Tumor-Activated Neutrophils Promote Metastasis in Breast Cancer via the G-CSF-RLN2-MMP-9 Axis

Youjing Sheng The First A liated Hospital of Anhui Medical University Weidong Peng The First A liated Hospital of Anhui Medical University Yan Huang Anhui Medical University Lanqing Cheng the Second A liated Hospital of Anhui Medical University Ye Meng the Second A liated Hospital of Anhui Medical University Louis Boafo Kwantwi The First A liated Hospital of Anhui Medical University Jiezhen Yang Anhui Medical University Jiegou Xu Anhui Medical University Han Xiao The First A liated Hospital of Anhui Medical University Julia Kzhyshkowska University of Heidelberg Qiang Wu (  aydjohn@yahoo.com ) The First A liated Hospital of Anhui Medical University


Introduction
Breast cancer is the most commonly diagnosed malignancy among women. Due to advances in early diagnosis and comprehensive treatment, the prognosis of breast cancer patients has improved, which the 5-year overall survival in breast cancer cases to 90% (1,2). However, the occurrence rate of metastasis is also increased. Recent studies had shown that 30% of breast cancer patients will develop metastases after surgical treatment which is a contributing factor of cancer related deaths among women (2)(3)(4). Therefore, exploration of the potential mark in predicting breast cancer metastasis and its underlying mechanisms is particularly important.
Neutrophils act as the body's rst line of defense against infection and respond to diverse in ammatory cues (5,6). In tumor cell line transplantation models and genetically engineered mouse models of cancer, tumor-associated neutrophils (TANs) have been reported to be a component of tumor-promoting in ammation (5)(6)(7). However, neutrophils can engage in pathways of antitumor resistance by killing tumor cells and/or by interacting with other components of immunity (8). In gastric cancer, TANs have been shown to foster immune suppression and disease progression via GM-CSF-PD-L1 pathway (9). Additionally, Zhou et al. found that TANs increase stem cell characteristics by secreting BMP2 and TGF-β and triggering miR-301-3p expression in hepatocellular carcinoma cells (10). However, in early stage of lung cancer, TANs inhibit cancer progression by activation T cells response (11). Therefore the role of TANs in cancer progression remains to be controversial, and can be speci c for each type of malignancy.
In this study, we investigated the prognostic value of CD66b+ neutrophils density in BC tissues and their correlation with clinical characteristic. Ex vivo, the ability of TANs to promote primary cancer growth and metastatic potential of malignant cells was examined. The signaling pathways that medicate cancer -promoting activity of TANs were identi ed.
For the supernatant collected, after the cells adhered, the medium was replaced with SFM (Lonza, Switzerland), and after 72 hours, the supernatants were collected (MDA231CS).

Patient characteristics, IHC and tumor tissue
Formalin-xed and para n-embedded (FFPE) surgical specimens were randomly selected from the First A liated Hospital of Anhui Medical University. The samples consisted of equal quantities for each subtype of patients who underwent surgical operations from 2015 to 2016. All these 300 samples were randomly separated into training (n=170) and validation (n=130) cohorts. The independent cohort consisted of 95 breast cancer patients FFPE tissues from the Second A liated Hospital of Anhui Medical University (Table S1). The last follow-up time was February 10, 2020. All patient were undergoing the treatment followed by CSCO guidance. This study was approved by the Clinical Research Ethics Committee of Anhui Medical University (20180096; 20200976). All procedures performed in this study were in accordance with the ethical standards of the institutional research committee and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Each tumor sample was cut into 2 μm section and used for IHC staining. Antigen retrieval was performed using a pressure cooker for 1.5 minutes in antigen unmasking solution (sodium citrate, pH 6.0). Afterward, samples were incubated with the primary antibody human CD66b (1:100; Cat. 555724.; BD Pharmingen, US) overnight at 4 °C and blocked with a biotin block kit (MXB biotechnologies, China) for 20 minutes at room temperature. This was followed by incubation with a secondary antibody (HRP rabbit/mouse Max Vision Kits, MXB biotechnologies) for 20 minutes at room temperature. Then immunodetection was performed by using DAB (MXB biotechnologies) for 3 minutes. Images were obtained by microscopy. After counting the number of neutrophils in 10 HPF, the median was selected as the criterion for dividing the density of TANs into 2 groups.

Neutrophil isolation and treatment
Fresh blood, donated by a healthy volunteer, was treated with EDTA and transferred to a new tube. Following magnetic labeling cells (MACSxpress Neutrophil Isolation Kit, Cat. 130-104-434; Miltenyi; Germany), the tube was put into MACSmix Rotator (Miltenyi) at 37 °C for 15 minutes, and neutrophils were suspended in the supernatant. After eliminating erythrocytes with RBC lysing buffer (Cat. 555899; BD Pharmingen; USA), neutrophils were isolated.

Proliferation assays
The assay was performed in 96-well plates. A total of 3×10 3 cancer cells were seeded in each well, and the culture medium was replaced with TANCS, NeuCS, SFM and MDA231CS the next day. After 24 hours, each well was incubated with 10 μl Cell Counting Kits 8 (CCK-8, Bestbio, China) solution at 37 °C for 2 hours. The absorbance was measured at 460 nm using a plate reader.

Flow cytometry
The viability of TANs and neutrophils were determined by annexin-V/PI apoptosis detection kits (Biobest). After 10 hours of treatment, TANs and neutrophils were centrifuged at 300 g. After discarding the supernatants, the cells were resuspended in 400 μl Annexin V binding buffer. After incubation with Annexin V-FITC at 4 °C for 15 minutes and PI-PE incubation at 4 °C for 5 minutes. The data were analyzed by Flowjo (Version. 7.6.1).

Invasion and migration assay
After cancer cells were pretreated with SFM for 12 hours, experiments were performed in an 8 μm-cell culture insert (Corning Company). For the invasion assay, Matrigel (BD Company) diluted with SFM in a 1:8 ratio was precoated in a chamber and solidi ed for over 2 hours at 37 °C. A total of 4×10 4 breast cancer cells suspended in SFM were added to the upper chamber, whereas TANCS or NeuCS were added to the lower chambers. The cancer cells migrated and invaded for both 6 hours. Afterward, cancer cells in the upper chamber were removed with a swab, and the cells attached to the underside of the chambers were xed with 4% paraformaldehyde for 15 minutes and then stained with 0.5% crystal violet for 1 minute. Quanti cation was performed by the mean number of cells in ve 200x microscopic elds per chamber. In some experiments, in the lower chamber, NeuCS with IL-1γ (2 ng/ml), M-CSF (1.5 ng/ml), MIF (2 ng/ml), RLN2 (1 ng/ml), VEGF (10 ng/ml) and CD31 (3 ng/ml) (Preprotein).

ELISA
Tissue homogenates were isolated from fresh invasive ductal carcinoma samples obtained during surgery. Brie y, 0.1 g tissues were washed with cold normal saline. After removing blood and suspending cold normal saline at a ratio of 1:9, tissues were grinded with a homogenizer at 4° C at 70 rpm for 10 minutes. The samples were centrifuged at 3000 rpm at 4° C, and the supernatants were harvested as the 10% tissue homogenates. G-CSF (Cat. ksk11017, Bioss, China) and RLN2 (Cat. ab243688, Abcam) ELISA kits were purchased from Bioss or Abcam. All experiments were performed according to the manufacturer's instructions.

RNA infection
To knock down speci c target genes, cells were plated at 5 × 10 5 cells/mL and transfected with speci c siRNA duplexes using Lipofectamine 3000 Transfection Reagent (Invitrogen, catalog no. L3000015) according to the manufacturer's instructions. siRNAs were provided by GenePharma Inc for 3 paired primers and selected due to the PCR (China, table 2S).

Cytokine Antibody Arrays
To evaluate cytokine pro les, cytokine antibody arrays (R&D) were used. TANCS, NeuCS, MDA231CS and SFM were collected and diluted with array buffer. After blocking with array buffer for 1 hour, the array membrane was incubated with 1.5 ml dilution overnight at 4 °C. After incubation with HRP-conjugated detection antibodies for 1 hour, outcomes were visualized by ECL (Thermo Fisher). The signals, as relative units of spot color density, were evaluated by ImageJ software.

Biomathematics analyze
The RNA-seq and clinical features data of breast invasive ductal carcinoma were download from (http://www.cbioportal.org/study/summary?id=brca_metabric). After excluding patients without ER statues, PR statues, HER2 statues, lymph nodes involvements, metastasis, stage, progression state and survival time messages, a total 1222 breast cancer RNA-seq were included. Data was analyzed by Perl (Version 5.30.2) and R software (Version 4.1.1). Levels of neutrophils in each breast cancer were calculated by "cibersort" R package (12). Multi-cox test was analyzed by "survival" and "survminer" R package.

Statistics
Statistical analysis was performed using Prism (version 8.0.1; GraphPad Software) as follows: paired 2-tailed Student's t test ( Fig. 1 A, B, C and D; Figure 2, B, C, D and E; Fig. 3 A, B and C; Figure 3 Fig. 6 C, D, E and F), survive analysis followed by log-rank test (Fig. 1B ), chi-square test followed (Tab. 1; Tab. 2 ). For all analyses performed, signi cance was de ned as P < 0.05.

Results
The high density of TANs was related to the poor prognosis of breast cancer patients.
To investigate the presence of TANs in breast cancer, clinical breast cancer surgical samples (table S1) were obtained from the two institutions and IHC was performed for CD66b. CD66b + neutrophils in ltrating in the parenchyma of breast cancer tissue counted (Fig.  1A). Median value of the density of TANs was chosen to be the cut-off value to divided into 2 groups.
The prognostic value was then analyzed by survival and Cox multivariate analysis. In all 3 cohorts, high density of TANs was positively correlated to poor PFS for BC patients (training cohort: Fig. 1B To further con rm our ndings, 1222 breast cancer patients RNA-seqs from Metabric were downloaded and analyzed. High level of TANs positively related to poor PFS (Fig. 1G, P=0.023<0.05). The univariate analysis found high level of TANs to be associated with worse PFS (P=0.023, HR= 1.29, Supplementary table S1). Cox multivariate analysis also demonstrated that high level of neutrophils was an independent prognostic factor (Fig. 1M, P=0.018, HR= 1.3).

Activated by BC cells, TANs promoted MCF-7 activities
Neutrophils were isolated from peripheral blood from healthy donates as reported previously (13). The purity of neutrophils isolated was more than 90% (Fig. S1). Compared with unstimulated neutrophils, a signi cant increase in the lifespan of neutrophils treated with either MCF7CS (MCF7TANs, P=0.0111) or MDA231CS (MDA231TANs, P=0.0003) was evident. However, the lifespan of MDA231-TANs was signi cantly higher than that of MCF7TANs (Fig 2A).
We further evaluated the role of TANs on breast cancer cells. After culturing breast cancer cells with supernatant from either MDA231TANCS and or MCF7TANCS, there was no signi cantly change was found in the migration and invasion of both MDA-MB-231 and MCF-7 cells respectively (Fig S2). However, MDA231TANCS was found to promote the proliferation (Fig 2B, P< 0.0001), migration (Fig 2C, P= 0.0043) and invasion (Fig 2D, P= 0.0011) abilities of MCF-7 cells (Fig 2B, 2C, 2D), hence MDA231TANCS was chosen for further investigations.
Neutrophils were activated by tumor-derived G-CSF and turned to TANs in breast cancer Based on the above ndings, the effective components of MDA231CS were then investigated by cytokine antibody arrays. Compared to MDA231CS, the level of G-CSF, CXCL1, GDF-15, and PDGF-AA was signi cantly lower in TANCS (Fig. 3A, Supplementary table 2).
With all 4 solute mediators, both viability and pro-invasive abilities of TANs were promoted. To identify the most key component, neutrophils were then stimulated by 3 of these 4 cytokines in turn. As results suggested, without rh-G-CSF stimulation, both viability and migration-promotion ability of neutrophils will not be promoted (Fig 3B, 3C, Fig S3, Fig S4). To further investigate the role of G-CSF, neutrophils were then stimulated by rh-G-CSF. As the result indicated, its viability and abilities of pro-invasive and pro-metastasis were signi cantly promoted. (Fig 3B, 3C).
To con rm the role of G-CSF being the main component in activating neutrophils, G-CSF vector was transfected into MCF-7 cells (GCSF high -MCF7) to up-regulate its expression of G-CSF. After the exposure to GCSF high -MCF7 supernatant, the viability of neutrophils was signi cantly enhanced. Signi cantly increased invasion and migration of MCF-7 cells were then observed after the cancer cells were cultured in the supernatant of GCSF high -MCF7-TANs (Fig 3D, 3E).
The relationships between G-CSF and TANs were then investigated in breast cancer surgical tissue. 20 fresh breast cancer tissues and their FFPS tissue were collected. The level of G-CSF was investigated by ELISA, and the density of TANs was evaluated by IHC. The results showed a positive correlation between these two factors (Fig. 3F, P=0.012, r s =0.553), which was also demonstrated in breast cancer RNA-seq from TCGA (Fig. 3G, P< 0.0001, r s =0.156).
After exposure to the MK2206 (AKT inhibitor), the lifespan of TANs was signi cantly decrease, while the ability to promote tumor activities was not affected. In contrast, after exposing to the JSH-23 (NFκB inhibitor), the ability to promote tumor activities of TANs was a signi cant decrease, while the lifespan was not affected. Exposure to both inhibitors, neutrophils cannot be activated to TANs by both MDA231CS and rh-G-CSF (Fig 4B, 4C).
The roles of PI3K-AKT and NFκB were further con rmed in female breast ca ncer tissues by using IF assay. The results showed positive staining signals of p-AKT (Fig. 4D) and nuclei-NFκB (Fig. 4E) located in CD66b+ neutrophils, which in ltrated in the parenchyma of breast cancer tissue.

TANs promoted the migration and invasion of MCF-7 cells by activating the RLN2-PI3K-AKT-MMP-9 pathway
The role of TANs on breast cancer cells were then investigated. According to the cytokine array assay, there was a signi cantly increased expression levels of IL-1ra, M-CSF, MIF, RLN2, VEGF and sCD31 in TANCS (Fig. 3A), which were con rmed by ELISA (Fig.  5A).
After stimulated by all these 6 cytokines together with NeuCS, the migration and invasion of MCF-7 were promoted. To identify key components, MCF-7 cells were then stimulated by NeuCS with 5 of these 6 cytokines in turn. As the results showed, without RLN2 stimulation, the invasion and migration of MCF-7 cells cannot be promoted (Fig 5B, S6). To identify the role of RLN2 in TANCS, MCF-7 cells were then treated with NeuCS + RLN2. Transwell assay showed, both of the migration and invasion of MCF-7 cells were signi cantly promoted, while treated with rh-RLN2 alone, no promoted migration and invasion were observed ( Fig 6A). As it had been known that RLN2 targeted on RXFP1 on breast cancer cells, its expression in MCF7 were then knocked down by (siR-MCF7). The MCF-7 cells transfected by si-NC was taken as control (siN-MCF7, Fig 6S). Treated with TANCS and NeuCS+rh-RLN2 respectively, the migration and invasive of siR-MCF7 cells cannot be promoted, while these two supernatants still promoted this behavior for siN-MCF7 ( Fig 6B).
As it had been known that the RLN2 promoted the breast cancer cell migration and invasive by activating the PI3K-AKT-MMP9 signaling pathway (19) , the role of RLN2 pathway were studied. Levels of expression for PI3K, p-AKT and MMP9 in siN-MCF7, TAN-siN-MCF7, Neu-siN-MCF7, Neu+rh-RLN2-siN-MCF7 and TAN-siR-MCF7 were evaluated by WB. As the results showed, levels of PI3K, p-AKT and MMP9, compared to the siN-MCF7 and Neu-siN-MCF7, were signi cantly increased in TAN-siN-MCF7, Neu+rRLN2hR-siN-MCF7 respectively. Signi cantly lower levels of PI3K, p-AKT and MMP9 were found in TAN-siR-MCF7 compared to those in TAN-siN-MCF7. After inhibiting AKT in TAN-siN-MCF7 by MK2206, the level of MMP-9 was signi cantly decreased (Fig. 7A). After knocking down MMP9 in MCF7 cells by siRNA (siM-MCF7), the migration and invasive of the cancer cells were not promoted, regardless of the treatment of TANCS or NeuCS+ RLN2 (Fig 7B).

Discussion
Accumulating evidence is available for the complex composition and cancer-promoting properties of breast cancer tumor immune microenvironment in patients and animal tumor models (20)(21)(22). Although some immune/in ammatory cells have been shown to interact with breast cancer cells, as the body's rst line of defense, the role of neutrophils in cancer microenvironment is largely unknown (5,23).
Due to the limited knowledge about TANs, its markers for IHC staining and prognostic value in BC patients are not fully de ned (5,11,24,25). Myeloperoxidase (MPO), one of the most abundant proteins in neutrophils (26), had been applied as a marker for TANs in some articles and was are reported to be independently associated with a favorable prognosis in both colorectal and breast cancer. However, MPO is known as an enzyme secreted by neutrophil granulocytes as a result of phagocytosis during in ammation.
Hence, MPO-expressing neutrophils are in ammation-activated rather than tumor-activated (27), which makes MPO as a maker for TANs in breast cancer requiring for IHC need further evaluation. CD66b, normally expressed only by neutrophils, had been proven to be an effective marker for neutrophils. Due to their, correlation with the progression of several cancers (28-39), it was applied as a marker of TANs in our study. Consistent with these previous ndings, our study found a high density of CD66b+ TANs in ltrating in in BC tissues parenchyma positively related with PFS in BC patients. According to the ROC and Cox analysis, the density of CD66b+ TANs see not only speci c, but also accuracy in predicting the poor PFS in BC patients. These results suggested that evaluating the density of TANs in BC tissue by CD66b immunostaining could be an easily accessible method for predicting progression in BC patients.
Although the density of TANs has been proven to be an effective biomarker for predicting BC metastasis and poor clinical outcomes, its role in cancer progression is still not remains unclear. TANs have had been ignored as a group of immune cells in tumor microenvironments due to their short lifespan (4 to 10 hours). As commonly recognized, the lifespan of in ammation-activated neutrophils is enhanced, which can survive for more than 10 hours (40). Can the lifespan of TANs be enhanced by cancer cells? Recent studies have shown that the viability of neutrophils to be is enhanced in tumor microenvironments to prolonged than previous known (5,25). For this reason, it can be regarded as a novel marker for neutrophil activation. However, the activation of neutrophils to TANs and the interplay with tumor cells remain unclear. It is becoming increasingly clear that TANs can promote tumor progression, the role way of of TANs in several solid tumor are con icting: their affecting on cancer cells seem to be different in different reports (8). In GC, TANs isolated from fresh surgical tumor tissues have been shown to promote immunosuppression and EMT process via GM-CSF-PD-L1(9) and IL-17a(36) pathways respectively. Through recruiting macrophages and T-regulatory cells, TANs have been demonstrated that can promote the growth, progression, and resistance to sorafenib in HCC (41). While TANs have been shown to regulate other immune cells to enhance their tumor promoting functions, others have reported a direct role of TANs on tumor cells with little evidence on how neutrophils are activated to TANs (36, [42][43][44]. In this study, we found TANs activated by breast cancer cells to promote the proliferation, invasion and migration of MCF-7 cells. These results indicated that, in breast cancer microenvironments, neutrophils activated by breast cancer cells can promote the biological behavior of cancer cells, suggesting an interaction between cancer cells and in ltrating neutrophils. Accumulating evidence have suggested that cytokines including but not limited to IL8(45), IL6(33), can alter the phenotype of neutrophils towards tumor promoting effects (46). Herein, we found G-CSF was the key component in MDA231CS prolonging the lifespan and metastasis abilities of TANs. Our study on breast cancer tissues and surgical tissues also showed a positive correlation between TANs and the expression of G-CSF. It is known that G-CSF is a critical regulator of the proliferation, differentiation, and survival of granulocytes (47,48). Previous study showed that G-CSF promoted mouse granulocytes, Ly6G cells, differentiation and decreased T cell proliferation and promoted pancreatic ductal adenocarcinoma growth in a mouse model (49). High levels of G-CSF expression have been observed and reported to be associated with poor OS in human triple-negative BC (24). However, G-CSF' role in TANs activation in BC were seldomly reported. In mouse models, 4T1 murine mammary tumors are known to produce G-CSF and increase the numbers of immunosuppressive CD11bGr1 myeloid-derived suppressor cells (MDSCs) in tissues such as the spleen and lungs of tumor-bearing mice (50). Studies in mice also showed that pretreatment with recombinant G-CSF promotes 4T1 cell lung metastasis by modifying Ly6G+Ly6C+ granulocytes (51). The above research supports the results of our study, which showed that tumor-derived G-CSF can activate neutrophils to enhance metastasis of breast cancer cells. However, in this study, both MDA231-TANs and MCF7-TANs cannot promote the invasion and migration of their own breast cancer cell lines which was same as it previous reported (10). A previous study has demonstrated a high production of G-CSF in MDA-MB-231 cells than in MCF-7 cells to trigger the activation of M2 TAMs (24). Accordingly, we observed that after the upregulation of G-CSF in MCF-7 cells, MCF7-TANs signi cantly promoted the migration and invasive of MCF-7 cells. In a mouse model, polarization of neutrophils by G-CSF has been shown to promote pulmonary and lymph node metastases (48). Taken together, our ndings and the aforementioned studies suggest that G-CSF plays an important role in the activation and tumor promoting functions of TANs in breast cancer tumor microenvironments.
Besides Wang's report on activation of neutrophils to induce PD-L1 expression via G-CSF/JAK/STAT-3 cell signaling pathway of PD-L1(9), the signaling pathway regulating TANs activation in breast cancer is unknown. Our study showed the expression of PI3K, p-AKT and nuclei NFκB p65 to be signi cantly upregulated in TANs. This suggested that the G-CSF-PI3K-AKT signaling pathway activated and prolonged the lifespan of TANs while G-CSF -NFκB pathway regulated the pro-metastatic ability of TANs in breast cancer cells. This nding reveals a new insight into the role of TANs in BC.
Our study showed that the level of RLN2 was signi cantly increased in TANCS and these TAN-derived RLN2 was the key component which promoted the invasion and migration of BC cells via PI3K-AKT-MMP-9 axis. These results were also proved in BC surgical tissues, where a positive correlation among the density of TANs and the expression of RLN2 and MMP-9 was observed. RLN2 is known to play an important role in pregnancies, including those of humans. However, its role in cancer still remains very controversial (52). Studies on endometrial cancer and prostatic carcinoma have proved that RLN2 promotes the metastasis and invasion of cancer cells and could be a biomarker to predict prognosis (53). In BC, although RLN2 is already known to promote in vitro invasiveness by upregulating MMP-9 expression in BC cell lines, the ndings regarding its functions still remain con icts (54). Binder et al. reported RLN2 serum levels to correlate positively to the metastatic of BC (55). Others have reported that RLN2 can promote the of breast cancer cells by upregulating S100A/MMP-9 signaling (54, 56). However, our study is the rst to show the presence of RLN2 in TANs-conditioned medium rather than RLN2 alone to promote the migration of BC cells. We also found that by targeting RLN2 receptor, RXFP1, TANs-derived RLN2 promoted the metastasis of BC cells by upregulating the expression of MMP-9 via PI3K-AKT signaling pathway. However, others have reported that RLN2 as a matrix-depleting agent, has the potential to improve penetration and consequently the e cacy of anticancer immunotherapies and chemotherapies (57,58). Therefore, targeting the RXFP1-RLN2 pathway appears to have both antitumor and protumor effects. A Study in HCC have demonstrated that the upregulation of RLN2 mitigates liver metastasis (58), while others found that both ligand-derived and receptor antagonists targeting RLN2 and RXFP1 respectively could result in antitumor activities in xenograft models of prostatic cancer (59). Recently, Sumera et al. explained that these two faces of the pathway is largely depended on the delivery method of RLN2 and tumor type (52). According to our study, RLN2 secreted by TANs promoted the migration of BC cells.
Our study indicated that high density of TANs in BC is positively related to cancer metastasis and poor outcomes, and as proved in