumor growth and increased tumor rejection in a murine early BC model [29].We observed a significant increase in circulating M-MDSC in our BC patients, but not in subjects with benign breast tumors, compared to controls (p = 0.001).Worth mentioning, earlier studies reported elevated circulating M-MDSC in patients with primary BC, loco-regional recurrence, metastasis to lymph nodes and visceral organs and in more advanced stage of the disease [30,31].In contrast to the aforementioned studies, Toor (2017) found no difference between M-MDSC levels in BC patients compared to controls [21].Remarkably, recent research demonstrated that elevated levels of M-MDSC in peripheral blood favours an immunosuppressive microenvironment that promotes metastasis and BC progression [32].
In line with earlier studies [33,34], no difference was found between our patients with BC or benign breast lesions and controls regarding G-MDSC.Nevertheless, significantly increased G-MDSC was found in our patients with positive family history of BC compared to patients without affected family members (p = 0.02).Moreover, we observed a significant increase in G-MDSC (p = 0.019) among our BC patients with tumor size > 2 cm.Worthmentioning, Toor (2017) demonstrated that expansion of tumor-infiltrating G-MDSC in BC was not reflected in peripheral blood.On the contrary, Safarzadeh (2019) [20] reported significantly elevated circulating G-MDSC in BC patients compared with controls.Remarkably, Mehmeti-Ajradini (2020) [35] presented evidence that G-MDSC were generated in patients with metastatic BC, as cells of the neutrophil lineage at a range of maturation stages [35], promoting BC growth and myeloid immune cell exclusion.
In the current work, e-MDSC were significantly decreased in patients with BC compared with subjects with benign tumors and controls (p <0.001), nonetheless, no significant difference was observed between patients with benign breast lesions and controls.In contrast to our findings, Yu (2013) demonstrated that e-MDSC were significantly increased in patients with BC compared with controls [33].The fact that their study included patients with invasive duct carcinoma unlike the current work, could be a plausible explanation, nevertheless, further studies are warranted to confirm this hypothesis.Despite the fact that e-MDSC have potent immunosuppressive capacity and play a vital role in carcinogenesis, nonetheless, the mechanism underlying their development in cancer remain undisclosed [36].
We found that abnormal MDSC were significantly increased in patients with BC compared to patients with benign breast lesions as well as controls (p <0.01), yet no significant difference was found between patients with benign tumors and the control group.In line with our findings, Safarzadeh (2019) [20] reported increased abnormal MDSC in patients with BC compared with healthy controls.Furthermore, we observed a significant decrease in abnormal MDSC (p = 0.037) among our BC patients with tumor size > 2 cm.
The results of the present work were in agreement with those of other reports, which demonstrated no differences regarding MDSC levels between BC patients with positive estrogen, progesterone or HER2/neu receptor subtypes compared to patients that were receptor-negative [33,20].There were no differences among our patients with BC with regards to MDSC levels in relation to BC history, tumor histopathology or Ki-67.There was no difference in MDSC levels between patients who received neoadjuvant therapy compared to those who didn't receive neoadjuvant therapy prior to surgery.
In conclusion, the present study showed that the frequencies of circulating total MDSC, M-MDSC and abnormal MDSC were significantly increased in an Egyptian cohort with BC.The frequency of G-MDSC positively correlated with tumor size and positive family history of BC, while abnormal MDSC negatively correlated with tumor size.The current study elaborated the importance of MDSCs in breast cancer follow up and prognosis.A deeper understanding of the myeloid derived suppressor cell populations' effects on cancer progression and response to therapies may contribute to the development of more effective treatments for BC.

Limitations of the study
1.The current work didn't study the MDSC in the tumor micro-environment which is important hence studies about whether peripheral blood MDSC really reflect their percentage in tumor microenvironment were controversial.
2. The relatively small number of the studied group.

Table 1 .
Comparison between Patient Groups with BC, benign Breast Tumors and Controls Regarding Circulating MDSC Levels.Data are presented as median and interquartile range.b , p-values < 0.05 were considered statistically significant.csignificant p value between breast cancer and control groups.dsignificant p value between breast cancer and benign breast tumors groups.Abbreviations: BC, breast cancer; e-MDSC, early-stage myeloid derived suppressor cells; G-MDSC, granulocytic myeloid derived suppressor cells; M-MDSC, monocytic myeloid derived suppressor cells; n, number.
a ,