Antidiabetic DPP-4 Inhibitors Reprogram Tumor Microenvironment That Facilitates Murine Breast Cancer Metastasis Through Interaction With Cancer Cells via a ROS–NF-кB–NLRP3 Axis

Improvement of understanding of the safety profile and biological significance of antidiabetic agents in breast cancer (BC) progression may shed new light on minimizing the unexpected side effect of antidiabetic reagents in diabetic patients with BC. Our recent finding showed that Saxagliptin (Sax) and Sitagliptin (Sit), two common antidiabetic dipeptidyl peptidase-4 inhibitors (DPP-4i) compounds, promoted murine BC 4T1 metastasis via a ROS–NRF2–HO-1 axis in nonobese diabetic–severe combined immunodeficiency (NOD-SCID) mice. However, the potential role of DPP-4i in BC progression under immune-competent status remains largely unknown. Herein, we extended our investigation and revealed that Sax and Sit also accelerated murine BC 4T1 metastasis in orthotopic, syngeneic, and immune-competent BALB/c mice. Mechanically, we found that DPP-4i not only activated ROS–NRF2–HO-1 axis but also triggered reactive oxygen species (ROS)-dependent nuclear factor kappa B (NF-κB) activation and its downstream metastasis-associated gene levels in vitro and in vivo, while NF-кB inhibition significantly abrogated DPP-4i-driven BC metastasis in vitro. Meanwhile, inhibition of NRF2–HO-1 activation attenuated DPP-4i-driven NF-кB activation, while NRF2 activator ALA enhanced NF-кB activation, indicating an essential role of ROS–NRF2–HO-1 axis in DPP-4i-driven NF-кB activation. Furthermore, we also found that DPP-4i increased tumor-infiltrating CD45, MPO, F4/80, CD4, and Foxp3-positive cells and myeloid-derived suppressor cells (MDSCs), and decreased CD8-positive lymphocytes in metastatic sites, but did not significantly alter cell viability, apoptosis, differentiation, and suppressive activation of 4T1-induced splenic MDSCs. Moreover, we revealed that DPP-4i triggered ROS-NF-κB-dependent NLRP3 inflammasome activation in BC cells, leading to increase in inflammation cytokines such as interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), IL-1β and IL-33, and MDSCs inductors granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and M-CSF, which play a crucial role in the remodeling of tumor immune-suppressive microenvironment. Thus, our findings suggest that antidiabetic DPP-4i reprograms tumor microenvironment that facilitates murine BC metastasis by interaction with BC cells via a ROS–NRF2–HO-1–NF-κB–NLRP3 axis. This finding not only provides a mechanistic insight into the oncogenic ROS–NRF2–HO-1 in DPP-4i-driven BC progression but also offers novel insights relevant for the improvement of tumor microenvironment to alleviate DPP-4i-induced BC metastasis.

Improvement of understanding of the safety profile and biological significance of antidiabetic agents in breast cancer (BC) progression may shed new light on minimizing the unexpected side effect of antidiabetic reagents in diabetic patients with BC. Our recent finding showed that Saxagliptin (Sax) and Sitagliptin (Sit), two common antidiabetic dipeptidyl peptidase-4 inhibitors (DPP-4i) compounds, promoted murine BC 4T1 metastasis via a ROS-NRF2-HO-1 axis in nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. However, the potential role of DPP-4i in BC progression under immune-competent status remains largely unknown. Herein, we extended our investigation and revealed that Sax and Sit also accelerated murine BC 4T1 metastasis in orthotopic, syngeneic, and immune-competent BALB/c mice. Mechanically, we found that DPP-4i not only activated ROS-NRF2-HO-1 axis but also triggered reactive oxygen species (ROS)-dependent nuclear factor kappa B (NF-kB) activation and its downstream metastasis-associated gene levels in vitro and in vivo, while NF-кB inhibition significantly abrogated DPP-4i-driven BC metastasis in vitro. Meanwhile, inhibition of NRF2-HO-1 activation attenuated DPP-4i-driven NF-кB activation, while NRF2 activator ALA enhanced NF-кB activation, indicating an essential role of ROS-NRF2-HO-1 axis in DPP-4i-driven NF-кB activation. Furthermore, we also found that DPP-4i increased tumor-infiltrating CD45, MPO, F4/80, CD4, and Foxp3-positive cells and myeloid-derived suppressor cells (MDSCs), and decreased CD8-positive

INTRODUCTION
Accumulating evidence indicates that diabetes enhances the incidence of human cancers including breast cancer (BC) (1). Long-term exposure of antidiabetic reagents may have unexpected effects on diabetic patients with BC. Thus, better understanding of the safety profile and biological significance of antidiabetic agents in BC progression is essential to minimize the side effect of antidiabetic agents in BC bearing-diabetic patients (2). Dipeptidyl peptidase-4 inhibitors (DPP-4i), as one of common antidiabetic reagents, are currently recommended for the first-line hypoglycemic treatment of type 2 diabetes mellitus (T2DM). Emerging evidence recently reported an unpredictable adverse effect of DPP-4i in cancer progression (3)(4)(5)(6). Our latest finding also revealed that Saxagliptin (Sax) and Sitagliptin (Sit), two common antidiabetic DPP-4i reagents, facilitated murine 4T1 BC cells metastasis in immune-deficient nonobese diabeticsevere combined immunodeficiency (NOD-SCID) mice (7). However, the risky effect of DPP-4i on BC progression under immune-competent status remains largely unknown.
The concept of cancer immunoediting offers a novel insight into the crosstalk between tumor cells and immune system during the cancer progression (8,9). Tumor microenvironment, including tumor immune microenvironment, has been recognized as a complex milieu where tumor cells interact with immune cells via numerous biochemical and physical signals that are crucial for cancer progression (10,11). Tumor cells, as a major orchestrator of tumor microenvironment, have been shown to reprogram tumor microenvironment by producing cytokines and even inducing or recruiting the immunosuppressive cells such as regulatory T (Treg) cells or myeloid-derived suppressor cells (MDSCs) during cancer progression (7)(8)(9)(10)(11)(12)(13). Although recent data suggest a potential role of DPP-4 inhibition in CXCL10-mediated lymphocyte trafficking in melanoma B16F10-bearing mice (12), very little information is available for the potential effect of DPP-4i on tumor immune microenvironment, especially on tumor-infiltrating immune-suppressive cells in BC progression.
In the present study, we utilized the orthotopic and syngeneic murine 4T1 BC metastasis model in immune-competent BALB/C mice, a well-known mice model to effectively mobilize MDSCs, to characterize the effect of Sax and Sit on BC metastasis under immune-competent conditions. Then, we further investigated whether and how DPP-4i can reprogram tumor microenvironment during the BC metastasis.

Reactive Oxygen Species Detection
Intracellular ROS and mitochondrial ROS (mROS) were measured by flow cytometry as described previously (7,15). Briefly, Sax-or Sit-treated cells were stained with dihydroethidium (DHE,10 mM) (Sigma-Aldrich) for 30 min at 37°C and were resuspended in ice-cold PBS for intracellular ROS analysis by flow cytometry. For mROS detection, Sax-or Sittreated cells were stained with MitoSoX Red probe (5.0 mM, Thermo Fisher Scientific) for 20 min at 37°C. After washing with PBS, mROS were analyzed by flow cytometry.

RNA Isolation and Quantitative
Real-Time PCR RNA isolation and quantitative real-time PCR (qRT-PCR) were performed as described previously (7,14,15). Briefly, total RNA was isolated from cells using Tripure Isolation Reagent (Roche, Mannheim, Germany). One microgram of total RNA was reverse transcribed into complementary DNA (cDNA) using the PrimeScript ™ RT reagent Kit with gDNA Eraser (Takara, Japan), and qRT-PCR was performed with QuantiNova SYBR Green PCR Kit (Qiagen, Germany) on CFX Connect ™ Real-Time System (BIO-RAD) according to the manufacturer's instructions. The relative gene expressions were normalized to the housekeeping b-actin gene and calculated using the 2 −DDCt method. The details of the primers are listed in Supplementary Table S1.

Western Blotting
4T1 cells were treated with indicated reagents and then subject to Western blotting analysis as described previously (7,(13)(14)(15)(16)(17)(18). In brief, protein lysates extracted using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Haimen, China) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by blotting onto polyvinylidene fluoride (PVDF) membranes, blocked in QuickBlock ™ Blocking Buffer (Beyotime, Haimen, China), and followed by primary antibody incubation at 4°C overnight. After washing with TBST buffer, blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 1.0 h, washed with TBST three times, and detected with the enhanced chemiluminescence (ECL) system. All antibodies used in this study are listed in Supplementary Table S2.

Luciferase Reporter Gene Assays
Luciferase reporter gene assay was performed as described previously (7,14,15

Immunofluorescence
Indirect or direct immunofluorescence (IF) staining was performed in paraffin-embedded liver or lung metastatic tissue sections (4 mm) as described previously (6,14,15). In brief, tissue sections were blocked with QuickBlock ™ Blocking Buffer (Beyotime, Haimen, China) for 15 min at room temperature.
Then, indirect IF staining was performed to detect CD45, CD4, CD8, MPO, and CD11b by incubating with primary antibodies at 4°C overnight, followed by incubation for 1-2 h at room temperature with AF555-or AF647-conjugated secondary antibody (Bioss, Beijing, China). Direct IF double staining was performed to detect CD11b/F4/80 and CD11b/Gr-1 by incubating with fluorescence-conjugated primary antibodies at 4°C overnight. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a Nikon AIR Confocal Laser Microscope (Nikon, Minato, Japan), and data were measured by a NIS elements AR analysis software version 5.21. All antibodies are listed in Supplementary  Table S3.

Statistics
Statistical analysis was carried out with the GraphPad Prism 7.0 (GraphPad Software) as previously described (7,(13)(14)(15)(16)(17)(18)(19). All data were expressed as means ± SD. The significance of difference between groups was determined by unpaired two-tailed Student's t-test or one-way analysis of variance (ANOVA). The value of p < 0.05 was considered statistically significant.

DPP-4i (Sax and Sit) Facilitates 4T1 BC Cells Metastasis in Immune-Competent BALB/c Mice
To understand the potential role of DPP-4i in BC metastasis, we investigated the effect of Sax and Sit, two DPP-4i compounds, on BC metastasis in vitro and in vivo. We found that Sax and Sit markedly promoted cell migration and cell invasion of BC cells ( Figures 1A, B), consistent with our previous finding (7). Meanwhile, metastasis-associated proteins MMP-2, MMP-9 and vascular endothelial growth factor (VEGF) were also significantly enhanced after DPP-4i treatment ( Figure 1C), consistent with their messenger RNA (mRNA) levels upon DPP-4i treatment (7). These results indicate that DPP-4i promotes BC metastasis in vitro.
Given an oncogenic role of Sax in immune-deficient NOD-SCID mice (7), we sought to know whether DPP-4i could accelerate 4T1 BC metastasis in immune-competent BALB/c mice. As shown in Figures 1D, E, we observed that treatment of Sax and Sit significantly enhanced lung and liver metastasis of BC cells in vivo as shown in HE and IHC staining for micro-metastasis marker vimentin. Moreover, metastasisassociated MMP-2, MMP-9, and VEGF expressions were further detected by IHC staining in lung and liver micrometastasis nodes ( Figures 1F-H). These results suggest that DPP-4i facilitates spontaneous metastasis of BC cells in immune-competent BALB/c mice.
Given the aberrant ROS in DPP-4i-treated BC cells (7), we further investigated whether ROS inhibition could reverse DPP-4i-driven NF-кB activation in BC cells. As shown in Figure 2G, we found that ROS scavenger NAC significantly reversed DPP-4i-induced p-IKKa/b, IKKa, p-IKBa, and IKBa expressions and p65 and p-p65 levels. Furthermore, NF-кB transcriptional activation and NF-кB-responsive targets levels were significantly attenuated after NAC treatment in DPP-4i-treated BC cells (Figures 2H, I). Collectively, these data indicate that DPP-4i induces aberrant NF-кB activation in BC cells via a ROSdependent manner.

Inhibition of ROS-NF-кB Activation Abrogates DPP-4i-Driven BC Metastasis
Next, we investigated whether ROS-NF-кB activation is critical to DPP-4i-induced BC metastasis. Using ROS scavenger NAC, we found that ROS inhibition significantly abrogated DPP-4idriven BC cell migration and invasion with a dose-dependent manner (Supplementary Figure S2), consistent with our previous finding (7), suggesting an oncogenic role of ROS in DPP-4i-driven BC metastasis. To further define the role of NF-кB activation in DPP-4i-driven BC metastasis, we used BAY 11-7082, a specific NF-кB inhibitor to explore whether pharmaceutical NF-кB inhibition could reverse DPP-4i-driven BC metastasis. As shown in Figure 3A, we found that NF-кB inhibition significantly attenuated p65 and p-p65 levels and NF-кB-responsive and metastasis-associated proteins in DPP-4itreated BC cells (Figures 3B, C). Notably, DPP-4i-driven cell migration and invasion were also significantly abrogated by NF-кB inhibition with a dose-dependent manner ( Figures 3D, E), indicating an essential role of NF-кB activation in DPP-4i-driven BC metastases in vitro. Therefore, these results suggest that DPP-4i drives BC metastasis via ROS-NF-кB activation.
Given the aberrant NRF2 activation in metastasis tissues of Sax-treated 4T1-bearing NOD-SCID mice (7), we investigated whether DPP-4i also could promote NRF2 activation in 4T1bearing BALB/c mice. As shown in Supplementary Figure S3, we found that Sax or Sit treatment also enhanced NRF2-HO-1 (E) NF-кB transcriptional activation was analyzed by luciferase reporter gene assay. (F) NF-кB-responsive targets were detected by Western blotting. b-Actin was a loading control. Data are presented as mean ± SD of three independent experiments. Representative images are shown. *p < 0.05, **p < 0.01, and ***p < 0.001 between the indicated groups determined by the one-way analysis of variance (ANOVA). activation in lung and liver metastasis tissues of 4T1-bearing BALB/c mice, suggesting that DPP-4i-induced NRF2-HO-1 activation is independent on immune status of 4T1-bearing mice model. Next, we used NRF2 activator ALA to test whether pharmaceutical NRF2 activation could promote NF-кB activation in vitro and in vivo. In vitro, we observed that ALA treatment significantly enhanced p65 and p-p65 levels and NF-кB transcriptional activation in BC cells (Figures 5A, B). Meanwhile, p-IKKa/b, IKKa, p-IKBa, and IKBa, and NF-кB-responsive genes expressions were also increased upon ALA treatment in vitro (Figures 5C, D). Moreover, in 4T1-bearing NOD-SCID mice, p65 and p-p65 expression and NF-кBresponsive protein levels were also enhanced after ALA treatment in lung and liver metastasis tissues ( Figures 5E-G), indicating that pharmaceutical NRF2 activation promotes NF-кB activation in vivo. Together, these data suggest that NRF2-HO-1 activation plays a critical role in DPP-4i-driven ROS-dependent NF-kB activation of BC cells.

DPP-4i Promotes the Recruitment of Tumor-Infiltrating Inflammatory and Immunosuppressive Cells in Metastatic Sites
Given a critical role of tumor-infiltrating T cells in the prediction of clinical outcomes in BC patients (13), we further investigated the effect of DPP-4i on tumor-infiltrating immune cells in metastatic sites. Therefore, we analyzed the expression of immune-cell-associated markers in metastatic tissues including pan-leukocyte marker CD45, neutrophil marker MPO, macrophage markers CD11b, F4/80, and T cell markers CD4 and CD8 (14). We observed that DPP-4i significantly promoted the infiltration of CD45 + , MPO + , and CD11b + /F4/80 + cells in lung and liver metastasis sites (Supplementary Figure S4). Furthermore, we also observed an increase in CD4 + cells but a decrease in CD8 + T cells in lung and liver metastasis sites ( Figures 6A, B), indicating that DPP-4i may induce tumorimmunosuppressive microenvironment in metastatic sites.
To further define whether DPP-4i is involved in the infiltration of immunosuppressive cells in metastatic sites (7)(8)(9)(10)(11)(12)(13), we further investigated the effect of DPP-4i on the infiltration of Treg cells and MDSCs in metastasis sites of 4T1-bearing BALB/c mice and observed a significant increase in tumor-infiltrating Foxp3 + cells in metastasis sites ( Figure 6C), indicating that immunosuppressive Treg cells may be responsible for the increased tumor-infiltrating CD4 + T cells after DPP-4i treatment. Furthermore, we also observed that tumor-infiltrating MDSCs were also increased in lung and liver metastasis tissues of DPP-4i-treated mice ( Figure 6D), indicating that DPP-4i may promote 4T1-induced recruitment or expansion of immunosuppressive cells in metastasis tissues. Given MDSCs as a major immunosuppressive population in 4T1-bearing BALB/c mice (13,20), we analyzed the effect of DPP-4i on MDSCs proliferation and differentiation in PBMCs and splenic cells of 4T1-bearing BALB/c mice. Interestingly, we did not observe an obvious increase in CD11b + GR-1 + MDSCs in PBMCs or splenic cells (Supplementary Figure S5A). Then, we evaluated MDSCs differentiation by analyzing the percentage of G-MDSCs and Mo-MDSCs, two major subtypes of MDSCs, but did not find a significant alteration of MDSCs differentiation in PBMCs and splenic cells of 4T1-bearing mice (Supplementary Figure S5B), indicating that therapeutic ranges of DPP-4i may not exert direct effects on biologic behavior of MDSCs in vivo. To obtain more direct evidence, next, we set up an in vitro co-culture system in which DPP-4i was cultured with 4T1-induced splenic MDSCs. However, MDSCs differentiation, cell viability, and cell apoptosis were not markedly changed after DPP-4i treatment (Supplementary Figures S5C and S6A, B). In addition, DPP-4i treatment did not significantly promote ROS release, NRF2 activation, and expression of suppressive molecules including ARG-1 (Arginase-1), NCF1 (NOX components P47 phox ), CYBB (NOX components gp91 phox ), TGF-b, and IL-10 in 4T1-induced splenic MDSCs (Supplementary Figures S6C-E). Thus, these data suggest that DPP-4i may induce tumor immunosuppressive microenvironment by promoting recruitment or expansion of tumor-infiltrating Treg and (or) MDSCs via an indirect manner.

DPP-4i Reprograms Tumor Microenvironment by Direct Interaction With BC Cells via ROS-NF-кB-NLRP3 Axis
Given an indirect role of DPP-4i in the remodeling of tumor immunosuppressive microenvironment, we sought to know whether DPP-4i could orchestrate tumor microenvironment by direct interaction with BC cells. To this end, we first investigated the effect of DPP-4i on NLRP3 activation, a critical inflammasome in the remodeling of tumor microenvironment (21,22). We observed that DPP-4i obviously promoted NLRP3 inflammasome activation and IL-1b and IL-33 expressions in vitro and 4T1-bearing BALB/c mice ( Figures 7A, B). Similar results were also observed in Sax-treated 4T1-bearing NOD-SCID mice (Supplementary Figure S7). However, we did not find that inhibition of NLRP3 inflammasome by MCC950 can inhibit DPP-4i-driven BC cell migration and invasion in vitro (Supplementary Figure S8), indicating that NLRP3 inflammasome may not be directly involved in BC metastasis in vitro. Thus, these data suggest that DPP-4i can trigger NLRP3 inflammasome activation by direct interaction with BC cells, thereby contributing to the remodeling of tumor microenvironment.
It has been shown that GM-CSF, G-CSF, and M-CSF cytokines can induce accumulation and expansion of MDSCs, leading to the enhancement of the 4T1 BC metastasis (20,23). Thus, we further investigated whether these cytokines were involved in DPP-4i-induced tumor immunosuppressive microenvironment in BC cells. As shown in Figure 8A, we observed that both Sax or Sit treatment markedly promoted transcription levels of G-CSF, M-CSF, and GM-CSF in BC cells, suggesting that DPP-4i may directly induce G-CSF, M-CSF, and GM-CSF secretion in BC cells. Given the essential role of GM-CSF in the recruitment and maintenance of MDSCs of tumorimmunosuppressive microenvironment (24), we then focused on how DPP-4i can regulate GM-CSF expression in vitro and in vivo. Using Western blotting and IHC staining, we found that DPP-4i significantly upregulated GM-CSF expression in vitro and in 4T1-bearing BALB/c mice ( Figures 8B, C). Meanwhile, in 4T1-bearing NOD-SCID mice, we also observed that Sax or ALA treatments also enhanced GM-CSF levels in lung and liver metastasis tissues ( Figures 8D, E), indicating that ROS-NRF2-HO-1-NF-кB axis play a crucial role in DPP-4i-driven GM-CSF secretion in BC cells. To further verify these results, we used a serial of chemical inhibitors to investigate whether ROS-NRF2-HO-1-NF-кB inhibition could reverse DPP-4i-driven GM-CSF secretion in BC cells. We found that no matter the ROS-NRF2-HO-1 inhibition or NF-кB inhibition, it significantly attenuated DPP-4i-driven GM-CSF expression in BC cells ( Figures 8F-I), suggesting an essential role of ROS-NRF2-HO-1-NF-кB axis in DPP-4i-driven GM-CSF production in BC cells. Together, these results suggest that DPP-4i reprograms tumor microenvironment by interaction with BC cells via the ROS-NF-кB-NLRP3 axis.

DISCUSSION
Better understanding the role of antidiabetic DPP-4i in BCinduced tumor microenvironment would not only offer novel insights into its potential role in BC progression but also may provide new strategies to alleviate the dark side of DPP-4i in diabetic patients with BC. Here, our results presented a novel finding that DPP-4i can reprogram tumor microenvironment that facilitates murine breast cancer metastasis by interacting with cancer cells via a ROS-NRF2-HO-1-NF-кB-NLRP3 axis, providing an immune mechanistic insight into the dark side of DPP-4i in BC progression.
Our finding demonstrates that DPP-4i promotes BC metastasis by triggering NF-кB activation via a ROS-NRF2-HO-1-dependent manner, offering more mechanistic insights into the oncogenic role of ROS-NRF2-HO-1 axis in DPP-4idriven BC metastasis. Our recent finding reveals that DPP-4i can facilitate murine BC metastasis by oncogenic ROS-NRF2-HO-1 axis via a positive NRF2-HO-1 feedback loop (7). However, the downstream signaling underlying ROS-NRF2-HO-1 axis mediates DPP-4i-induced BC metastasis has not yet been completely elucidated. Of note, we noted that ROS-NRF2-HO-1 axis promoted DPP-4i-induced MMP-2, MMP-9, and VEGF levels (7), three well-known NF-кB-responsive targets (14), promoting us to investigate whether NF-кB activation is involved in DPP-4i-induced BC metastasis. Here, our data showed that DPP-4i triggered aberrant NF-кB activation in both immune-deficient NOD-SCID and immune-competent BALB/C mice. Subsequently, we also revealed that ROS-NF-кB inhibition abrogated DPP-4i-driven BC metastasis, while abrogation of NRF2-HO-1 attenuated DPP-4i-driven ROSdependent NF-kB activation in BC cells. Moreover, pharmaceutical NRF2 activation by ALA also promoted NF-кB activation in vitro and in 4T1-bearing NOD-SCID mice. Thus, our results strongly suggest that aberrant NF-кB activation, as a downstream signaling of ROS-NRF2-HO-1 axis, plays an essential role in DPP-4i-driven BC metastasis, further improving our understanding of the role of DPP-4i in the BC progression. However, the regulation of DPP-4i-driven NRF2 to NF-кB activation has not been completely demonstrated in 4T1 cells, and further study is need to dissect the more mechanistic details. Our present finding reveals that DPP-4i promotes tumorinfiltrating inflammation and immune-suppressive cells in metastatic sites, offering new strategies to develop effective immunotherapeutic approaches to alleviate DPP-4i-driven BC metastasis. A previous report suggested a potential role of DPP-4 inhibition in CXCL10-mediated lymphocyte trafficking in melanoma B16F10-bearing mice (5). However, tumorinfiltrating T cells (TILs) rather than circulating T cells were shown to play a critical role in the prediction of clinical outcomes of BC patients (13), promoting us to focus on tumor-infiltrating immune cells in metastatic tissues. 4T1 cells originally from BALB/c mice share many characteristics with naturally occurring human BC and can metastasize to distant lung and liver organs, providing an ideal mice model for mimicking the metastatic and advanced stages of human BC (13). MDSCs, as a major immunosuppressive population in 4T1-bearing BALB/c mice (13,20), contribute to tumor-immuno suppressive microenvironment not only by producing a serial of suppressive molecules such as Arg-1, NCF1, CYBB, TGF-b, and IL-10 (25, 26) but also by promoting recruitment and expansion of Treg cells via TGF-b and IL-10 (26). Our current finding revealed that DPP-4i enhanced tumor-infiltrating MPO + , CD4 + , F4/80 + , Foxp3 + cells, and MDSCs, but decreased CD8 + T lymphocytes in metastatic sites, indicating that DPP-4i may induce tumor-immunosuppressive microenvironment by enhancing tumor-infiltrating immune-suppressive cells. However, our current finding showed no direct effects of DPP-4i on cell viability, apoptosis, differentiation, and even immunesuppressive molecule levels in 4T1-induced PBMC or splenic MDSCs. Thus, these findings indicate that DPP-4i may induce the recruitment or expansion of tumor-infiltrating MDSCs via an indirect mechanism.
Our finding further highlights that DPP-4i as a potential orchestrator may contribute to the tumor-immune-suppressive microenvironment by direct interaction with BC cells via ROS-NF-кB-NLRP3 axis, providing more immune mechanistic insights into the DPP-4i-driven infiltration of immuno suppressive cells in BC metastasis. Our finding suggest an indirect role of DPP-4i in the recruitment of tumor-infiltrating MDSCs, raising a possibility that DPP-4i may reprogram tumor microenvironment by direct interaction with BC cells, thereby promoting infiltration of immune-suppressive cells in metastatic sites. Our previous finding revealed that NF-кB inhibition in human gastric cancer cells inhibited tumor-infiltrating CD11c, F4/80, CD11b, and Gr-1-positive cells in lung and liver metastatic tissues of BALB/c nude (nu/nu) mice (14), indicating a critical role of NF-кB activation in the remodeling of tumor microenvironment. Here, our finding revealed that DPP-4i-induced NF-кB activation not only enhanced metastasisassociated MMP-2, MMP-9, IL-6, and VEGF levels but also increased adhesion proteins ICAM-1 and VCAM-1. Noteworthy, among these downstream targets, VEGF was reported to induce inflammatory neovascularization for pathological hemangiogenesis and lymphangiogenesis by recruiting inflammation monocytes and (or) macrophages (14,23), while ICAM-1 and VCAM-1, as ligands by LFA-1 and Mac-1 (CD11b) expressed in leukocytes, were also shown to contribute to the recruitment of circulating leukocytes into the inflammation sites (14,21,23). Furthermore, our finding also showed that DPP-4i promoted NF-кB-dependent secretion of G-CSF, M-CSF, and GM-CSF, three well-known cytokines for the accumulation and expansion of MDSCs (20,23), while a combination of GM-CSF/IL-6 or other cytokines such as TGFb and VEGF was also shown to induce immune-suppressive MDSCs (25,27). Therefore, these data suggest that DPP-4i may contribute to the modification of tumor microenvironment by releasing a serial of adhesion or cytokines via NF-кB activation in BC cells.
More significantly, our finding revealed that DPP-4i also triggered NF-кB-dependent NLRP3 inflammasome activation, leading to caspase-1-mediated processing of IL-1b and IL-33, two critical proinflammatory cytokines for the tumor-immunesuppressive microenvironment (21,22). IL-1b has been shown to facilitate BC tumor metastasis by multiple routes, including modulating the immune cell milieu, promoting the recruitment of MDSCs, and increasing adhesion molecules levels at the metastasis sites (21). While IL-33, a novel member of the IL-1 family of cytokines, also plays a critical role in the modulation of the metastatic immune microenvironment by facilitating intratumoral accumulation of immunosuppressive and innate lymphoid cells (22,28,29). Here, our finding showed that DPP-4i promoted caspase-1-dependent processing of IL-1b and IL-33 by activating NF-кB-NLRP3 activation, indicating that DPP-4i may reprogram tumor microenvironment by promoting 4T1 cells-derived IL-1b and IL-33 via NF-кB-NLRP3 pathway. However, due to complex cell types or cytokines in tumor microenvironment, besides MDSCs, our present finding has not completely demonstrated the effect of DPP-4i on the development of others tumor-immunosuppressive cells like Treg cells, which should be further clarified in ongoing study. Overall, these results suggest that DPP-4i can reprogram tumor microenvironment by direct interaction with BC cells via ROS-NF-кB-NLRP3 axis, offering novel insights relevant for the development of effective immunotherapeutic approaches to alleviate DPP-4i-driven BC metastasis.
In summary, our study suggests that antidiabetic DPP-4i as a potential orchestrator reprograms tumor microenvironment that facilitates murine BC metastasis by interacting with BC cells via a ROS-NRF2-HO-1-NF-kB-NLRP3 axis. This finding not only provides a mechanistic insight into the oncogenic role of ROS-NRF2-HO-1 in DPP-4i-driven BC progression but also offers novel insights relevant for the development of effective immunotherapeutic approaches to alleviate the dark side of DPP-4i in BC progress.
NLRP3 inflammasome-associated proteins were detected by western blotting. bactin was a loading control. (B) ALA enhances NLRP3 inflammasome of BC cells in vivo. 4T1-bearing NOD-SCID mice were treated with or without ALA (80 mg/kg) via intraperitoneal (i.p.) administration. NLRP3, IL-1b and IL-33 expressions were detected by IHC staining in lung and liver metastatic tissues. Data are presented as mean ± SD of three independent experiments. Scale bar: 50 mm. *p < 0.05, **p < 0.01 and ***p < 0.001 between the indicated groups determined by unpaired student's t-test.