Myeloid-Derived Suppressor Cells as Therapeutic Targets in Uterine Cervical and Endometrial Cancers

Uterine cervical and endometrial cancers are the two most common gynecological malignancies. As demonstrated in other types of solid malignancies, an increased number of circulating or tumor-infiltrating myeloid-derived suppressor cells (MDSCs) have also been observed in uterine cervical and endometrial cancers, and increased MDSCs are associated with an advanced stage, a short survival, or a poor response to chemotherapy or radiotherapy. In murine models of uterine cervical and endometrial cancers, MDSCs have been shown to play important roles in the progression of cancer. In this review, we have introduced the definition of MDSCs and their functions, discussed the roles of MDSCs in uterine cervical and endometrial cancer progression, and reviewed treatment strategies targeting MDSCs, which may exhibit growth-inhibitory effects and enhance the efficacy of existing anticancer treatments.


Introduction
Uterine cervical and endometrial cancers are the two most common gynecological malignancies. In the United States, 13,800 and 65,620 new cases of cervical and endometrial cancers, respectively, were reported in 2020 [1]. Although surgery followed by tailored adjuvant treatment is potentially curative, a considerable number of patients develop recurrence and die due to disease progression; 4290 and 12,590 deaths due to cervical and endometrial cancers, respectively, were reported in 2020 in the United States [1].
Cervical cancer has been considered an immunogenic tumor, as it is induced by persistent infection with human papillomavirus. Due to the existence of polymerase epsilon-ultramutated and microsatellite instability-hypermutated tumors, endometrial cancer has also been considered immunogenic and a reasonable candidate for active and/or passive immunotherapy [2]. Although immunotherapy (such as that with the programmed death [PD]-1 antibody pembrolizumab) has recently become a viable treatment for cervical and endometrial cancers, it has limited clinical efficacy.
Suppression of tumor immune surveillance is a main mechanism that prevents the destruction of tumor cells by the immune system and limits the efficacy of existing cancer treatments, including radiotherapy, chemotherapy, and immunotherapy [3]. Myeloidderived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells (IMCs) that play a central role in suppressing antitumor immunity. Additionally, MDSCs can directly stimulate tumor cell proliferation, metastasis, and angiogenesis [4]. As all of these can lead to tumor progression during radiotherapy or chemotherapy and limit the potency of current immunotherapy that targets cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or PD-ligand 1 (PD-L1)/PD-1 [5], MDSCs are considered promising therapeutic targets and predictive biomarkers of treatment outcomes in patients with solid malignancies, including gynecological cancers [4,6].
Under normal circumstances, IMCs differentiate into macrophages, neutrophils, and DCs. However, under pathological conditions such as infection, inflammation, or cancer, the differentiation of IMCs is impaired, leading to the formation of MDSCs [4,8]. The development of MDSCs is a complex phenomenon consisting of increased production of IMCs in the bone marrow, inhibition of the terminal differentiation of IMCs, and pathological activation of MDSCs. Multiple factors secreted from cancer or stromal cells, such as macrophage colony-stimulating factor, granulocyte colony-stimulating factor (G-CSF), granulocyte monocyte colony-stimulating factor, vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha, prostaglandin E2 (PGE2), interleukins (IL-1β, IL-10, IL-4, and IL-6), and noncoding RNAs (microRNAs and long noncoding RNAs) are involved in these processes [4,8,10]. In addition to these, recent investigations have suggested that tumor-derived exosomes are involved in the development of MDSCs through the communication with bone marrow cells [11].

Effect of Cancer Treatment on Tumor-Infiltrating MDSC
Radiotherapy and surgery have been curative treatment options in patients with uterine cervical or endometrial cancer. Recent investigations have suggested that radiotherapy has two opposite effects on MDSC recruitment into TME: conventional fractionated radiotherapy increases MDSCs, while ablative hypofractionated radiotherapy decreases MDSCs. In a mouse model of prostate cancer, a fractionated radiotherapy (3 Gy × 5) has been shown to increase MDSC in the tumor, spleen, or lymph nodes via the production of colony stimulating factor 1 [14]. On the other hand, in mice models of colon tumors, a single, high-dose irradiation (30 Gy) has been shown to reduce MDSC infiltration into the TME [15]. This high dose is at the upper end used clinically to treat advanced or metastatic colorectal, liver, and non-small cell lung tumors. Although fractionated radiotherapy has been employed in the treatment, so far, no studies have investigated the effect of radiotherapy on MDSC recruitment in uterine endometrial and cervical cancer.

Immunosuppressive Functions of MDSCs
MDSCs suppress T cells in both antigen-specific and antigen-nonspecific ways by utilizing several mechanisms. The most prominent factors include arginase-1 (Arg-1), nitric oxide (NO), and reactive oxygen species (ROS). Of these, ROS is responsible for antigen-specific suppression that requires close contact of MDSCs and T cells, as ROS are unstable and active only for a very short period. In contrast, NO and Arg-1 that have relatively longer half-life, are responsible for antigen-nonspecific suppression.
Both PMN-and M-MDSCs produce Arg-1 (Figure 1), which causes the removal of L-arginine, an essential amino acid for T cell differentiation, from the TME. The depletion of L-arginine subsequently causes the downregulation of CD247 (the ζ-chain of the T cell receptor) expression in T cells. As CD247 is a subunit of the natural killer (NK) receptors NKp46, NKp30, and TcγIII in NK cells, the depletion of L-arginine leads to the inhibition of T cell and NK cell proliferation [4,8].
PMN-MDSCs have increased NADPH oxidase activity and produce large amounts of ROS, which lead to the production of peroxynitrite (PNT). As ROS and PNT are unstable and have very short half-life, PMN-MDSCs require close cell-to-cell contact to exert their effect on T cells. During the close interaction between MDSCs and CD8+ T cells via antigen recognition, PNT causes nitration and conformational changes of the TCR complex. CD8+ T cells consequently lose their binding ability to peptide-MHC class I complex and become nonresponsive to specific peptide presented by tumor cells. PNT may also induce nitration and structural changes of MHC class I molecules on tumor cells, leading to reduced capacity of antigenic peptide binding and impairment of recognition of tumor cells by CD8+ T cells [16,17]. M-MDSCs show low ROS production; however, they express high levels of iNOS, which produces NO that nitrates signaling molecules downstream of FcgRIIIA, resulting in the inhibition of the activities of T cells and NK cells [4,8]. NO also downregulates JAK3/STAT5 signaling, which is crucial for the survival of T cells and NK cells, leading to apoptosis or diminished interferon response [18]. Owing to the fact that NO has a much longer half-life than ROS, it is believed that M-MDSCs have higher suppressive activity than PMN-MDSCs when assessed on a per-cell basis [19].
Other roles of MDSCs in immune suppression include the production of indoleamine-2,3-dioxygenase, which decreases tryptophan levels in the TME, leading to the induction of cell cycle arrest or apoptosis of T cells. MDSCs can also produce immunosuppressive cytokines such as IL-10 and TGF-β, affect NK cell function, and induce regulatory T cell (Treg) expansion [4]. Lastly, MDSCs have an increased expression of PD-L1, which leads to the downregulation of T cell function via engagement of cell surface PD-1 [20].

MDSCs in Patients with Solid Cancers
An increased number of circulating MDSCs has been detected in various patients with cancers. In most cancers, including lung, breast, colon, renal, head and neck, and pancreatic cancers, PMN-MDSCs represent the major population of MDSCs. However, M-MDSCs show low ROS production; however, they express high levels of iNOS, which produces NO that nitrates signaling molecules downstream of FcgRIIIA, resulting in the inhibition of the activities of T cells and NK cells [4,8]. NO also downregulates JAK3/STAT5 signaling, which is crucial for the survival of T cells and NK cells, leading to apoptosis or diminished interferon response [18]. Owing to the fact that NO has a much longer half-life than ROS, it is believed that M-MDSCs have higher suppressive activity than PMN-MDSCs when assessed on a per-cell basis [19].
Other roles of MDSCs in immune suppression include the production of indoleamine-2,3-dioxygenase, which decreases tryptophan levels in the TME, leading to the induction of cell cycle arrest or apoptosis of T cells. MDSCs can also produce immunosuppressive cytokines such as IL-10 and TGF-β, affect NK cell function, and induce regulatory T cell (Treg) expansion [4]. Lastly, MDSCs have an increased expression of PD-L1, which leads to the downregulation of T cell function via engagement of cell surface PD-1 [20].

MDSCs in Patients with Solid Cancers
An increased number of circulating MDSCs has been detected in various patients with cancers. In most cancers, including lung, breast, colon, renal, head and neck, and pancreatic cancers, PMN-MDSCs represent the major population of MDSCs. However, patients with melanoma, multiple myeloma, and prostate cancer have a substantially higher proportion of M-MDSCs in the peripheral blood than that of PMN-MDSCs [25].

Author/Year/Type of Cancer Findings from In Vitro/In Vivo Studies of Uterine Cervical and Endometrial Cancer
Mabuchi, S., et al. 2014 [21] Cervical cancer MDSC inhibited the activity of CD8 + T cells and stimulated angiogenesis. MDSCs were responsible for the rapidly progressive and radioresistant nature of cervical cancer. The administration of anti-Gr-1-neutralizing antibody or the depletion of MDSCs by splenectomy inhibited tumor growth and enhanced radiosensitivity in cervical cancer.
Consistent with the findings of in vitro and in vivo experiments, an increase in the number of circulating or tumor-infiltrating MDSCs was associated with a decrease in the number of tumor-infiltrating CD8 + T cells [29,31,35]. An increased number of MDSCs was also associated with unfavorable clinicopathological parameters, including advanced clinical stage [35], visceral or lymph node metastases [22,35,38], deep stromal invasion [35], poor sensitivity to anticancer treatments (radiotherapy or chemotherapy) [21,28,29], high recurrence rate [35], and short survival [21,28,29,31] in patients with uterine cervical and endometrial cancers (Table 2). Moreover, it has been recently demonstrated that MDSC-mediated premetastatic niche formation in the lymph nodes induces 18F-fluorodeoxyglucose (FDG) uptake during FDG-positron emission tomography/computed tomography and causes false-positive detection of nodal metastasis [23].
However, abovementioned studies have limitations that warrant further investigationsmall sample size, inconsistent histological subtypes, use of inconsistent MDSC surface markers, and limited clinical information.

Pregnant Condition
During pregnancy, maternal plasma levels of estradiol increase up to 100-fold compared to the nonpregnant status. A previous study demonstrated that the exogenous E2 treatment stimulated the mobilization of MDSC from bone marrow and directly augmented their suppressive activities, leading to the progression of cervical cancer [33]. Consistent with this, a significantly increased number of tumor-infiltrating MDSCs was observed in pregnant women with cervical cancer or in pregnant mice bearing human cervical cancer, which can be attributed to the increased E2 levels during pregnancy [33]. These results indicate that E2 facilitates the progression of female cancers, including cervical cancer, under pregnant condition by inducing MDSC.

Rationale for Targeting MDSCs in Cancer Treatment
Accumulating preclinical evidence has shown that MDSC inhibition has therapeutic efficacy against various solid malignancies as a monotherapy or as in combination with existing anticancer treatments [4,6]. Although conventional fractionated radiotherapy increases MDSCs, and ablative hypofractionated radiotherapy decreases MDSCs, inhibition of MDSCs has consistently enhanced the antitumor effect of radiotherapy in preclinical studies, regardless of radiotherapy scheme [40]. Moreover, recent preclinical investigations have suggested that the efficacy of ICIs can be enhanced by MDSC inhibition [26]. As some ICIs have been approved or are being tested in clinical trials in patients with uterine cervical and endometrial cancers, MDSC inhibition can be a promising strategy to extend the benefits of chemotherapy, radiotherapy, or immunotherapy in such patients.

Preclinical Investigation of MDSC-Targeting Therapies in Uterine Cancer
Various MDSC-targeting strategies have been evaluated in murine models of uterine cervical and endometrial cancers (Table 2), such as anti-Gr-1 antibody [22,29,31], anti-IL-6 antibody [32], COX-2 inhibitor [29], STAT3 inhibition [32], and splenectomy [21,28]. They demonstrated significant activity in reducing the number of MDSCs from the TME or inhibiting their suppressive activity against CD8 + T cells, which leads to the inhibition of tumor growth or metastasis [21,22,29,33], prolongation of survival [31], attenuation of the growth-promoting effect of E2 [33], or enhancement of the efficacies of existing anticancer treatments, including cisplatin therapy [28] or radiotherapy [21]. In addition to the inhibition of the immunosuppressive activity of MDSCs, depletion of MDSCs has been shown to successfully attenuate premetastatic niche formation and inhibit visceral organ metastasis in uterine cervical and endometrial cancers [22]. Moreover, MDSC depletion has been shown to attenuate the induction of cancer stem-like cells and enhance chemosensitivity in uterine cervical [30] and endometrial cancer [29]. In contrast, MDSC increment in TME using either G-CSF or swainsonine (an alpha-mannosidase inhibitor) has been shown to stimulate the progression of uterine cervical or endometrial cancer [21,22,28,29,33,34]. Collectively, these results strongly indicate the significance of MDSCs as therapeutic targets in this patient population.

Predictive Biomarkers for MDSC-Targeting Therapy
In previous investigations including uterine cervical and endometrial cancer patients, an increased number of MDSCs were observed only in those who displayed tumor-related leukocytosis (TRL) [21,22,28]. In addition, recently, a ribonucleic acid sequencing analysis revealed that the MDSC signature in patients with cervical cancer in the Cancer Genome Atlas database is associated with leukocytosis [39]. These findings are partially in line with previous studies showing that uterine cervical or endometrial cancer patients exhibiting TRL, neutrophilia, increased neutrophil-to-lymphocyte ratio, or those with tumor expressing G-CSF are associated with decreased survival rate or resistance to radiotherapy or chemotherapy [21,28,29]. Moreover, an increased number of MDSCs were detected in patients with uterine cervical and endometrial cancers whose tumors overexpressed G-CSF [21,22,28]. Consistent with these findings, MDSC-targeting treatments, such as anti-Gr-1 antibody treatment or splenectomy, had significant antitumor effects in mouse models of G-CSF-expressing, TRL-positive cervical and endometrial cancers that exhibited increased MDSC [21,28,29]. These results strongly indicate that leukocyte count, neutrophil count, neutrophil-to-lymphocyte ratio or tumor G-CSF expression, which can be easily assessed by peripheral blood cell count or immunohistochemistry, can be used as a biomarker to predict the sensitivity of MDSC-targeting treatments.
Moreover, recently, it was found that an increased number of MDSCs was associated with increased bone marrow FDG uptake in patients with uterine cervical cancer [31]. Thus, by evaluating bone marrow FDG uptake, we might be able to identify a group of patients with increased MDSC who are candidates for MDSC-targeting agents. To the best of our knowledge, these are the only studies that have attempted to identify biomarkers for MDSC-targeting therapy.
Theoretically, other tumor-derived substances (including cytokines, chemokines, noncoding RNAs, or exosomes) that stimulate the production of MDSC can also be predictive biomarkers. We hope the clinical utility of such biomarkers be evaluated preclinically and clinically in the future, which would enable physicians to identify patients who might benefit from MDSC-targeting therapy.

Clinical Trials Targeting MDSCs in Patients with Solid Cancers
Although various MDSC-targeting strategies have been proposed in preclinical investigations (Table 3), only a few of them are tested in ongoing clinical trials. These agents include capecitabine, gemcitabine, ibrutinib (Bruton tyrosine kinase inhibitor), IPI-549 (PI3K inhibitor), and SX-682 (CXCR1/2 inhibitor) ( Table 3). The activity of MDSC inhibition has also been tested in a setting of combination therapy to establish a strategy to overcome resistance to ICIs. The safety, efficacy, and immunobiological effects of the CXCR4 antagonist BL-8040 (motixafortide) with pembrolizumab have recently been evaluated in a phase IIa study of metastatic pancreatic ductal adenocarcinoma (PDAC) [73]. In the study, BL-8040 increased the number of tumor-infiltrating CD8 + effector T cells and decreased the number of MDSCs in PDAC tumors, suggesting that CXCR4 inhibition may enhance the therapeutic efficacy of PD-1 blockade in patients with PDAC and warrants confirmation in subsequent randomized trials.
Activating mutation of PIK3CA and the resulting activation of PI3K is frequently observed in both uterine endometrial and cervical cancer, and, thus, PI3K-inhibition has been regarded as promising treatment [77]. Moreover, as CXCR2 has been shown to be involved in the MDSC recruitment into TME of uterine endometrial and cervical cancer [22,29], we hope that the activity of IPI-549 or SX-682 will be evaluated in this patient population. Positive clinical data on MDSC-targeting therapies are anticipated in the future.

Conclusions
An increased number of MDSCs is observed in patients with uterine cervical and endometrial cancers. MDSCs play a significant role in disease progression. To inhibit their tumor-promoting effects, the efficacy of MDSC-targeting therapies (either as monotherapies or in combination with existing treatments) against uterine cervical and endometrial cancers is currently being evaluated preclinically. We believe that increasing our understanding of MDSC biology will aid in the development of optimal MDSC-targeting therapies for patients with uterine cervical and endometrial cancers.

Conflicts of Interest:
The authors declare that they have no conflict of interest.