Background
An estimated 90% of cancer-related deaths are associated with tumor metastases [
1,
2], highlighting the need for new and effective therapeutic strategies to treat metastatic disease. The contribution of host tissues to the development of metastatic tumor growth was first postulated by Stephen Paget in the late 1800s, with his “seed-and-soil” hypothesis [
3] suggesting that metastatic tumor cells (seeds) must enter suitable host tissues (soil) in order to grow into tumor metastases. More recent evidence suggests that primary tumors can “fertilize” the metastatic soil within some tissues to promote metastatic tumor growth. Localized microenvironments can form in metastatic target organs, consisting of supportive stromal cells, pro-tumorigenic proteins, and a variety of different bone marrow-derived cells. These “pre-metastatic niches” are thought to represent fertile regions of tissue that facilitate the subsequent invasion, survival, and proliferation of metastatic tumor cells [
4,
5]. Pre-metastatic niches develop prior to the arrival of metastatic tumor cells and can be induced by injection of tumor cell-derived conditioned medium into tumor-free mice [
6‐
8]. Exosomes secreted by tumor cells can contribute to pre-metastatic niche development [
9], providing a mechanism for the delivery of proteins from primary metastatic tumors to distant tissues [
10,
11]. The tumor-derived factors and cells present in pre-metastatic niches differ between model tumor systems [
5], and evidence in immunocompetent mice indicates an important role for immunosuppressive cells in promoting metastatic growth in distant tissues.
Bone marrow-derived cells have been detected in metastatic tissues of cancer patients [
7,
8], and cells that express the cell surface marker CD11b may be particularly important for promoting breast cancer metastasis [
7]. CD11b (Mac-1) is an α
M integrin expressed on a variety of myeloid cells (granulocytes, monocytes, and macrophages), natural killer (NK) cells, and a subset of B cells. CD11b
+ myeloid cells, often co-expressing Gr1, are increased in some primary tumors and have been implicated in enhancing tumor cell invasion [
12,
13], angiogenesis [
14,
15], and vasculogenesis [
16]. Granulocyte-colony stimulating factor (G-CSF) secreted by 4T1 mammary tumor cells has been shown to induce CD11b
+Gr1
+ cell expansion [
17], and we, along with others, have shown that CD11b
+Gr1
+ cells accumulate in the spleens and lungs of mice bearing metastatic 4T1 murine mammary tumors [
17‐
20]. CD11b
+Gr1
+ cells represent a heterogeneous mixture of myeloid cells, including neutrophils and myeloid-derived suppressor cells (MDSCs) [
21].
MDSCs accumulate in response to inflammatory stimuli and normally function to prevent auto-immunity and resolve inflammation [
22]. MDSCs can be distinguished from other CD11b
+Gr1
+ myeloid cells by their ability to inhibit T cell- and NK cell-mediated immune responses [
22‐
24]. Aberrantly elevated levels of MDSCs have been described in tumor-bearing mice and cancer patients [
25] and are thought to be important mediators of tumor development and progression by actively suppressing the activity of cytotoxic T cells. Two sub-types of MDSCs have been identified in mice [
26], with CD11b
+Ly6G
+Ly6C
mid/lo granulocytic MDSCs (G-MDSCs) exhibiting less immunosuppressive potency than the less abundant CD11b
+Ly6G
−Ly6C
hi monocytic MDSCs (M-MDSCs) [
22]. While the presence of CD11b
+Gr1
+ cells in the lungs has been associated with enhanced growth of metastatic tumor cell foci [
17‐
19], CD11b
+Gr1
+ cells have also been shown to accumulate in non-metastatic target organs of tumor-bearing mice [
27] and in the peripheral blood of breast cancer patients [
28‐
30]. Primary tumor resection in mice is known to decrease MDSC levels in the spleen [
31,
32], although the longevity of MDSCs in the lungs after primary tumor resection, and the potential impact of these MDSCs on metastatic growth in the lungs, is less well-understood.
We have found that in addition to mice bearing 4T1 tumors, mice orthotopically implanted with metastatic 4T07 murine mammary tumors, but not non-metastatic 67NR tumors, have high levels of functional, immunosuppressive CD11b+Gr1+ MDSCs in the lungs. In addition to MDSCs in the lungs of 4T1 tumor-bearing mice, we also found elevated inflammatory macrophages, eosinophils, and NK cells. G-MDSCs, M-MDSCs, and macrophages rapidly decrease in the lungs within 48 h of primary 4T1 tumor resection, although G-MDSCs in the lungs remain higher than naïve mice for 2 weeks following tumor resection. These residual pulmonary G-MDSCs retain immunosuppressive function and are associated with enhanced metastatic tumor cell colonization in the lungs, indicative of a pro-metastatic environment in lung tissue that persists after primary tumor resection. Treating mice with gemcitabine after surgery decreases residual G-MDSCs and tumor colonization of the lungs, suggesting that targeting MDSCs after primary tumor resection may improve the treatment of metastatic breast cancer.
Methods
Tumor cells and mice
4T1, 4T07, and 67NR murine mammary carcinoma cells were a kind gift from Dr. Fred Miller (Karmanos Cancer Institutes, Detroit, MI). These cell lines were originally derived from a spontaneous mammary tumor in a Balb/cfC3H mouse and represent different levels of metastatic propensity [
33]. 4T1 tumor cells metastasize to the lung, liver, bone, and brain; 4T07 cells metastasize to the lungs and liver, but fail to grow into macroscopic metastases; 67NR cells do not metastasize. MSC2 cells are an immortalized MDSC cell line obtained from BALB/C Gr1
+ splenocytes and were provided from Dr. François Ghiringhelli (University of Burgundy, Dijon, France). All cells were maintained in RPMI-1640 medium + sodium pyruvate, HEPES, and 10% FCS and used within 20 passages.
Female Balb/c mice (8–10 weeks old) were purchased from Simonsen Laboratories (Gilroy, CA). All mice were housed under specific pathogen-free conditions in the Animal Resource Centre at the BC Cancer Research Centre. All animal experiments were performed in accordance with Institutional and Canadian Council on Animal Care Guidelines. For orthotopic mammary tumor implantation, mice were injected with 105 4T1 cells, 106 4T07 cells, or 2 × 105 67NR cells in the fourth mammary fat pad. We have found that these cell numbers produce consistent tumor growth rates, with tumor volumes that approach ethical restrictions (1–1.25 cm3) 4 weeks after implantation. For the intravenous (iv) studies, mice were injected with 1.2 × 104 4T1 cells in a 200-μl injection volume of PBS into the lateral tail vein.
Primary tumor resections
For resection of orthotopically implanted 4T1 tumors, mice were anesthetized with 2.5% isoflurane in oxygen prior to shaving over the left side of the body (adjacent to the tumor implantation site in the fourth mammary fat pad). The skin was scrubbed with 4% germi-stat and 70% alcohol prior to injection of 10–20 μL of 0.5% lidocaine as a subcutaneous line block along the intended incision site. A ~ 6-mm incision was made adjacent to the tumor using sterile surgical scissors. The blunt end of a trocar was used to gently separate the tumor from the overlying skin, and the tumor was gently pulled through the incision using sterilized forceps. After tumor resection, the incision site was closed with nylon monofilament non-absorbable sutures. Mice were injected with 5 mg/kg meloxicam into the dorsal neck pouch followed by 10 mL/kg warmed saline. Tumor-bearing mice exposed to sham surgery underwent all of the above procedures other than tumor resection; incisions were open for a total of 5–6 min per mouse, and mice received all anesthetics and analgesics.
Gemcitabine and antibody treatments
A single dose of 60 mg/kg of gemcitabine (Sandoz, Boucherville, QC) was injected ip into mice bearing 17-day-old 4T1 tumors or 1 day following 4T1 primary tumor excision. Gemcitabine was diluted to a 15 mg/ml working solution in physiological saline. Mice were euthanized and tissues were harvested 24, 48, 72, and 96 h post drug administration.
For immunological depletion of MDSCs, 100 μg of anti-Gr1 antibody (clone 1A8; BioXCell) or IgG2b isotype control (clone LTF-2; BioXCell) was administered via ip injection or intranasally every 4 days beginning 7 days after primary tumor implant. We found that 200 μg of anti-Gr1 antibody was lethal by the third injection. For immunological depletion of eosinophils, anti-IL-5 antibody (TRFK5, BioXCell) or isotype control (TNP6A7, BioXCell) was administered by weekly ip injection at 1 mg/kg in PBS.
Blood processing
For analysis of circulating CD11b+Gr1+ cells, 100 μl of murine peripheral blood was collected once per week by inserting a 26-G needle into the lateral tail vein to collect blood into a heparin-coated capillary tube. Blood samples were transferred to K2EDTA-treated tubes (BD Microtainer, Franklin Lakes, NJ), centrifuged at 1000×g for 10 min at room temperature, and the plasma removed. The cellular fraction of each sample was treated with NH4Cl for 9 min on ice to induce erythrocyte lysis prior to antibody incubation for subsequent flow cytometry analyses. For antibody array or enzyme-linked immunosorbent assay (ELISA) analyses, plasma was collected by terminal cardiac puncture using a heparin-coated syringe with a 26-G needle prior to processing as outlined above.
Antibody array and mG-CSF quantification
Plasma was collected from naïve and 4T1 tumor-bearing mice as previously described, and chemokines were analyzed with an R&D Systems Mouse Cytokine Array, Panel A (Catalog # ARY006) according to the manufacturer’s instructions. Array images were developed onto X-ray film and digitized with a flatbed scanner.
G-CSF serum levels were quantified using a mouse G-CSF Quantikine ELISA (R&D Systems, Minneapolis, MN) as per the manufacturer’s protocol. ELISA plates were analyzed using a Tecan Safire2 at 450 nm with wavelength correction at 540 nm.
Tissue processing
The spleens and livers were pushed through 100-μm and 40-μm mesh filters with PBS to create single-cell suspensions. For clonogenic and immune suppression assays, lungs and kidneys were finely minced with crossed scalpels prior to agitation for 40 min at 37 °C with an enzyme suspension containing 0.5% trypsin and 0.08% collagenase I in PBS (for clonogenic assays). After incubation, 0.06% DNase was added and the cell suspension was gently vortexed and filtered through 30-μm nylon mesh. Single-cell suspensions were treated with NH4Cl for 9 min on ice to induce erythrocyte lysis. For flow cytometry analyses, lungs were processed as above except with 1 mg/mL collagenase II (Gibco Life Technologies) in RPMI medium for the tissue digestion step (no trypsin or DNase).
Clonogenic assays from disaggregated lung tissue were performed as previously reported [
34,
35]. Briefly, single-cell suspensions derived from lung tissue were washed in PBS, and aliquots of 3 × 10
3 to 10
6 cells were plated in triplicate in medium containing 60 μM 6-thioguanine to select for the 6-thioguanine-resistant 4T1 tumor cells. Plates were incubated for 10–12 days prior to staining cell colonies with malachite green for manual enumeration.
Mass cytometry
Antibody labeling with the indicated metal tag was performed using the MaxPAR antibody conjugation kit (Fluidigm), and concentration was assessed after metal conjugation using a Nanodrop (Thermo Scientific). Single-cell suspensions of lung cells were fixed with 1.6% paraformaldehyde (PFA; Electron Microscopy Sciences) for 10 min at room temperature. Cells were washed in PBS + 2% FBS and resuspended in blocking buffer (PBS + 5% FBS) and 1.5 μg/mL anti-mouse CD32 antibody at a concentration of 3 × 106 cells/50 μL for 10 min. Cells were then stained for 45 min on ice with antibodies at a concentration of 3 × 106 cells/100 μL. The cells were subsequently washed twice with MaxPar Cell Staining Buffer (Fluidigm) before being permeabilized and fixed by incubation in 1 mL of MaxPar Fix and Perm Buffer for 1.5 h. Cells were subsequently washed twice with MaxPar Perm-s Buffer and stained with intracellular antibody at 3 × 106 cells/100 μL in MaxPar Perm-s Buffer before being washed twice with MaxPar Cell Staining Buffer (Millipore). EQ™ Four Element Calibration Beads (DVS Sciences) were added at a concentration of 3.3 × 104 beads/mL to the cells in milli-Q H2O at a cell concentration of 1 × 106 cells/mL. Cells were then filtered and run on a CyTOF 2 (Fluidigm) with a flow speed of 0.045 mL/min, a 30-s acquisition delay, and 10-s detector stability delay.
Data files were concatenated using the FCS file concatenation tool available from Cytobank (
https://www.cytobank.org/) and normalized using software in MatLab (MathWorks) [
36]. Normalized data was debarcoded using a debarcoding tool with cell and sample-specific stringency adjustment [
37]. Data were analyzed in R using the package “cytofkit”: a total of 10,000 cells were downsampled from each sample without replacement for ArcSinh transformation and subsequent t-SNE analysis for PhenoGraph clustering and viSNE visualization. Other analyses were completed using FlowJo VX (Treestar). Cell surface markers used to identify each immune cell subset in the lungs are listed in Additional file
1: Table S1.
T cell proliferation assay
Spleen or lung tissue of naïve mice or mice 3 weeks after primary mammary tumor implant were harvested and CD11b
+Gr1
+ cells were isolated from single-cell suspensions via Gr1-PE positive selection using the EasySep system (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions. CD11b
+Gr1
+ cell purity of the isolated cells was > 95% as determined by subsequent flow cytometry analysis. Immunosuppression assays were performed using HL-1 medium (BioWhittaker; Basel, Switzerland), supplemented with 1% penicillin, 1% streptomycin, 1% Glutamax, and 50 μM 2-mercaptoethanol. We did not use serum in these assays, as we have previously found that the use of serum in immunosuppression assays can mask the immunosuppressive function of CD11b
+Gr1
+ cells [
34]. Erythrocyte-depleted splenocytes (an abundant source of T cells) from naïve mice stimulated ex vivo with 1 μg/ml anti-CD3 + 5 μg/ml anti-CD28 (eBioscience, San Diego, CA) were used as responder cells in the assay and cultured at 2 × 10
5 cells/well ± isolated CD11b
+Gr1
+ cells. Co-cultured cells were incubated at 37 °C for 72 h, and 1 μCi/well
3H-thymidine (2 Ci/mM, PerkinElmer, Woodbridge, ON, Canada) was added for the last 18 h of the assay. Cells were harvested onto filtermats, and radioactivity was measured using a Betaplate liquid scintillation counter (Wallac, Waltham, MA). Data are expressed as mean ± SEM of the
3H counts per minute (cpm) from triplicate cultures, or as cell proliferation relative to control samples (stimulated splenocytes alone).
Flow cytometry
5 × 105 freshly harvested cells (or rehydrated alcohol-fixed cells for BrdU analyses) were washed in PBS + 4% FCS prior to incubation with primary antibodies. Cells were stained with the following antibodies: CD11b-PE, Gr1-Alexa 488 (Invitrogen), Ly6G-PE, Ly6C-FITC (BD Pharmigen), and unconjugated F4/80 (eBioscience). When unconjugated F4/80 was used, cells were incubated with Alexa-488 or Alexa-594 secondary antibodies (Invitrogen). Where indicated, mice were given 90 mg/kg 5-bromo-2′-deoxyuridine (BrdU; Sigma-Aldrich, Oakville, ON) intraperitoneally (ip) 90 min before tissue harvest. For BrdU analysis, cells were denatured with HCl prior to neutralization and anti-BrdU (Abcam, Toronto, ON, Canada) contact in PBS + 4% FCS + 0.1% Triton-X. List mode files were collected using a dual laser Epics Elite-ESP flow cytometer (Coulter Corp., Hialeah, FL) and were subsequently reprocessed for analysis. Doublet correction and bitmap gating were used to select the cell populations of interest with the WINLIST software package (Verity Software House Inc., Topsham, ME).
Peripheral blood samples were stained with Fixable Viability Dye eFluor 780 (eBioscience, San Diego, CA) after NH4Cl treatment. In addition to CD11b-PE and Gr1-Alexa 488, blood samples were stained with CD45-APC (eBioscience, San Diego, CA), samples were run on a FACSCalibur (DxP 6-color Upgrade), and events were acquired/analyzed using FlowJo CE software. For cell cycle analysis data, ethanol fixed lung and spleen samples were rehydrated and 5 × 105 to 1 × 106 cells were stained with propidium iodide (PI) in order to generate DNA profiles. FlowJo CE software was used to analyze cell cycle profiles.
For 12–15 color flow cytometry panels, single-cell suspensions from the lungs were washed with PBS and stained for 30 min on ice with eFluor® 780 fixable-viability dye (eBioscience). Cells were washed and resuspended in Hanks balanced salt solution with 10 mM HEPES (StemCell Technologies) + 2% FBS + 0.05% NaN3. Anti-murine CD16/32 (clone 2.4G2, eBioscience) was used to block cells prior to antibody staining. Cells were stained with the following antibodies on ice for 30 min: CD45-APC, CD8α-FITC, CD3ε-PE, CD11b-e450 (eBioscience), F4/80-PE, SiglecF-TexasRed, FoxP3-V421, Ly6C-PerCP-Cy5.5 (BD Biosciences), Gr1-FITC, MHCII-V500, CD11c-BV605, CD25-APC, CD4-BV605, CD11b-APC, FasL-PE, CD19-PECy7, Ly6G-AF700, and NKp46-BV711 (Biolegend). Cells were fixed and permeabilized for 30 min using a transcription factor buffer set (eBioscience). For T regulatory cell identification, cells were stained with FoxP3-PECy7 or FoxP3-V421 for 1 h. All samples were acquired on a BD LSRFortessa (FACSDiva software, BD) and analyzed with FlowJo (TreeStar).
Resazurin assay
The metabolic activity of 4T1 and MSC2 cells was measured using a colorimetric resazurin assay. Resazurin sodium salt (Sigma, Oakville, ON) was made up in 0.9% NaCl saline to a concentration of 4.4 μM. 1 × 104 4T1 or MSC2 cells were seeded in 24-well TC-treated plates and treated with gemcitabine at 10, 1, 0.5, 0.1, and 0.01 μM for 48 h. Physiological saline was used as a vehicle control. Resazurin was added to cells at a final concentration of 218 nM, and plates were read after 3–4 h by a 29 TECAN GENios plate reader using a 535-nm excitation and 590-nm emission filter. Experimental values are reported as normalized to cells grown in physiological saline.
Statistical analyses
Student’s t tests with Welch’s correction were used for all comparisons using GraphPad Prism with *p < 0.05, **p < 0.01, and ***p < 0.001 indicating statistical significance. NS = data are not significantly different.
Discussion
A variety of bone marrow-derived cells have been implicated in supporting primary tumor growth and metastasis, and a better understanding of the expansion, phenotype, and longevity of these cells is required for the development of improved therapies to treat metastatic disease. Our findings indicate that, in addition to mice with 4T1 tumors, 4T07 tumor-bearing mice produce G-CSF and systemically induce the expansion of functional, immunosuppressive CD11b
+Gr1
+ MDSCs in the spleen and accumulation in the lungs. CD11b
+Gr1
+ cells isolated from mice implanted with non-metastatic 67NR tumors exhibited minimal T cell suppression at levels that were comparable to CD11b
+Gr1
+ cells from naïve mice. Profound proteomic differences have been reported between CD11b
+Gr1
+ cells isolated from the spleens of 4T1 or 67NR tumor-bearing mice [
49], providing further evidence that CD11b
+Gr1
+ cells can be phenotypically and functionally distinct in mice with different mammary tumor types. The expansion and functional activation of CD11b
+Gr1
+ cells are influenced by tumor-derived factors [
22], and recent work demonstrates that environmental factors can also induce the accumulation of CD11b
+Gr1
+ cells in tissues [
50]. Low-grade chronic inflammation associated with obesity has been shown to drive the expansion of CD11b
+Gr1
+ cells within the lung in an IL-5 and GM-CSF-dependent manner, resulting in an increase in breast cancer pulmonary metastasis [
50].
The lungs are a common site for breast cancer metastasis, and it is tempting to relate MDSC accumulation with pre-metastatic niche formation in the lungs. We found that MDSCs were detectable in the lungs prior to metastatic tumor cells, which is consistent with previously published pre-metastatic niche development kinetics [
7,
8]. However, we found MDSCs accumulating systemically in metastatic and non-metastatic tissues (Additional file
5: Figure S4C-D), suggesting that immunosuppressive MDSCs are not specific to pre-metastatic niches or metastatic target organs in mice bearing 4T1 tumors. A similar phenotype was recently observed using the metastatic MMTV-polyoma middle T (PyMT) mammary tumor mouse model, where CD11b
+Ly6G
+ myeloid cells were mobilized to both metastatic and non-metastatic target organs [
27]. In this model, accumulation of CD11b
+Ly6G
+ cells also occurred prior to tumor cell detection in the lungs and was abrogated in the absence of G-CSF.
Tumor-secreted factors, such as G-CSF, GM-CSF, TGF-β, and various interleukins, have been shown to drive the expansion of MDSCs, which then contribute to both an immunosuppressive tumor microenvironment and systemic dampening of the immune system [
22]. Studies carried out in several mouse tumor models have shown that G-CSF is an important tumor-derived factor capable of altering myelopoiesis and inducing aberrant granulocytic MDSC expansion [
17,
18,
38]. G-CSF loss- and gain-of-function approaches have shown that abrogating G-CSF production significantly diminishes MDSC accumulation in tissues, while over-expressing G-CSF or treatment of naïve mice with recombinant G-CSF induces MDSC accumulation [
51]. We found that serum G-CSF levels decreased dramatically after surgical resection of primary 4T1 tumors, implicating the primary tumor as the main source of circulating G-CSF in these mice. Tumor resection also reduced MDSC levels in the spleen, peripheral blood, and lungs within 48 h. However, functional G-MDSCs remained significantly elevated relative to naïve mice for 2 weeks after tumor resection, indicating that continued production of G-CSF by the primary tumor is not required to maintain aberrantly high MDSC levels. Interestingly, the lungs of 4T1 tumor-bearing mice also contained elevated M-MDSCs, infiltrating macrophages, NK cells, and eosinophils (Fig.
1, Additional file
2: Figure S1), with M-MDSCs and macrophages returning to control levels 48 h after tumor resection (Fig.
5b–d). Eosinophils remained elevated in the lungs after primary tumor resection, which may be related to the high levels of IL-33 release we have previously observed in the lungs of 4T1 tumor-bearing mice [
52] since IL-33 is known to activate eosinophils and induce eosinophilic airway inflammation [
53]. Despite the increased eosinophil content in the lungs after tumor resection, we did not find that eosinophils affected 4T1 metastatic tumor growth in the lungs (Additional file
12: Figure S11).
One limitation of our study is that we were unable to extend our experimental timeline beyond 2 weeks post-tumor resection. We do not use radiation or chemotherapy after tumor resection, and due to the highly aggressive nature of the 4T1 tumor line, we observe regrowth of primary tumors in the surgical field 14 days after tumor resection. Whether this regrowth is due to tumor cells that were missed during the resection or due to metastasis of 4T1 cells from the lungs to the site of wound healing is an open question. Regardless, the propensity for tumor regrowth limits the timeframe of our experiments, and we are therefore unable to speculate on the longevity of suppressive lung MDSCs past the 2-week time point. Taken together, our data indicate that G-CSF-producing metastatic primary tumors create a pro-metastatic environment in the lungs consisting of several immunosuppressive myeloid cell types and that G-MDSCs persist in the lungs after primary tumor resection and are capable of promoting the growth of metastatic tumor foci.
Interestingly, the reduction in splenic MDSCs after tumor resection is consistent with some reports [
31,
32], but depending on the surgical method, MDSC levels can increase in the spleen and bone marrow after 4T1 tumor resection when combined with abdominal nephrectomy [
54,
55]. We did not observe a change in MDSCs or other immune cell types in the lungs of 4T1 tumor-bearing mice exposed to ‘sham’ surgery (i.e., without tumor resection; Additional file
10: Figure S9), confirming that the changes in the lung immune microenvironment found after tumor resection (Figs.
3 and
5) were due to removal of the tumor rather than the surgical procedure. With the metastasis promoting effects of MDSCs, it is important to determine whether MDSC populations persist in patients following surgical resection of primary breast tumors. Monitoring MDSC levels in the blood appears to be a suitable indication of MDSCs in tissues, and assessing circulating MDSCs in patients following surgical resection could identify patients at increased risk of developing metastatic disease. Indeed, elevated MDSC levels are observable in the peripheral blood of patients with metastatic cancer [
56,
57], and increased MDSCs in the circulation of breast cancer patients correlates with clinical stage and decreased survival [
58]. Circulating MDSC levels may be useful for screening and monitoring purposes, both before and after treatment, since breast cancer patients with elevated MDSCs may harbor immunosuppressive environments in peripheral tissues that could promote the development of secondary metastases after surgery.
Identifying therapeutic strategies that selectively target MDSCs could be used in patients after surgery to prevent subsequent metastatic growth. We found that a single dose of gemcitabine was sufficient to reduce G-MDSC, M-MDSCs, infiltrating macrophages, and eosinophils (Fig.
5b–d) in the lungs without affecting other myeloid or lymphoid cell types (Additional file
11: Figure S10). Gemcitabine-mediated depletion of G-MDSCs that persist in the lungs after primary tumor resection dramatically decreased tumor cell engraftment in an experimental model of lung metastasis. These data support the development of therapies that target G-MDSCs in concert with primary tumor removal for improved treatment of metastatic breast cancer. Clinical studies have demonstrated the efficacy of pharmacological strategies to reduce MDSC number (e.g., sunitinib) [
59], to inhibit MDSC suppressive function (e.g., sildenafil) [
60], or to differentiate MDSCs into mature myeloid cells (e.g., all-trans retinoic acid or 25-hydroxyvitamin D
3) [
28‐
30] in a variety of human cancers. We have previously shown that ATRA-mediated differentiation of MDSCs can promote metastatic tumor growth by generation of highly immunosuppressive macrophages [
35], and therefore, strategies to target or inhibit MDSCs may produce more predictable outcomes. Directly targeting MDSCs with 5-fluorouracil [
61] or gemcitabine [
62] in various murine models of cancer significantly enhances T cell-dependent antitumor immunity, suggesting that therapeutics which target MDSCs may work synergistically with T cell-targeted therapies. Treatment with gemcitabine has been shown to deplete MDSCs in the peripheral blood of pancreatic cancer patients, as well as increase the ratio of T effector cells to T regulatory cells, indicating that targeting MDSCs can have additional downstream effects on immune cell populations critical for tumor rejection [
63]. Combining cyclophosphamide or gemcitabine analogue treatment with adoptive dendritic cell therapy in the 4T1 breast carcinoma model was shown to increase activation of NKT cells, decrease tumor burden, and enhance protection against metastatic recurrence in the lungs [
64]. Although treatment with cyclophosphamide or gemcitabine analogues decreased the frequency of MDSCs, these chemotherapeutics also promoted immunogenic cell death of 4T1 tumor cells and enhanced 4T1 immunogenicity by inducing the release and expression of immunogenic cell death-associated proteins [
64]. Taken together, these studies suggest that targeting immunosuppressive cells in conjunction with immunotherapies that target T cells, such as anti-CTLA4 and anti-PD-L1, could be effective treatment strategies for tumor metastases.
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