Clinical relevance of systemic monocytic-MDSCs in patients with metastatic breast cancer

Breast cancer is the most common form of malignancy in women [1] and although survival has improved, due to enhanced diagnostics and adjuvant therapies, breast cancer can recur after many years [2, 3]. When there is progression to metastatic breast cancer (MBC), there is in most cases no curable treatment and the 5-year survival rate is merely 20–30% [2, 4].

Over the past decades, the role of the immune system in cancer development and progression has gained increased attention. Although the exact mechanisms are still being investigated, it is known that some immune cells are able to recognize and eliminate tumor cells, a mechanism called immunosurveillance [5, 6]. In order for cancer to develop and progress, malignant cells must escape this surveillance and hijack normal physiological processes typically involved in tissue remodeling, angiogenesis, and cell regeneration [6-8]. One mechanism by which tumors can elude immunosurveillance is through myeloid-derived suppressor cells (MDSCs). MDSCs are a heterogeneous group of highly immunosuppressive cells that accumulate during severe pathological conditions such as sepsis, trauma and cancer [9-11]. Physiologically, they function to dampen excessive immune responses and limit or promote repair of tissue damage [9, 10]. In cancer, however, the net result would be tumor persistence and progression due to the production of immunosuppressive and pro-angiogenic cytokines and inhibition of anti-tumor T cell responses [10, 12]. The role of MDSCs in breast cancer patients remains relatively unexplored.

Depending on cell surface antigen expression, MDSCs have historically been roughly divided into two groups; granulocytic-MDSCs (G-MDSCs; CD11b⁺CD15⁺CD33⁺Lin⁻HLA-DR^low/−) and monocytic-MDSCs (Mo-MDSCs; CD11b⁺CD14⁺CD33⁺HLA-DR^low/−Co-receptor^low/−) [12]. Generally, MDSCs correlate with tumor progression, angiogenesis and poor prognosis in different cancer forms [13-15]. Accordingly, in breast cancer patients, MDSCs are enriched in the peripheral blood and has been proposed to correlate with clinical stage and metastatic burden [16-19]. In patients with stage IV disease, higher levels of MDSCs have also been associated with reduced overall survival [19, 20]. However, the surface definition of MDSCs has varied substantially between studies and many do not distinguish between G-MDSCs and Mo-MDSCs. A recent study proposes that G-MDSCs (CD11b⁺CD33⁺HLA-DR⁻CD15⁺ cells) may correlate with triple negative breast cancer and inversely with complete pathologic response after neo-adjuvant chemotherapy [21]. Historically, Mo-MDSCs have been less studied. Enrichment of circulating Mo-MDSCs has previously been identified in patients with melanoma [22, 23], prostate cancer [24], glioblastoma [25], non-Hodgkin lymphoma [26], and bladder cancer [27]. Depending on the type of malignancy, amongst other factors, the presence of Mo-MDSCs correlated with more active [22] or aggressive disease [26] as well as increased tumor size and grade [27]. Whether Mo-MDSCs may provide prognostic or predictive value in patients with breast cancer is currently unknown.

We were recently first to show that Mo-MDSCs are enriched in the peripheral blood of breast cancer patients, primarily in patients with metastatic disease [28]. Here, we aim to further delineate the role of Mo-MDSCs in patients with MBC. By analyzing the frequency of CD14⁺HLA-DR^low/−Co-receptor^low/− Mo-MDSCs in the peripheral blood of patients with MBC we show that Mo-MDSCs significantly correlate to estrogen receptor (ER)-negative disease, liver- and bone metastases, and de novo MBC (metastatic disease at initial diagnosis). Furthermore, in patients with distant recurrent MBC, high levels of Mo-MDSCs were associated with more circulating tumor cells (CTCs), more metastatic sites and disease progression at 3 month’s radiology assessment. In patients with ER-positive distant recurrent MBC, there was a trend towards an association between high levels of Mo-MDSCs and worse overall survival (P = 0.053). Altogether this indicates that Mo-MDSCs may be a potential biomarker related to a more aggressive disease and worse outcome in patients with MBC.

In the present study we evaluated the role of systemic Mo-MDSCs in patients with newly diagnosed MBC. A high level of Mo-MDSCs was significantly correlated with ER-negative primary tumors, as well as with liver- and bone metastases, which is in accordance with our previous study, comprising the first 23 patients enrolled in this study [28]. This is also in line with a previous study proposing that circulating G-MDSCs may be more predominant in triple-negative breast cancer compared to other breast cancer subtypes [21]. ER-negative breast cancer has been associated with a stronger inflammatory response, where more inflammatory cytokines (e.g., IL-8 and IL-6) have been observed in tumors of patients with ER-negative breast cancer or in ER-negative breast cancer cell-lines [33, 34]. Pro-inflammatory stimulus is one important component of MDSC generation and accumulation [12]. The higher inflammatory activity associated with ER-negative breast cancer may consequently promote the accumulation of Mo-MDSCs.

Interestingly, the vast majority of patients with de novo MBC displayed high levels of Mo-MDSCs. De novo MBC differs from other breast cancer forms in terms of both characteristics and treatment strategies. Compared to early breast cancer, de novo MBC is a disease with systemic involvement already at first diagnosis. Though, unlike patients with distant recurrent MBC, where the primary tumor had been resected at initial diagnosis, the primary tumor is still present in the subgroup of patients with de novo MBC. This implies that the primary tumor might impact the levels of Mo-MDSCs and that different biological mechanisms may be at play in patients with de novo MBC and distant recurrent MBC. Indeed, the levels of MDSCs decrease after removing the primary tumor in breast cancer mouse models and patients with MBC [19, 35]. Studies have shown that cytokines such as GM-CSF, produced by tumor cells, may induce expansion of MDSCs [12, 13]. It is also interesting to note that patients with early, localized, breast cancer generally display a modest enrichment in circulating Mo-MDSCs as compared to patients with locoregional recurrence or MBC [28]. The enrichment of Mo-MDSCs may thus be related to the metastatic or immunoregulatory switch during the transition to a more systemic disease [36]. This is supported by a study proposing that Mo-MDSCs themselves may induce a pro-metastatic phenotype of 4T1 murine breast tumor cells [37].

The strong relationship between Mo-MDSCs and liver- and bone metastases, as well as the observed associations with more CTCs, more metastatic sites, disease progression, and shorter progression-free and overall survival, indicate that Mo-MDSCs are related to a more aggressive metastatic disease and worse prognosis. This association was especially apparent in patients with distant recurrent MBC and is in line with previous studies where MDSCs in general are associated with clinical stage, metastatic burden and poor overall survival in breast cancer patients [17, 19]. The potential correlation between Mo-MDSCs and CTCs was hitherto unexplored in breast cancer patients, although it has been proposed that MDSCs (CD11b⁺CD33⁺Lin⁻HLA-DR⁻ cells) may correlate with CTCs [16]. Interestingly, CTCs have been suggested to preferably cluster together with leukocytes and that these clusters might be associated with worse prognosis in breast cancer patients [38]. The specific leukocyte population(s) was until recently uncharacterized. However, MDSC-like pro-tumor N2 neutrophils were recently shown to support the metastatic potential of CTCs in patients with metastatic breast cancer [39]. Whether also Mo-MDSCs, specifically, may play a role will be of interest to delineate in the future.

Tumor metastasis is a complex process and the outcome depends on both tumor properties and the host’s response, accounting for the fact that only 0.01% of the malignant cells are estimated to succeed in establishing micro-metastases [40]. The process of establishing new metastatic sites is not fully understood, but has been proposed to involve formation of pre-metastatic niches. Intriguingly, several MDSC-associated mediators (such as S100A8/A9, MMP9 and IL-1β) have been identified as important in establishment of pre-metastatic niches [40, 41]. Accordingly, mouse models of malignant melanoma have shown that both Mo-MDSCs and G-MDSCs are found in pre-metastatic sites weeks before the actual metastasis could be detected [41]. In fact, Mo-MDSC-derived cytokines have been proposed to facilitate adhesion of CTCs to the endothelium in the pre-metastatic organ [41]. How and why Mo-MDSCs would localize to pre-metastatic niches is not fully characterized, nor is it known whether this also occurs in human patients [42]. It is tempting to speculate that correlation between high levels of circulating Mo-MDSCs and specific metastatic sites, predominantly liver and bone, may be related to establishment of pre-metastatic niches by MDSCs [43]. This would also be in line with our hypothesis that enrichment of Mo-MDSCs is related to the metastatic and immunoregulatory switch in these patients. Further we show that high Mo-MDSC levels may be associated with impaired survival. This seems to be particularly relevant in patients with ER-positive MBC, a patient group with generally better prognosis compared to ER-negative MBC, implying that Mo-MDSC levels could be a prognostic factor in this subgroup of patients representing a more aggressive disease. Thus, although the inflammatory environment in ER-negative tumors promotes accumulation of Mo-MDSCs to a greater extent, the impact of Mo-MDSCs on survival may be limited due to the overall worse prognosis in this subgroup of patients.

Immune therapies are currently revolutionizing the field of oncology and check point inhibitors are being implemented as systemic therapies for different tumor types including breast cancer [44, 45]. For MBC the PD-L1 inhibitor atezolizumab was recently shown to improve PFS and OS in patients with metastatic TNBC [46]. Mo-MDSCs are cells with capability to modulate the immune response in MBC patients and could hence be a potential target for immune therapies in the future. Our study is limited by the sample size, and some analyses thus fail to reach statistical significance. Despite this, strong trends are observed shedding light on the clinical impact of systemic Mo-MDSCs in breast cancer patients. An extended knowledge of the systemic immune response is, therefore, extremely relevant in understanding what patients will benefit most from immune therapies.