Journal of Leukocyte Biology, 2023, 113, 383–399

https://doi.org/10.1093/jleuko/qiad004
Advance access publication 18 January 2023
Article

Tumor-activated neutrophils promote metastasis in

breast cancer via the G-CSF-RLN2-MMP-9 axis
Youjing Sheng,1,2,† Weidong Peng,1,† Yan Huang,3,† Lanqing Cheng,4 Ye Meng,5 Louis Boafo Kwantwi,1 Jiezhen Yang,6 Jiegou Xu,7
Han Xiao,1 Julia Kzhyshkowska,2,8,9,10,* and Qiang Wu1,6,9,*
1
Department of Pathology, The First Affiliated Hospital of Anhui Medical University, 218 Jixi Road, Shushan District, Hefei 230022, Anhui, PR China
2
Institute of Transfusion Medicine and Immunology, Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, University of Heidelberg,

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Ludolf-Krehl Strasse 13-17, 68167 Mannheim, Germany
3
Department of Pharmacy, Anhui Medical University, Hefei, Anhui 230032, PR China
4
Department of Pathology, The Second Affiliated Hospital of Anhui Medical University, 678 Furong Road, Shushan District, Hefei, Anhui 230601, PR China
5
Department of Hematology, The Second Affiliated Hospital of Anhui Medical University, 678 Furong Road, Shushan District, Hefei, Anhui 230601, PR China
6
Department of Pathology, Anhui Medical University, 81 Meishan Road, Shushan District, Hefei, Anhui 230032, PR China
7
Department of Immunology, Anhui Medical University, 81 Meishan Road, Hefei, Anhui 230032, PR China
8
German Red Cross Blood Donor Service Baden-Württemberg–Hessen, Friedrich-Ebert Strasse 107, 68167 Mannheim, Germany
9
Laboratory of Translational Cellular and Molecular Biomedicine, Tomsk State University, Lenin Ave, 36, 634050 Tomsk, Tomsk Oblast, Russia
10
Laboratory of Genetic Technologies, Siberian State Medical University, Moskovskiy Trakt, 2, 634050 Tomsk, Tomsk Oblast, Russia
Corresponding authors: Qiang Wu, Department of Pathology, the First Affiliated Hospital of Anhui Medical University, 218 Jixi Rd, Shushan District Hefei, Anhui
230022, PR China. Email: aydjohn@yahoo.com; Julia Kzhyshkowska, Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg
University; Ludolf-Krehl Strasse 13-17, D-68167 Mannheim, Germany. Email: julia.Kzhyshkowska@medma.uni-heidelberg.de

Abstract
The immune component of the tumor microenvironment is essential for the regulation of cancer progression. In breast cancer (BC), a
patient’s tumor mass is frequently infiltrated by neutrophils (tumor-associated neutrophils, TANs). Our study addressed the role of
TANs and their mechanism of action in BC. Using quantitative IHC, ROC, and Cox analysis, we demonstrated that a high density of
TANs infiltrating the tumor parenchyma was predictive of poor prognosis and of decreased progression-free survival of patients with
BC, who underwent surgical tumor removal without previous neoadjuvant chemotherapy, in 3 different cohorts: training, validation,
and independent cohorts. Conditioned medium from human BC cell lines prolonged the lifespan of healthy donor neutrophils ex vivo.
Neutrophils activated by the supernatants of BC lines demonstrated an increased ability to stimulate proliferation, migration, and
invasive activity of BC cells. Cytokines involved in this process were identified using antibody arrays. The relationship between these
cytokines and the density of TANs was validated by ELISA and IHC in fresh BC surgical samples. It was determined that tumor-
derived G-CSF significantly extended the lifespan and increased the metastasis-promoting activities of neutrophils via the PI3K-AKT
and NF-κB pathways. Simultaneously, TAN-derived RLN2 promoted the migratory abilities of MCF7 cells via PI3K-AKT-MMP-9.
Analysis of tumor tissues from 20 patients with BC identified a positive correlation between the density of TANs and the activation of
the G-CSF-RLN2-MMP-9 axis. Finally, our data demonstrated that TANs in human BC have detrimental effects, supporting malignant
cell invasion and migration.

Keywords: tumor-associated neutrophils, breast cancer, NF-κB pathway, migration, PI3K-AKT pathway
Abbreviations: AUC, Area under the curve; BC, Breast cancer; EMT, Epithelial-mesenchymal transition; FFPE, Formalin-fixed and
paraffin-embedded; G-CSF, Granulocyte-colony stimulating factor; HCC, Hepatocellular carcinoma; HR, Hazard ratio; IHC,
immunohistochemistry; MCF7CS, MCF7 cell culture supernatant; MCF7TANCS, MCF7CS-treated neutrophils cell cultured
supernatant; MCF7TANs, MCF7CS-treated neutrophils; MDA231CS, MDA-MB-231 cell culture supernatant; MDA231-TAN-CS,
MDA231CS-treated neutrophils cell cultured supernatant; MDA231TANs, MDA231CS-treated neutrophils; MDSCs, Myeloid-derived
suppressor cells; MEM, Minimum essential medium; METABRIC, Breast Cancer Gene Expression Profiles; MMP-9, Matrix
metallopeptidase 9; MPO, Myeloperoxidase; NeuCS, Neutrophil cell culture supernatant; NLR, Neutrophil to lymphocyte ratio; OS,
Overall survival; p-AKT, Phospho-Akt; PFS, Progression-free survival; rh-, Recombination human cytokine-; RLN2, Relaxin-2;
RNA-seq, RNA sequencing; ROC curve, Receiver operating characteristic curve; RXFP1, Relaxin family peptide receptor 1; SFM,
Serum-free medium; siR-MMP9-MCF7, siR-MMP-9 transfected MCF7 cells; siR-NC-MCF7, siR-NC transfected MCF7 cells;
siR-RXFP-MCF7, siR-RXFP1 transfected MCF7 cells; STAT3, Signal transducer and activator of transcription 3; TANCS, TAN cell
culture supernatant; TANs, Tumor-associated neutrophils; TME, Tumor microenvironment.

1 Introduction reaching a 5-year overall survival (OS) in 90% of cases in the

Breast cancer (BC) is the most commonly diagnosed malignancy in
women. Owing to advances in early diagnostics and comprehen­
sive treatment, the prognosis of patients with BC has improved,
†
Contributed equally first authors.
United States and China.1,2 However, the rate of metastasis in­
creased from 2008 to 2017. Recent studies have shown that 30%
of patients with BC develop metastases after surgical treatment,
which is a critical factor in cancer-related deaths among wom­
en.2–4 It is also well established that the tumor microenvironment

Received: July 8, 2022. Editorial Decision: January 16, 2023. Corrected and Typeset: March 17, 2023
© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-
nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use,
please contact journals.permissions@oup.com
384 | Journal of Leukocyte Biology, 2023, Vol. 113, No. 4
(TME), particularly its immune component, controls the metastat­
ic spread of cancer cells.5,6 Therefore, exploring potential markers
for predicting BC metastasis and its underlying mechanisms is
essential.
Neutrophils act as the body’s first line of defense against infec­
tion and respond to diverse inflammatory cues.7–10 Neutrophils
are characterized by functional heterogeneity based on their dis­
tinct transcriptional programs.11 In addition to the physiological
role of neutrophils, tumor-associated neutrophils (TANs) play a
crucial role in tumor development and progression in cancer mi­
croenvironments.12–14 Several lines of evidence have shown the
dual role of neutrophils in the TME.6,15 TANs can affect the biology
of both cancer and intratumoral immune cells. Direct effects of
TANs on cancer cells have been reported in lung cancer,16 hepato­
cellular carcinoma (HCC),17 gastric cancer,18 and oral squamous
cell carcinoma.19 In several animal models of HCC, TANs have
been reported to be a component of tumor-promoting inflamma­
tion.7,8,20 TANs can also promote cancer progression by support­
ing the immune suppressive activity of T cells in gastric
cancer.13,21
However, neutrophils can also exhibit antitumor activities.22 In
the early stages of cancer, TANs directly kill cancer cells by releas­
ing nitric oxide synthase23 or neutrophil elastase.24 TANs can also
suppress tumor development by activating cytotoxic T cell re­
sponses in the TME of lung cancer25 and sarcoma.26 Therefore,
the role of TANs in cancer progression is complex and depends
on the type and stage of the cancer. However, the mechanism
that determines whether TANs support or inhibit tumors in a spe­
cific context remains to be established.
In addition to the controversial role of neutrophils, their value
in predicting the prognosis of patients with cancer is controver­
sial.7 In BC, the blood neutrophil-to-lymphocyte ratio (NLR)
showed prognostic value for poor clinical outcomes only in triple-
negative and HER-2 positive populations, but not in hormone
receptor-positive groups.27,28 Other studies have reported that
NLR does not significantly correlate with OS in patients with
BC.29 The predictive value of neutrophils infiltrating the TME
has been reported in several cancer types.21 A bioinformatics
study showed that TANs could be predictors of shorter survival
in patients with BC.30 Thus, the prognostic value of tumor-
infiltrating neutrophils requires further analysis in independent
cohorts of patients with different BC types.
In this study, we investigated the prognostic value of CD66b+
neutrophil density in BC tissues and its correlation with the clinical
characteristics of the patients. The ability of TANs to promote pri­
mary cancer growth and the metastatic potential of malignant cells
was examined ex vivo. Mechanistically, signaling pathways that
mediate the cancer-promoting activity of TANs were identified.
2 Materials and methods
2.1 Tumor cell culture
MDA-MB-231 and MCF7 cell lines were provided by the Cell Bank of
the Chinese Academy of Sciences. MDA-MB-231 cells were cultured
in L15 medium (HyClone, USA) supplemented with 10% FBS (fetal
bovine serum; Gibco, USA) and 100 U/mL penicillin/streptomycin
(HyClone). MCF7 cells were cultured in MEM (minimum essential
medium; HyClone) supplemented with 10% FBS (Gibco), 100 U/mL
penicillin/streptomycin (HyClone), and 0.01 µg/mL insulin. After
the cells adhered to the supernatant, the medium was replaced
with SFM (serum-free medium; Lonza, Switzerland), and after
72 h the supernatant was collected (MDA231CS).
2.2 Patient characteristics,
immunohistochemistry (IHC) assays,
and tumor tissues
Formalin-fixed and paraffin-embedded (FFPE) surgical specimens
were randomly selected from the First Affiliated Hospital of Anhui
Medical University. Equal number of samples from each BC subtype
was included; they belonged to patients who underwent surgical op­
erations between 2015 and 2016. In total, 302 samples were randomly
separated into training (n = 170) and validation (n = 132) cohorts. The
independent cohort consisted of 94 samples from patients with BC
whose FFPE tissues were obtained from the Second Affiliated
Hospital of Anhui Medical University (Supplementary Table 1). The
last follow-up was conducted on 10 February 2020. All patients
underwent treatment following the CSCO guidelines. This study
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was approved by the Clinical Research Ethics Committee of Anhui
Medical University (20180096; 20200976). All procedures performed
in this study followed the ethical standards of the institutional re­
search committee and the 1964 Declaration of Helsinki and its later
amendments or comparable ethical standards.
Each tumor sample was cut into 2-μm sections and used for IHC
staining. Antigen retrieval was performed using a pressure cooker
for 1.5 min in an antigen unmasking solution (sodium citrate, pH
6.0). Afterward, samples were incubated overnight at 4 °C with
human CD66b (1:100; Cat. 555724; BD Pharmingen, USA) as pri­
mary antibody and blocked using a biotin block kit (MXB
Biotechnologies, China) for 20 min at room temperature. This
was followed by incubation with a secondary antibody (HRP rab­
bit/mouse max vision kit, MXB Biotechnologies) for 20 min at
room temperature. Immunodetection was performed using DAB
(MXB biotechnologies) for 3 min. Images were captured using a
microscope. 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.
2.3 Neutrophil isolation and treatment
Fresh blood samples donated by healthy volunteers were treated
with EDTA and transferred to new tubes. Following cell magnetic
labeling (MACSxpress neutrophil isolation kit, Cat. 130-104-434;
Miltenyi, Germany), tube contents were mixed using a MACSmix
Rotator (Miltenyi) at 37 °C for 15 min, and neutrophils were sus­
pended in the supernatant. After eliminating erythrocytes with
RBC lysis buffer (Cat. 555899; BD Pharmingen, USA), neutrophils
were isolated.
Isolated neutrophils were cultured in 6-well culture plates
(Corning) with MDA231CS and SFM (1:1) or with SFM only. After
cultivation of neutrophils in 50%/50% MDA231 conditioned me­
dium (supernatant, CS)/serum-free medium for 10 h, the final
conditioned medium was harvested and named TANCS. For the
neutrophils that were cultured in 100% serum-free medium for
10 h, the harvested conditioned medium was named NeuCS. For
cytokine production, neutrophils were stimulated with G-CSF
(2 ng/mL), CXCL-1 (1.8 ng/mL), GDF-15 (1.4 ng/mL), and PDGF-AA
(1 ng/mL) (Peprotech, USA). To validate the functions of G-CSF,
we used TAN cell culture supernatant (TANCS) combined with a
neutralized antibody (1.6 mg/mL) (R&D Systems) to treat neutro­
phils. To evaluate the signaling pathway, neutrophils were treated
with TANCS in the presence of MK2206 (65 nM) and/or JSH-23
(7.1 μM) (Med Chem Express, USA).
2.4 Proliferation assays
Proliferation assays were performed in 96-well plates. In total,
3 × 103 cancer cells were seeded in each well, and the culture

medium was replaced after 24 h with TANCS, neutrophil cell cul­
ture supernatant (NeuCS), SFM, or MDA231CS. Then, after 24 h,
10 μL CCK8 solution (cell counting kit 8, CCK-8; Bestbio, China)
was added to each well, and plates were incubated at 37 °C for
2 h. The absorbance was measured at 460 nm using a plate reader.
2.5 Flow cytometry
The viability of TANs and neutrophils was determined using an
annexin V/PI apoptosis detection kit (Biobest). TANs and neutro­
phils were centrifuged at 300 × g for 5 min at 4 °C. After collecting
the supernatants and washing the cells twice, the cells were re­
suspended in 400 μL annexin V binding buffer. The cells were in­
cubated with annexin V-FITC at 4 °C for 15 min, followed by
incubation with PI-PE at 4 °C for 5 min. Annexin- and PI-positive
cells were detected using a FACSVerse flow cytometer (BD
Biosciences). Data were analyzed using FlowJo (version. 7.6.1).
2.6 Invasion and migration assays
After cancer cells were pretreated with SFM for 12 h, experiments
were performed in an 8 μm cell culture insert (Corning Company).
For the invasion assay, Matrigel (BD Company) diluted with SFM at
a 1:8 ratio was precoated in a chamber and solidified for over 2 h
at 37 °C. In total, 4 × 104 BC cells suspended in SFM were added to
the upper chamber, whereas TANCS or NeuCS were added to the
lower chambers. Cancer cells were left to migrate and invade for
6 h. Afterward, the cancer cells in the upper chamber were removed
with a swab, and the cells attached to the underside of the chambers
were fixed with 4% paraformaldehyde for 15 min and stained with
0.5% crystal violet for 1 min. Quantification was performed using
the mean number of cells in five 200× microscopic fields per cham­
ber. In some experiments, in the lower chamber, NeuCS was mixed
with IL-1γ (2 ng/mL), M-CSF (1.5 ng/mL), MIF (2 ng/mL), RLN2 (1 ng/
mL), VEGF (10 ng/mL), or CD31 (3 ng/mL) (Preprotein).
2.7 Western blotting
Proteins were extracted using a nuclear and cytoplasmic extrac­
tion kit (Bestbio). For phosphorylation protein analysis, all lysates
were treated with a phosphatase inhibitor cocktail (MCE). Proteins
were loaded onto SDS polyacrylamide gels and transferred to
PVDF membranes. The membranes were blocked with 5% BSA
in TBST at room temperature for 1 h. Afterward, the membranes
were incubated with the following primary antibodies: anti
p-AKT (1:1,000; Cat. 4060T; CST, USA), anti-p-STAT3 (1:1,000;
Cat. 4074S; CST, USA), p-ERK (1:1,000; Cat. 4370T; CST, USA),
PI3K (1:1,000; Cat. 4249S; CST, USA), P65 (1:1,000; Cat. 3031S;
CST, USA), β-catenin (1:1,000; Cat. 4970T; CST, USA), MMP-9
(1:1,000; Cat. 13667T; CST, USA), GAPDH (1:1,000; Cat. 5174S;
CST, USA), and histone H3 (1:1,000; Cat. 4499T; CST, USA) at 4 °C
overnight. Membranes were incubated with peroxidase-
conjugated secondary antibodies (CST), and the antigen-antibody
reaction was visualized using an enhanced chemiluminescence
assay (ECL, Thermo). The signals, as relative units of each lane col­
or density, were evaluated using ImageJ software.
2.8 ELISA
Tissue homogenates were isolated from fresh invasive ductal car­
cinoma samples obtained during patient surgeries. Briefly, 0.1 g of
each tumor was washed several times with cold PBS. After remov­
ing the blood, the tissue was placed in 0.9 mL cold PBS and homo­
genized using a homogenizer (NingBo Scientz Biotechnology,
Shanghai) at 4 °C and 70 rpm for 10 min. The samples were then
centrifuged at 3,000 rpm at 4 °C, and the supernatants were
Sheng et al. | 385
harvested. Protein concentration was determined using bicincho­
ninic acid (Cat. P0010, Beyotime, China). 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 and measured at
450 nm using a smart microplate reader (USCAN kit, Inc., China).
2.9 RNA transfection
Cells were plated at 5 × 105 cells/mL to knock down specific target
genes and transfected with specific siRNA duplexes using
Lipofectamine 3000 transfection reagent (Invitrogen, Cat. L3000015)
according to the manufacturer’s instructions. The siRNAs were
provided by GenePharma, Inc. (China; Supplementary Table 3).
Transfection efficiency was confirmed using qRT-PCR.
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2.10 qRT-PCR
We used qRT-PCR to evaluate the mRNA levels of RXFP1 and
MMP-9. Total RNA was isolated from cultured cells using TRIzol
reagent. RNA was reverse-transcribed into cDNA using a
PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd.,
Dalian, China). qRT-PCR was performed using a SYBR Prime
Script kit (Takara Bio, Inc., Otsu, Japan). The primers were pro­
vided by GenePharma, Inc. (China; Supplementary Table 3). PCR
was conducted at 95 °C for 15 min, followed by 40 cycles at 94 °C
for 15 s, 55 °C for 30 s, and 64 °C for 30 s. The experiment was re­
peated three times, and the expression of RXFP1 and MMP-9 was
quantified using the 2−ΔΔCq method.31
2.11 Cytokine antibody arrays
Cytokine antibody arrays (R&D Systems) were used to evaluate the
cytokine profiles of different cell culture supernatants. TANCS,
NeuCS, MDA231CS, and SFM were collected and diluted with array
buffer. After blocking with array buffer for 1 h, the array membrane
was incubated with 1.5 mL dilutions overnight at 4 °C. After incuba­
tion with HRP-conjugated detection antibodies for 1 h, the signals
were visualized using an ECL kit (Thermo Fisher) and quantified,
as relative units of spot color density, using ImageJ software.
2.12 Statistical analysis
Statistical analysis was performed using Prism (version 8.0.1;
GraphPad Software) as follows: paired 2-tailed Student’s t-test
(Figs. 2A–D, 3B–E, 4B–D, 5A, B, 7B, C, and Supplementary Fig. 5);
multiple and universal Cox tests (Fig. 1D, G, J, and M; Table 1)
were performed using R software. For all analyses, significance
was defined as P < 0.05.
2.13 Bioinformatics analysis
RNA-sequencing (RNA-seq) expression data and clinical charac­
teristics of patients with breast cancer were downloaded from
METABRIC database in cBioPortal (http://www.cbioportal.org/).
CIBERSORT (http://cibersort.stanford.edu/) was applied to esti­
mate and analyze the immune cell infiltration through RNA-seq
expression profiles. The average value of neutrophil levels was
then applied to divide the cohorts into 2 groups: high levels of
TANs and low levels of TANs.
3 Results
3.1 A high density of TANs is related to poor
prognosis of patients with BC
To investigate the presence of TANs in BC, clinical BC surgical
samples (Supplementary Table 1) were obtained from 2
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Figure 1 TANs, infiltrating in tumor parenchyma, are associated with poor PFS in patients with breast cancer (BC). (A) CD66b+ neutrophils in the
parenchyma tissues of patients with BC, detected by IHC staining. Scale bars, 50 μm. (B–D) Kaplan–Meier survival curves showing that patients
with high density of TANs had a poor PFS in all 3 training (P < 0.001, HR = 2.036; B), validation (P < 0.001, HR = 3.113; C), and independent (P = 0.0221,
HR = 2.078; D) cohorts. (E–G) Multivariate Cox test results suggesting that a high density of TANs is an independent risk factor in BC progression in all 3
training (HR = 2.8; E), validation (HR = 4.0; F), and independent (HR = 3.4; G) cohorts. (H–J) ROC analyses showing that TANs have a high prognostic value
for predicting PFS in the training (P < 0.001, AUC = 0.80; H), validation (P < 0.001, AUC = 0.82; I), and independent (P = 0.0004, AUC = 0.72; J) cohorts.
(K) TCGA dataset analyses showing that high levels of neutrophils negatively correlate with overall survival (n = 1100, P = 0.028, HR = 1.21). (L)
METABRIC database analyses showing that a high level of neutrophils negatively correlated with release-free survival in patients with BC (n = 1222,
P = 0.0233, HR = 1.297). (M) Multivariate Cox analyses showing that a high density of TANs is an independent risk factor in release-free survival of
patients with BC (P = 0.018, HR = 1.3).
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Figure 2 After being treated with BC cell cultured supernatant, neutrophils are activated to TANs and promote biological behaviors of BC cells. (A)
After being treated with MCF7CS or MDA231CS for 10 h, the lifespan of neutrophils was evaluated by flow cytometry. Result showed that the lifespan of
MCF7TANs (P = 0.0111) and MDA231TANs (P = 0.0003) was significantly extended. (B) After treatment with TANCS, compared to NC, NeuCS, and
MDA231 CS the proliferation of MCF7 cells was independently evaluated by CCK8 at 24, 48, and 96 h. MCF7 cell proliferation was significantly enhanced
after 24 and 48 h (P < 0.0001). (C) After treatment with TANCS, the invasion (P = 0.0011) and migration (P = 0.0043) abilities of MCF7 cells were
significantly promoted. Magnification: 200×.
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Figure 3 Tumor-derived G-CSF activates neutrophils into TANs in BC. Antibody array assays showed that, compared with those from CM, NeuCS, and
TANCS, the expression levels of G-CSF, CXCL-1, GDF-15, and PDGF-AA were higher in MDA231CS. (A) These results were confirmed by ELISA (n = 3). The
viability of neutrophils exposed to four mediators was then analyzed. As shown, the four-mediator treatment (B) extended the lifespan (P < 0.0001) and
(C) promoted invasion and migration (P < 0.0001) abilities of neutrophils. A three-mediator treatment, lacking G-CSF, could not promote the viability of
neutrophils, whereas G-CSF-stimulated neutrophils showed increased viability and invasion and migration abilities (P < 0.0001).

institutions, and an IHC assay was performed for CD66b. CD66b+
neutrophils infiltrating the parenchyma of the BC tissues were
counted (Fig. 1A). The median value of the density of TANs was
chosen as the cutoff value to divide the patients into 2 groups.
The prognostic value was then analyzed using survival and Cox
multivariate analyses. In all 3 cohorts, a high density of TANs was
positively correlated with poor progression-free survival (PFS) in
BC patients (training cohort: Fig. 1B, P < 0.0001; validation cohort:
Fig. 1C, P < 0.0001; independent cohort: Fig. 1D, P = 0.0221 < 0.05).
Univariate analyses revealed a significant positive association be­
tween TANs and PFS in all 3 cohorts (Table 1). Cox multivariate
analyses revealed that a high density of TANs was an independent

Figure 4 After G-CSF upregulation, MCF7 cells promote procancer activity in neutrophils. (A) G-CSF upregulation in MCF7significantly extended
neutrophil lifespan (P = 0.0325). (B) Treated with G-CSF-MCF7TANCS, both the invasion (P = 0.0014) and migration (P = 0.0008) abilities of MCF7 cells
were significantly promoted (n = 3). (C) The expression of G-CSF positively correlated with the density of TANs in BC tissues (n = 20, P = 0.012, rs = 0.553).
prognostic factor for poor PFS in all 3 cohorts (training cohort:
Fig. 1E, hazard ratio (HR) = 2.8, P < 0.001; validation cohort:
Fig. 1F, HR = 4.0, P < 0.001; independent cohort: Fig. 1G, HR = 3.4,
P < 0.001).
The prognostic value of TANs was then assessed using ROC
analysis. TANs showed a high prognostic value in predicting
poor PFS in all 3 training (Fig. 1H, area under the curve [AUC] =
0.80, 95% confidence interval [CI]: 0.74 to 0.87), validation
(Fig. 1I, AUC = 0.82, 95% CI: 0.75 to 0.90), and independent
(Fig. 1J, AUC = 0.72, 95% CI: 0.61 to 0.82) cohorts. The prognostic
value of TANs for OS was validated using TCGA datasets.
According to TIMER, a high density of TANs was positively corre­
lated with poor OS (Fig. 1K, P = 0.018, HR = 1.36).
To further confirm our findings, RNA-seq data from 1,222
patients with BC from METABRIC were downloaded and analyzed.
High levels of TANs were positively correlated with poor PFS
(Fig. 1L, P = 0.023 < 0.05). Univariate analyses revealed that high lev­
els of TANs were associated with a worse PFS (P = 0.023, HR = 1.29,
Supplementary Table 2). Cox multivariate analyses also demon­
strated that a high neutrophil count was an independent prognostic
factor (Fig. 1M, P = 0.018, HR = 1.3).
3.2 TAN activation by BC cells promotes
different MCF7 activities
Neutrophils were isolated from the peripheral blood of healthy
donors as reported previously.32 The purity of isolated neutrophils
was >90% (Supplementary Fig. 1A).
Neutrophils were cultured in the presence of 50% of condi­
tioned supernatant (CS) of MDA231 (MDA231CS) or conditioned
Sheng et al. | 389
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supernatant of MCF7 (MCF7CS). After 10 h of neutrophil cultiva­
tion, both cells and conditioned medium were harvested. The har­
vested cells were named MDA231TAN or MCF7TAN, respectively,
and conditioned medium of neutrophils was named
MDA231-TAN-CS or MCF7-TAN-CS, respectively. For neutrophils
cultured in 100% serum-free medium for 10 h, the harvested cells
and conditioned medium were named Neu and NeuCS, respect­
ively. Significantly increased lifespan of MCF7TANs (P = 0.0111)
or MDA231TANs (P = 0.0003) was detected when compared to
Neu. However, the lifespan of MDA231TANs was significantly lon­
ger than that of MCF7TANs (Fig. 2A).
We further evaluated the role of TANs in BC cells. After cultur­
ing MDA-MB-231 and MCF7 cells with either MDA231-TAN-CS or
MCF7-TAN-CS; no significant changes were identified for migra­
tion and invasion activity of cells (Supplementary Fig. 1B).
However, MDA231-TAN-CS promoted the proliferation (Fig. 2B,
P < 0.0001), migration (Fig. 2C, P = 0.0043), and invasion (Fig. 2C,
P = 0.0011) abilities of MCF7; hence, MDA231-TAN-CS was chosen
for further investigation.
3.3 Neutrophils are activated by tumor-derived
G-CSF and converted into TANs in BC
Based on the above findings, the active components of MDA231CS
were investigated using cytokine antibody arrays. Compared with
those in MDA231CS, the levels of G-CSF, CXCL-1, GDF-15, and
PDGF-AA were significantly lower in TANCS (Fig. 3A).
These 4 solute mediators enhanced both the viability and
proinvasive abilities of TANs. To identify the key component,
neutrophils were stimulated by alternated mixtures of 3 of
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Figure 5 G-CSF activate TANs (GCSFhigh-MCF7-TANs) in BC via the PI3K-AKT and NF-κB signaling pathways. (A) The expression of PI3K, p-AKT, and
nuclei-p65 was enhanced in TANs and GCSFhigh-MCF7-TANs, compared to that in neutrophils. (B) When treated with MK2206 or JSH-23, the viability or
invasion and migration abilities, respectively, of GCSFhigh-MCF7-TANs cannot be promoted. (C) When treated with both inhibitors, viability-enhanced
and tumor-promoting activities cannot be seen in GCSFhigh-MCF7-TANs.
 Sheng et al. | 391

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Figure 6 TANs promote the migration and invasion of MCF7 cells by activating the RLN2-PI3K-AKT-MMP-9 pathway. (A) The presence of high levels
of the mediators IL-1ra, M-CSF, MIF, RLN2, VEGF, and sCD31 in TANCS was determined by ELISA. (B) When treated with TANCS or NeuCS + 6 mediators,
the invasion and migration of MCF7 cells are significantly promoted, whereas when treated with NeuCS + 5 mediators (excluding RLN2), neither
invasion nor migration of MCF7 cells can be promoted.

these 4 cytokines. The results suggested that, without rh-G-CSF Supplementary Figs. 2 and 3). Moreover, to further investigate
stimulation, both the viability and migration-promoting the role of G-CSF, neutrophils were stimulated with G-CSF.
ability of neutrophils were not stimulated (Fig. 3B and C, The results indicated that the viability and proinvasive and
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Figure 7 TANs promote MCF7 cell invasion and migration via RLN2-RXFP1. (A) Neutrophils treated with either MDA231CS or NueCS + RLN2 promote
both invasion and migration of MCF7 cells. (B) After knocking down RXFP1 in MCF7, both the invasion and migration abilities of these cells cannot be
promoted, neither by TANCS nor NeuCS + RLN2.

prometastatic properties of the neutrophils were significantly
promoted (Fig. 3B and C).
To confirm the role of G-CSF as the main component in the
neutrophil activation phenotype, a G-CSF-overexpression vector
was transfected into MCF7 cells (GCSFhigh-MCF7). After exposure
to the GCSFhigh-MCF7 supernatant, the viability of neutrophils
was significantly enhanced. Moreover, significantly increased in­
vasion and migration by MCF7 cells were observed after cultur­
ing them in the supernatant of GCSFhigh-MCF7-TANs (Fig. 4A
and B).
 Sheng et al. | 393

Figure 8 TAN-derived RLN2 promotes MCF7 cell invasion and migration via the PI3K-AKT-MMP-9 axis. (A) Western blot analysis of PI3K, p-AKT, and Downloaded from https://academic.oup.com/jleukbio/article/113/4/383/6991105 by guest on 12 May 2023
MMP-9 in siR-NC-MCF7, TAN-siR-NC-MCF7, Neu-siR-NC-MCF7, Neu + RLN2-siR-NC-MCF7, and TAN-siR-MCF7. (B) After knocking down MMP-9 in MCF7
cells, the invasion and migration abilities of these cells cannot be promoted by TANCS or NueCS + RLN2. (C) Relationship among TAN density and RLN2
and MMP-9 expression in BC surgical tissues.

The relationship between G-CSF and TANs was investigated in
BC surgical tissues. Twenty fresh BC and FFPE tissue samples
were collected. The levels of G-CSF were investigated using ELISA,
and the density of TANs was evaluated using IHC. The
results showed a positive correlation between these 2 factors
(Fig. 4C, P = 0.012, rs = 0.553).
3.4 PI3K-AKT and NF-κB signaling pathways are
activated in TANs by G-CSF
The mechanism underlying the effect of G-CSF on neutrophil ac­
tivation was also investigated. Several signaling pathways, such
as PI3K- AKT, JAK-STAT3, MAPK-ERK1, NF-κB, and Wnt–
β-catenin, have been shown to regulate the effect of G-CSF on
the activation of neutrophils.33–37 Compared with those in nonsti­
mulated neutrophils, the levels of PI3K, p-AKT, and nuclear NF-κB
were significantly increased in TANs and GCSFhigh-MCF7-TANs
(Fig. 5A and Supplementary Fig. 4).
After exposure to MK2206 (an AKT inhibitor), the lifespan of
TANs was significantly decreased, while their ability to promote
tumor activities was not affected. In contrast, after exposure to
JSH-23 (an NF-κB inhibitor), the ability of TANs to promote tumor
activity was significantly decreased, while the lifespan was not
394 | Journal of Leukocyte Biology, 2023, Vol. 113, No. 4

Table 1. Univariate analysis for training, validation, and independent cohorts.

Training cohorts Validation cohorts Independent cohorts
HR 95% CI P value HR 95% CI P value HR 95% CI P value

High density of TANs 3.07 (2.017,4.685) 0.00 3.77 (2.111,6.73) 0.00 3.02 (1.545,5.888) 0.00
HER2(+) 1.89 (1.206,2.971) 0.01 0.95 (0.403,2.235) 0.90 0.92 (0.444,1.92) 0.83
ER(+) 0.84 (0.561,1.266) 0.41 1.62 (0.875,2.991) 0.13 0.59 (0.301,1.142) 0.12
PR(+) 0.90 (0.594,1.357) 0.61 2.27 (1.177,4.38) 0.01 0.56 (0.286,1.098) 0.09
Age <50 1.06 (0.714,1.584) 0.76 1.08 (0.609,1.926) 0.79 1.01 (0.519,1.964) 0.98
ki-67 ≥30% 1.47 (0.972,2.228) 0.07 0.95 (0.528,1.713) 0.87 1.53 (0.748,3.12) 0.25
LN(+) 1.99 (1.314,3.025) 0.00 1.00 (0.557,1.778) 0.99 1.56 (0.803,3.033) 0.19
Stage 3 2.01 (1.316,3.084) 0.00 1.79 (0.643,5) 0.26 3.55 (1.81,6.944) 0.00
T ≥3 cm 1.30 (0.846,1.988) 0.23 1.00 (0.557,1.778) 0.99 3.30 (1.552,7.034) 0.00

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affected. After exposure to both MK2206 and JSH-23, neutrophils
could not be activated into TANs, neither by MDA231CS nor by
rh-G-CSF (Fig. 5B and C).
3.5 TANs promote the migration and
invasion of MCF7 cells by activating the
RLN2-PI3K-AKT-MMP-9 pathway
The role of TANs in BC cells was also investigated. According to
the cytokine array assay, there were significantly increased ex­
pression levels of IL-1ra, M-CSF, MIF, RLN2, VEGF, and sCD31 in
TANCS (Fig. 3A); these results were confirmed by ELISA (Fig. 6A).
After stimulation by all 6 cytokines together with NeuCS, the
migration and invasion of MCF7 cells were promoted. To identify
key components, MCF7 cells were stimulated by NeuCS with alter­
nated mixtures of 5 of the 6 cytokines. The results showed that,
without RLN2 stimulation, the invasion and migration of MCF7
cells could not be promoted (Fig. 6B and Supplementary Fig. 5A).
Additionally, to identify the role of RLN2 in TANCS, MCF7 cells
were treated with NeuCS + RLN2. Transwell assays showed that
both the migration and invasion of MCF7 cells were significantly
promoted in the presence of NeuCS + RLN2, whereas treatment
with rh-RLN2 alone did not promote any migration or invasion
(Fig. 7A). As it is known that RLN2 targets RXFP1 in BC cells, the
expression of the latter was knocked down by siR-RXFP1 in
MCF7 cells (siR-RXFP-MCF7). MCF7 cells transfected with siR-NC
were used as controls (siR-NC-MCF7, Supplementary Fig. 5B).
Treatment with TANCS and NeuCS + rh-RLN2 did not promote
the migration and invasion of siR-RXFP-MCF7 cells. As expected,
these 2 supernatants promoted both migration and invasion of
siR-NC-MCF7 cells (Fig. 7B).
RLN2 promotes BC cell migration and invasion by activating the
PI3K-AKT-MMP-9 signaling pathway.38 Therefore, we examined
the specific role of RLN2 in the pathway. The levels of PI3K,
p-AKT, and MMP-9 were analyzed by Western blotting.
Elevated levels of PI3K, p-AKT, and MMP-9 were detected in
TANCS-stimulated MCF7 compared to NeuCS-stimulated MCF7
(Fig. 8A). Although added purified RLN2 did not increase the ex­
pression of PI3K, MMP9 and phosphorylation of AKT (level of
p-AKT) in MCF7 stimulated by conditioned medium of normal
neutrophils, blocking of RLN2 with antibodies in MCF7 stimulated
by conditioned medium of tumor-associated neutrophils (TANCS)
had a clear inhibitory effect on MMP9 expression level, and also on
p-AKT levels (Fig. 8A). These data suggest that RLN2, despite being
critical for the activation of p-AKT-dependent MMP9 production,
still needs to cooperate with other TAN-derived factors to activate
this pathway in cancer cells. Inhibition of p-AKT with MK2206
MCF7 stimulated by TANCS additionally demonstrated that
p-AKT mediates MMP9 production (Fig. 8A). After knocking
down MMP-9 in MCF7 cells using a siRNA (siR-MMP9), migration
and invasion were not promoted in cancer cells, regardless of
treatment with TANCS or NeuCS + RLN2 (Fig. 8B).
The relationships among TANs, RLN2, and MMP-9 were eval­
uated in 20 BC surgical tissues by IHC and ELISA. The results
showed that the density of TANs was positively related to the
level of expression of RLN2 (rs = 0.4805, 95% CI: 0.01754 to
0.7738, P = 0.0436) and MMP-9 (rs = 0.8425, 95% CI: 0.6098 to
0.9415, P < 0.0001). A positive correlation was also observed be­
tween the expression of RLN2 and MMP-9 (rs = 0.7495, 95% CI:
0.4225 to 0.9038, P = 0.0003) (Fig. 8C).
4 Discussion
Accumulating evidence is available for the complex composition
and cancer-promoting properties of the BC tumor immune micro­
environment in patients and animal tumor models.39–41 Although
some immune/inflammatory cells have been shown to interact
with BC cells as the body’s first line of defense, the role of neutro­
phils in the cancer microenvironment is largely unknown.7,42
Due to limited knowledge about TANs, markers for IHC stain­
ing and of prognostic value in patients with BC have not been fully
defined.7,25,43,44 However, it is known that CD66b is exclusively ex­
pressed by neutrophils, and it was accepted as specific for these
white blood cells. Given that the presence of activated neutrophils
correlates with the progression of several cancers,18,45–55 CD66b
was used here as a marker of TANs. Consistent with the findings
of previous studies, we found that a high density of CD66b+
TANs infiltrating the BC tissue parenchyma positively correlated
with PFS of patients. According to ROC and Cox analyses, the
density of CD66b+ TANs may be both specific and accurate in pre­
dicting poor PFS in patients with BC. These results suggest that
evaluating the density of TANs in BC tissues by CD66b immunos­
taining could be an easily accessible method for predicting BC
progression.
Although the density of TANs has proven to be an effective bio­
marker for predicting BC metastasis and poor clinical outcomes, its
role in cancer progression remains unclear. TANs have been
ignored as a group of immune cells in TMEs because of their short
lifespan (4 to 10 h). It is commonly recognized that the lifespan of
inflammation-activated neutrophils can be enhanced by overex­
pression of PCNA56,57 (>10 h).58 However, it is unknown whether
the lifespan of TANs is enhanced by cancer cells. Recent studies
have shown that the viability of neutrophils can be enhanced in
TMEs.7,44 Therefore, an extended lifespan can be regarded as a
novel marker of neutrophil activation. However, the activation of
neutrophils to TANs and their interplay with tumor cells remain
unclear. It is becoming increasingly clear that TANs can promote
tumor progression. The role of TANs in several solid tumors is

conflicting; their effects on cancer cells seem to differ among dif­
ferent reports.22 In gastric cancer, TANs isolated from fresh surgi­
cal tumor tissues have been shown to promote
immunosuppression and EMT via the GM-CSF-PD-L113 and
IL-17a18 pathways, respectively. By recruiting macrophages and
T-regulatory cells, TANs have been demonstrated to promote
growth, progression, and resistance to sorafenib in HCC.59
While TANs have been shown to regulate other immune cells
to enhance their tumor-promoting functions, some reports
have demonstrated a direct role of TANs in tumor cells, with lit­
tle evidence on how neutrophils are activated by TANs.18,60–62 In
this study, we found that TANs activated by BC cells promoted
the proliferation, invasion, and migration of MCF7 cells. These
results indicate that, in BC microenvironments, neutrophils acti­
vated by BC cells can promote the biological behavior of cancer
cells, suggesting an interaction between cancer cells and infil­
trating neutrophils.
Accumulating evidence suggests that cytokines, including but
not limited to IL-863 and IL-6,50 can alter the phenotype of neutro­
phils toward tumor-promoting effects.64 Herein, we found that
G-CSF was the key component in MDA231CS, prolonging the life­
span and metastatic abilities of TANs. Our study on BC and surgi­
cal tissues also showed a positive correlation between TANs and
G-CSF expression. It is known that G-CSF is a critical regulator
of the proliferation, differentiation, and survival of granulo­
cytes.65,66 A previous study showed that G-CSF promoted mouse
granulocyte proliferation and Ly6G cell differentiation, decreased
T cell proliferation, and stimulated pancreatic ductal adenocar­
cinoma growth in a mouse model.67 Moreover, high levels of
G-CSF expression have been associated with poor OS in human
triple-negative BC.43 However, the role of G-CSF in TAN activation
in BC has seldom been reported. In mouse models, 4T1 murine
mammary tumors are known to produce G-CSF and increase the
number of immunosuppressive CD11b+ Gr1+ myeloid-derived
suppressor cells (MDSCs) in tissues such as the spleen and lungs
of tumor-bearing mice.68 Studies in mice have also shown that
pretreatment with recombinant G-CSF promotes 4T1 cell lung
metastasis by modifying Ly6G+Ly6C+ granulocytes.69 These
works support the results of our study, which showed that tumor-
derived G-CSF could activate neutrophils to enhance BC cell me­
tastasis. However, in this study, neither MDA231TANs nor
MCF7TANs promoted the invasion and migration of their own
BC cell lines, as previously reported.17 A previous study demon­
strated higher production of G-CSF in MDA-MB-231 cells than in
MCF7 cells, triggering the activation of M2 TAMs.43 Accordingly,
we observed that, after G-CSF upregulation in MCF7 cells,
MCF7TANs significantly promoted the migration and invasion of
MCF7 cells. In a mouse model, the polarization of neutrophils by
G-CSF has been shown to promote pulmonary and lymph node
metastases.66 Taken together, our findings and those of the afore­
mentioned studies suggest that G-CSF plays an important role in
the activation and tumor-promoting functions of TANs in BC
TMEs.
In addition to Wang’s report on the activation of neutrophils to
induce PD-L1 expression via the G-CSF/JAK/STAT-3 cell signaling
pathway,13 the signaling pathway regulating TAN activation in BC
is unknown. Our study showed that the expression levels of PI3K,
p-AKT, and nuclear NF-κB p65 were significantly upregulated in
TANs. This suggests that the G-CSF-PI3K-AKT signaling pathway
is activated and prolongs the lifespan of TANs and that the
G-CSF-NF-κB pathway regulates the prometastatic ability of
TANs in BC cells. These findings provide new insights into the
role of TANs in BC.
Sheng et al. | 395
Our study showed that the level of RLN2 was significantly in­
creased in TANCS and that TAN-derived RLN2 was the key compo­
nent that promoted the invasion and migration of BC cells via the
PI3K-AKT-MMP-9 axis. These results were also confirmed in BC
surgical tissues, where a positive correlation among the density
of TANs, the expression RLN2, and the expression of MMP-9 was
observed. RLN2 is known to play an important role in pregnancy,
including that in humans. However, its role in cancer remains
very controversial.70 Studies on endometrial and prostate cancers
have shown that RLN2 promotes the metastasis and invasion of
cancer cells and could be a biomarker to predict prognosis.71 In
BC, although RLN2 is known to promote in vitro invasiveness by
upregulating MMP-9 expression in BC cell lines, findings regarding
its functions are still polemical.72 Binder et al. reported that RLN2
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serum levels correlate positively with BC metastasis.73 Others have
reported that RLN2 can promote the formation of BC cells by upre­
gulating S100A/MMP-9 signaling.72,74 However, our study is the
first to show the presence of RLN2 in TAN-conditioned medium ra­
ther than RLN2 alone to promote the migration of BC cells. We also
found that, by targeting the RLN2 receptor RXFP1, TAN-derived
RLN2 promoted the metastasis of BC cells by upregulating the ex­
pression of MMP-9 via the PI3K-AKT signaling pathway. However,
others have reported that RLN2, as a matrix-depleting agent, has
the potential to improve penetration and, consequently, the effi­
cacy of anticancer immunotherapies and chemotherapies.75,76
Therefore, targeting the RXFP1-RLN2 pathway appears to have
both antitumor and protumor effects. A study in HCC demon­
strated that the upregulation of RLN2 mitigated liver metastasis,76
while others found that both ligand-derived and receptor antago­
nists targeting RLN2 and RXFP1, respectively, could result in anti­
tumor activities in xenograft models of prostate cancer.77 Recently,
Sumera et al. explained that these 2 faces of the pathway are large­
ly dependent on the delivery method of RLN2 and tumor type.70
According to our study, RLN2 secreted by TANs promotes the mi­
gration of BC cells.
Our study indicated that a high density of TANs in BC is posi­
tively correlated with cancer metastasis and poor outcomes and
that, as proven in vitro, TANs promote the migration and invasion
of BC cells via the G-CSF-RLN2-MMP-9 axis. CD66b + TANs in BC
tissues could be useful markers for predicting BC metastasis and
PFS. Targeting either RLN2-RXFP1 or G-CSF may provide a new
therapeutic strategy for metastatic BC.
Acknowledgments
We thank Professor Xu Jiegou for advice on isolating neutrophils.
We thank Xiaowen Han, Yongsheng Fang, Yu Xia, Jingbo Fan, and
Dachao Liu for donating the blood. We thank the Flow Cytometry
Center at the Laboratory Department of the First Affiliated
Hospital of Anhui Medical University and the Laser Scanning
Confocal Microscopy Center at the Center for Scientific Research
of Anhui Medical University.
Author Contributions
Qiang Wu and Youjing Sheng designed the experiments; Youjing
Sheng performed most of the experiments and analyzed the
data; Youjing Sheng initiated the experimental work. Weidong
Peng and Lanqin Cheng contributed to key experiments. Youjing
Sheng and Ye Meng prepared the figures; Youjing Sheng con­
ducted the mathematical modeling; Jiegou Xu, Han Xiao, and
Jiezhen Yang were involved in specific experiments; Qiang Wu
provided resources and Yan Huang advised on experimental
396 | Journal of Leukocyte Biology, 2023, Vol. 113, No. 4
protocols; Youjing Sheng, Louis, Julia Kzhyshkowska, and Qiang
Wu analyzed the data and wrote the manuscript; Qiang Wu
funded the work and provided overall research supervision.
Supplementary material
Supplementary materials are available at Journal of Leukocyte
Biology online.
Funding
This work was supported by the Hefei Natural Science Funds
(2022037), the Anhui Institute of Translational Medicine
(2017zhyx36), by the Special Fund for Discipline Leaders of
Anhui Province (2019H230), by the state contract of the Ministry
of Science and Higher Education of the Russian Federation
“Genetic and epigenetic editing of tumor cells and microenviron­
ment in order to block metastasis” o. 075-15-2021-1073 and by
Tomsk State University Development Programme (Priority 20-30).
Conflict of interest
The authors have to conflict of interest.
Data availability
The datasets used and/or analyzed during the current study are
available from the corresponding author upon reasonable
request.
Study approval
Tumor resections were performed in patients with breast cancer
at the First Affiliated Hospital of Anhui Medical University. All pa­
tients had untreated primary tumors. Before resection, informed
consent was obtained, and the collection of patient samples was
approved by the ethical review board of the Anhui Medical
University Institute (20180096; 20200976) and followed the
Declaration of Helsinki. Peripheral blood samples were obtained
from 5 healthy volunteers (20200976).
References
1. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu
XQ, He J. Cancer statistics in China, 2015. CA Cancer J Clin.
2016:66(2):115–132. https://doi.org/10.3322/caac.21338
2. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2021.
CA Cancer J Clin. 2021:71(1):7–33. https://doi.org/10.3322/caac.
21654
3. Harbeck N, Penault-Llorca F, Cortes J, Gnant M, Houssami N,
Poortmans P, Ruddy K, Tsang J, Cardoso F. Breast cancer. Nat
Rev Dis Primers. 2019:5(1):66. https://doi.org/10.1038/s41572-019-
0111-2
4. Liang Y, Zhang H, Song X, Yang Q. Metastatic heterogeneity of
breast cancer: molecular mechanism and potential therapeutic
targets. Semin Cancer Biol. 2020:60(Feb):14–27. https://doi.org/10.
1016/j.semcancer.2019.08.012
5. Yin S, Wang N, Riabov V, Mossel DM, Larionova I, Schledzewski
K, Trofimova O, Sevastyanova T, Zajakina A, Schmuttermaier
C, et al. SI-CLP inhibits the growth of mouse mammary adenocar­
cinoma by preventing recruitment of tumor-associated macro­
phages. Int J Cancer. 2020:146(5):1396–1408. https://doi.org/10.
1002/ijc.32685
6. Long W, Chen J, Gao C, Lin Z, Xie X, Dai H. Brief review on the roles
of neutrophils in cancer development. J Leukoc Biol. 2021:109(2):
407–413. https://doi.org/10.1002/JLB.4MR0820-011R
7. Shaul ME, Fridlender ZG. Tumour-associated neutrophils in pa­
tients with cancer. Nat Rev Clin Oncol. 2019:16(10):601–620.
https://doi.org/10.1038/s41571-019-0222-4
8. Coffelt SB, Wellenstein MD, de Visser KE. Neutrophils in cancer:
neutral no more. Nat Rev Cancer. 2016:16(7):431–446. https://doi.
org/10.1038/nrc.2016.52
9. Galdiero MR, Varricchi G, Loffredo S, Mantovani A, Marone G.
Roles of neutrophils in cancer growth and progression. J Leukoc
Biol. 2018:103(3):457–464. https://doi.org/10.1002/JLB.3MR0717-
292R
10. Rosales C, Lowell CA, Schnoor M, Uribe-Querol E. Neutrophils:
Downloaded from https://academic.oup.com/jleukbio/article/113/4/383/6991105 by guest on 12 May 2023
their role in innate and adaptive immunity 2017. J Immunol Res.
2017:2017(Jan 12):9748345. https://doi.org/10.1155/2017/9748345
11. Maskarinec SA, McKelvy M, Boyle K, Hotchkiss H, Duarte ME,
Addison B, Amato N, Khandelwal S, Arepally GM, Lee GM.
Neutrophil functional heterogeneity is a fixed phenotype and is
associated with distinct gene expression profiles. J Leukoc Biol.
2022:112(6):1485–1495. https://doi.org/10.1002/JLB.4A0322-164R
12. Mizuno R, Kawada K, Itatani Y, Ogawa R, Kiyasu Y, Sakai Y. The
role of tumor-associated neutrophils in colorectal cancer. Int J
Mol Sci. 2019:20(3):529. https://doi.org/10.3390/ijms20030529
13. Wang T-T, Zhao Y-L, Peng L-S, Chen N, Chen W, Lv Y-P, Mao F-Y,
Zhang J-Y, Cheng P, Teng Y-S, et al. Tumour-activated neutro­
phils in gastric cancer foster immune suppression and disease
progression through GM-CSF-PD-L1 pathway. Gut. 2017:66(11):
1900–1911. https://doi.org/10.1136/gutjnl-2016-313075
14. Hiramatsu S, Tanaka H, Nishimura J, Sakimura C, Tamura T,
Toyokawa T, Muguruma K, Yashiro M, Hirakawa K, Ohira M.
Neutrophils in primary gastric tumors are correlated with neu­
trophil infiltration in tumor-draining lymph nodes and the sys­
temic inflammatory response. BMC Immunol. 2018:19(1):13.
https://doi.org/10.1186/s12865-018-0251-2
15. Shaul ME, Fridlender ZG. The dual role of neutrophils in cancer.
Semin Immunol. 2021:57(Oct):101582. https://doi.org/10.1016/j.
smim.2021.101582
16. Peng W, Sheng Y, Xiao H, Ye Y, Kwantwi LB, Cheng L, Wang Y, Xu
J, Wu Q. Lung adenocarcinoma cells promote self-migration and
self-invasion by activating neutrophils to upregulate Notch3 ex­
pression of cancer cells. Front Mol Biosci. 2021:8(Jan 18):762729.
https://doi.org/10.3389/fmolb.2021.762729
17. Zhou S-L, Yin D, Hu Z-Q, Luo C-B, Zhou Z-J, Xin H-Y, Yang X-R,
Shi Y-H, Wang Z, Huang X-W, et al. A positive feedback loop be­
tween cancer stem-like cells and tumor-associated neutrophils
controls hepatocellular carcinoma progression. Hepatology.
2019:70(4):1214–1230. https://doi.org/10.1002/hep.30630
18. Li S, Cong X, Gao H, Lan X, Li Z, Wang W, Song S, Wang Y, Li C,
Zhang H, et al. Tumor-associated neutrophils induce EMT by
IL-17a to promote migration and invasion in gastric cancer cells.
J Exp Clin Cancer Res. 2019:38(1):6. https://doi.org/10.1186/s13046-
018-1003-0
19. Hu X, Xiang F, Feng Y, Gao F, Ge S, Wang C, Zhang X, Wang N.
Neutrophils promote tumor progression in oral squamous cell
carcinoma by regulating EMT and JAK2/STAT3 signaling through
chemerin. Front Oncol. 2022:12(Jan 28):812044. https://doi.org/10.
3389/fonc.2022.812044
20. Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils
in the activation and regulation of innate and adaptive immun­
ity. Nat Rev Immunol. 2011:11(8):519–531. https://doi.org/10.
1038/nri3024

21. Kwantwi LB, Wang S, Zhang W, Peng W, Cai Z, Sheng Y, Xiao H,
Wang X, Wu Q. Tumor-associated neutrophils activated by
tumor-derived CCL20 (C-C motif chemokine ligand 20) promote
T cell immunosuppression via programmed death-ligand 1
(PD-L1) in breast cancer. Bioengineered. 2021:12(1):6996–7006.
https://doi.org/10.1080/21655979.2021.1977102
22. Jaillon S, Ponzetta A, Di Mitri D, Santoni A, Bonecchi R, Mantovani
A. Neutrophil diversity and plasticity in tumour progression and
therapy. Nat Rev Cancer. 2020:20(9):485–503. https://doi.org/10.
1038/s41568-020-0281-y
23. Finisguerra V, Di Conza G, Di Matteo M, Serneels J, Costa S,
Thompson A, Wauters E, Walmsley S, Prenen H, Granot Z.
MET is required for the recruitment of anti-tumoural neutro­
phils. Nature. 2015:522(7556):349–353. https://doi.org/10.1038/
nature14407
24. Cui C, Chakraborty K, Tang XA, Zhou G, Schoenfelt KQ, Becker
KM, Hoffman A, Chang YF, Blank A, Reardon CA, et al.
Neutrophil elastase selectively kills cancer cells and attenuates
tumorigenesis. Cell. 2021:184(12):3163–3177.e21. https://doi.org/
10.1016/j.cell.2021.04.016
25. Eruslanov EB, Bhojnagarwala PS, Quatromoni JG, Stephen TL,
Ranganathan A, Deshpande C, Akimova T, Vachani A, Litzky L,
Hancock WW, et al. Tumor-associated neutrophils stimulate T
cell responses in early-stage human lung cancer. J Clin Invest.
2014:124(12):5466–5480. https://doi.org/10.1172/JCI77053
26. Ponzetta A, Carriero R, Carnevale S, Barbagallo M, Molgora M,
Perucchini C, Magrini E, Gianni F, Kunderfranco P, Polentarutti
N, et al. Neutrophils driving unconventional T cells mediate re­
sistance against murine sarcomas and selected human tumors.
Cell. 2019:178(2):346–360.e24. https://doi.org/10.1016/j.cell.2019.
05.047
27. Gianni C, Palleschi M, Schepisi G, Casadei C, Bleve S, Merloni F,
Sirico M, Sarti S, Cecconetto L, Di Menna G, et al. Circulating in­
flammatory cells in patients with metastatic breast cancer: im­
plications for treatment. Front Oncol. 2022:12(Aug 8):882896.
https://doi.org/10.3389/fonc.2022.882896
28. Ethier JL, Desautels D, Templeton A, Shah PS, Amir E. Prognostic
role of neutrophil-to-lymphocyte ratio in breast cancer: a sys­
tematic review and meta-analysis. Breast Cancer Res. 2017:19(1):
2. https://doi.org/10.1186/s13058-016-0794-1
29. Philip A, Jose M, Jose WM, Vijaykumar DK, Pavithran K.
Pretreatment neutrophil-to-lymphocyte ratio predicts lymph
node metastasis in triple-negative breast cancer. Indian J Cancer.
21 March 2021. https://doi.org/10.4103/ijc.IJC_914_19, preprint:
not peer reviewed.
30. Gentles AJ, Newman AM, Liu CL, Bratman SV, Feng W, Kim D,
Nair VS, Xu Y, Khuong A, Hoang CD, et al. The prognostic land­
scape of genes and infiltrating immune cells across human can­
cers. Nat Med. 2015:21(8):938–945. https://doi.org/10.1038/nm.
3909
31. Livak KJ, Schmittgen TD. Analysis of relative gene expression
data using real-time quantitative PCR and the 2(-Delta Delta
C(T)) method. Methods. 2001:25(4):402–408. https://doi.org/10.
1006/meth.2001.1262
32. Thanasupawat T, Glogowska A, Nivedita-Krishnan S, Wilson B,
Klonisch T, Hombach-Klonisch S. Emerging roles for the relax­
in/RXFP1 system in cancer therapy. Mol Cell Endocrinol.
2019:487(May 1):85–93. https://doi.org/10.1016/j.mce.2019.02.
001
33. Aliper AM, Frieden-Korovkina VP, Buzdin A, Roumiantsev SA,
Zhavoronkov A. A role for G-CSF and GM-CSF in nonmyeloid can­
cers. Cancer Med. 2014:3(4):737–746. https://doi.org/10.1002/
cam4.239
Sheng et al. | 397
34. Mehta HM, Malandra M, Corey SJ. G-CSF and GM-CSF in neutro­
penia. J Immunol. 2015:195(4):1341–1349. https://doi.org/10.4049/
jimmunol.1500861
35. Al-Rashed F, Thomas R, Al-Roub A, Al-Mulla F, Ahmad R. LPS in­
duces GM-CSF production by breast cancer MDA-MB-231 cells
via long-chain acyl-CoA synthetase 1. Molecules. 2020:25(20):
4709. https://doi.org/10.3390/molecules25204709
36. Akira S, Kishimoto T. IL-6 and NF-IL6 in acute-phase response
and viral infection. Immunol Rev. 1992:127(1):25–50. https://doi.
org/10.1111/j.1600-065X.1992.tb01407.x
37. Brasier AR. Therapeutic targets for inflammation-mediated
airway remodeling in chronic lung disease. Expert Rev Respir
Med. 2018:12(11):931–939. https://doi.org/10.1080/17476348.
2018.1526677
Downloaded from https://academic.oup.com/jleukbio/article/113/4/383/6991105 by guest on 12 May 2023
38. Valkovic AL, Bathgate RA, Samuel CS, Kocan M. Understanding
relaxin signalling at the cellular level. Mol Cell Endocrinol.
2019:487(May 1):24–33. https://doi.org/10.1016/j.mce.2018.12.
017
39. Vikas P, Borcherding N, Zhang W. The clinical promise of im­
munotherapy in triple-negative breast cancer. Cancer Manag
Res. 2018:10(Dec 10):6823–6833. https://doi.org/10.2147/CMAR.
S185176
40. Larionova I, Tuguzbaeva G, Ponomaryova A, Stakheyeva M,
Cherdyntseva N, Pavlov V, Choinzonov E, Kzhyshkowska J.
Tumor-associated macrophages in human breast, colorectal,
lung, ovarian and prostate cancers. Front Oncol. 2020:10(Oct 22):
566511. https://doi.org/10.3389/fonc.2020.566511
41. Ma R-Y, Zhang H, Li X-F, Zhang C-B, Selli C, Tagliavini G, Lam
AD, Prost S, Sims AH, Hu H-Y, et al. Monocyte-derived macro­
phages promote breast cancer bone metastasis outgrowth. J
Exp Med. 2020:217(11):e20191820. https://doi.org/10.1084/jem.
20191820
42. Safarulla S, Madan A, Xing F, Chandrasekaran A. CXCR2
mediates distinct neutrophil behavior in brain metastatic breast
tumor. Cancers (Basel). 2022:14(3):515. https://doi.org/10.3390/
cancers14030515
43. Hollmén M, Karaman S, Schwager S, Lisibach A, Christiansen AJ,
Maksimow M, Varga Z, Jalkanen S, Detmar M. G-CSF regulates
macrophage phenotype and associates with poor overall survival
in human triple-negative breast cancer. Oncoimmunology.
2016:5(3):e1115177. https://doi.org/10.1080/2162402X.2015.
1115177
44. Giese MA, Hind LE, Huttenlocher A. Neutrophil plasticity in the
tumor microenvironment. Blood. 2019:133(20):2159–2167.
https://doi.org/10.1182/blood-2018-11-844548
45. Posabella A, Kohn P, Lalos A, Wilhelm A, Mechera R, Soysal S,
Muenst S, Guth U, Stadlmann S, Terracciano L, et al. High dens­
ity of CD66b in primary high-grade ovarian cancer independ­
ently predicts response to chemotherapy. J Cancer Res Clin
Oncol. 2020:146(1):127–136. https://doi.org/10.1007/s00432-019-
03108-6
46. Yamada Y, Nakagawa T, Sugihara T, Horiuchi T, Yoshizaki U,
Fujimura T, Fukuhara H, Urano T, Takayama K, Inoue S, et al.
Prognostic value of CD66b positive tumor-infiltrating neutro­
phils in testicular germ cell tumor. BMC Cancer. 2016:16(1):898.
https://doi.org/10.1186/s12885-016-2926-5
47. Rakaee M, Busund LT, Paulsen EE, Richardsen E, Al-Saad S,
Andersen S, Donnem T, Bremnes RM, Kilvaer TK. Prognostic ef­
fect of intratumoral neutrophils across histological subtypes of
non-small cell lung cancer. Oncotarget. 2016:7(44):72184–72196.
https://doi.org/10.18632/oncotarget.12360
48. Matsumoto Y, Mabuchi S, Kozasa K, Kuroda H, Sasano T, Yokoi E,
Komura N, Sawada K, Kimura T. The significance of tumor-
398 | Journal of Leukocyte Biology, 2023, Vol. 113, No. 4
associated neutrophil density in uterine cervical cancer treated
with definitive radiotherapy. Gynecol Oncol. 2017:145(3):469–475.
https://doi.org/10.1016/j.ygyno.2017.02.009
49. Kitano Y, Okabe H, Yamashita YI, Nakagawa S, Saito Y, Umezaki
N, Tsukamoto M, Yamao T, Yamamura K, Arima K, et al.
Tumour-infiltrating inflammatory and immune cells in patients
with extrahepatic cholangiocarcinoma. Br J Cancer. 2018:118(2):
171–180. https://doi.org/10.1038/bjc.2017.401
50. Cheng Y, Li H, Deng Y, Tai Y, Zeng K, Zhang Y, Liu W, Zhang Q,
Yang Y. Cancer-associated fibroblasts induce PDL1 + neutrophils
through the IL6-STAT3 pathway that foster immune suppression
in hepatocellular carcinoma. Cell Death Dis. 2018:9(4):422. doi:10.
1038/s41419-018-0458-4
51. Huang X, Pan Y, Ma J, Kang Z, Xu X, Zhu Y, Chen J, Zhang W,
Chang W, Zhu J. Prognostic significance of the infiltration of
CD163(+) macrophages combined with CD66b(+) neutrophils in
gastric cancer. Cancer Med. 2018:7(5):1731–1741. https://doi.org/
10.1002/cam4.1420
52. Dabrosin N, Sloth Juul K, Baehr Georgsen J, Andrup S, Schmidt H,
Steiniche T, Heide Ollegaard T, Bonnelykke Behrndtz L. Innate
immune cell infiltration in melanoma metastases affects sur­
vival and is associated with BRAFV600E mutation status.
Melanoma Res. 2019:29(1):30–37. https://doi.org/10.1097/CMR.
0000000000000515
53. Ye L, Zhang T, Kang Z, Guo G, Sun Y, Lin K, Huang Q, Shi X, Ni Z,
Ding N, et al. Tumor-infiltrating immune cells act as a marker for
prognosis in colorectal cancer. Front Immunol. 2019:10(Oct 17):
2368. https://doi.org/10.3389/fimmu.2019.02368
54. Shaul ME, Eyal O, Guglietta S, Aloni P, Zlotnik A, Forkosh E, Levy
L, Weber LM, Levin Y, Pomerantz A, et al. Circulating neutrophil
subsets in advanced lung cancer patients exhibit unique im­
mune signature and relate to prognosis. FASEB J. 2020:34(3):
4204–4218. https://doi.org/10.1096/fj.201902467R
55. Mandelli GE, Missale F, Bresciani D, Gatta LB, Scapini P,
Caveggion E, Roca E, Bugatti M, Monti M, Cristinelli L, et al.
Tumor infiltrating neutrophils are enriched in basal-type urothe­
lial bladder cancer. Cells. 2020:9(2):291. https://doi.org/10.3390/
cells9020291
56. Witko-Sarsat V, Mocek J, Bouayad D, Tamassia N, Ribeil JA,
Candalh C, Davezac N, Reuter N, Mouthon L, Hermine O, et al.
Proliferating cell nuclear antigen acts as a cytoplasmic platform
controlling human neutrophil survival. J Exp Med. 2010:207(12):
2631–2645. https://doi.org/10.1084/jem.20092241
57. De Chiara A, Pederzoli-Ribeil M, Mocek J, Candalh C, Mayeux P,
Millet A, Witko-Sarsat V. Characterization of cytosolic proliferat­
ing cell nuclear antigen (PCNA) in neutrophils: antiapoptotic role
of the monomer. J Leukoc Biol. 2013:94(4):723–731. https://doi.org/
10.1189/jlb.1212637
58. Fridlender ZG, Albelda SM. Tumor-associated neutrophils: friend
or foe? Carcinogenesis. 2012:33(5):949–955. https://doi.org/10.
1093/carcin/bgs123
59. Zhou S-L, Zhou Z-J, Hu Z-Q, Huang X-W, Wang Z, Chen E-B, Fan J,
Cao Y, Dai Z, Zhou J. Tumor-associated neutrophils recruit mac­
rophages and T-regulatory cells to promote progression of hepa­
tocellular carcinoma and resistance to sorafenib.
Gastroenterology. 2016:150(7):1646–1658.e17. https://doi.org/10.
1053/j.gastro.2016.02.040
60. Chao T, Furth EE, Vonderheide RH. CXCR2-dependent accumula­
tion of tumor-associated neutrophils regulates T-cell immunity
in pancreatic ductal adenocarcinoma. Cancer Immunol Res.
2016:4(11):968–982. https://doi.org/10.1158/2326-6066.CIR-16-
0188
61. Governa V, Trella E, Mele V, Tornillo L, Amicarella F, Cremonesi
E, Muraro MG, Xu H, Droeser R, Däster SR, et al. The interplay be­
tween neutrophils and CD8(+) T cells improves survival in hu­
man colorectal cancer. Clin Cancer Res. 2017:23(14):3847–3858.
https://doi.org/10.1158/1078-0432.CCR-16-2047
62. Wang Y, Chen J, Yang L, Li J, Wu W, Huang M, Lin L, Su S.
Tumor-contacted neutrophils promote metastasis by a
CD90-TIMP-1 juxtacrine-paracrine loop. Clin Cancer Res.
2019:25(6):1957–1969. https://doi.org/10.1158/1078-0432.CCR-
18-2544
63. Bakouny Z, Choueiri TK. IL-8 and cancer prognosis on immuno­
therapy. Nat Med. 2020:26(5):650–651. https://doi.org/10.1038/
s41591-020-0873-9
64. Mukaida N, Sasaki SI, Baba T. Two-faced roles of tumor-
Downloaded from https://academic.oup.com/jleukbio/article/113/4/383/6991105 by guest on 12 May 2023
associated neutrophils in cancer development and progres­
sion. Int J Mol Sci. 2020:21(10):3457. https://doi.org/10.3390/
ijms21103457
65. Mouchemore KA, Anderson RL, Hamilton JA. Neutrophils, G-CSF
and their contribution to breast cancer metastasis. FEBS J.
2018:285(4):665–679. https://doi.org/10.1111/febs.14206
66. Coffelt SB, Kersten K, Doornebal CW, Weiden J, Vrijland K, Hau
CS, Verstegen NJM, Ciampricotti M, Hawinkels L, Jonkers J, et al.
IL-17-producing γδ T cells and neutrophils conspire to promote
breast cancer metastasis. Nature. 2015:522(7556):345–348.
https://doi.org/10.1038/nature14282
67. Pickup MW, Owens P, Gorska AE, Chytil A, Ye F, Shi C, Weaver
VM, Kalluri R, Moses HL, Novitskiy SV. Development of aggres­
sive pancreatic ductal adenocarcinomas depends on granulocyte
colony stimulating factor secretion in carcinoma cells. Cancer
Immunol Res. 2017:5(9):718–729. https://doi.org/10.1158/2326-
6066.CIR-16-0311
68. Bosiljcic M, Cederberg RA, Hamilton MJ, LePard NE, Harbourne
BT, Collier JL, Halvorsen EC, Shi R, Franks SE, Kim AY, et al.
Targeting myeloid-derived suppressor cells in combination
with primary mammary tumor resection reduces metastatic
growth in the lungs. Breast Cancer Res. 2019:21(1):103. https://
doi.org/10.1186/s13058-019-1189-x
69. Kowanetz M, Wu X, Lee J, Tan M, Hagenbeek T, Qu X, Yu L, Ross J,
Korsisaari N, Cao T, et al. Granulocyte-colony stimulating factor
promotes lung metastasis through mobilization of Ly6G + Ly6C+
granulocytes. Proc Natl Acad Sci U S A. 2010:107(50):21248–21255.
https://doi.org/10.1073/pnas.1015855107
70. Rizvi S, Gores GJ. The two faces of relaxin in cancer: antitumor or
protumor? Hepatology. 2020:71(3):1117–1119. https://doi.org/10.
1002/hep.30998
71. Fue M, Miki Y, Takagi K, Hashimoto C, Yaegashi N, Suzuki T, Ito
K. Relaxin 2/RXFP1 signaling induces cell invasion via the
β-catenin pathway in endometrial cancer. Int J Mol Sci. 2018:19­
(81):2438. https://doi.org/10.3390/ijms19082438
72. Cao W-H, Liu H-M, Liu X, Li J-G, Liang J, Liu M, Niu Z-H. Relaxin
enhances in-vitro invasiveness of breast cancer cell lines by up­
regulation of S100A4/MMPs signaling. Eur Rev Med Pharmacol Sci.
2013:17(5):609–617.
73. Binder C, Simon A, Binder L, Hagemann T, Schulz M, Emons G,
Trümper L, Einspanier A. Elevated concentrations of serum re­
laxin are associated with metastatic disease in breast cancer pa­
tients. Breast Cancer Res Treat. 2004:87(2):157–166. https://doi.org/
10.1023/B:BREA.0000041622.30169.16
74. Binder C, Hagemann T, Husen B, Schulz M, Einspanier A. Relaxin
enhances in-vitro invasiveness of breast cancer cell lines by up-
regulation of matrix metalloproteases. Mol Hum Reprod. 2002:8(9):
789–796. https://doi.org/10.1093/molehr/8.9.789

75. Hu M, Zhou X, Wang Y, Guan K, Huang L. Relaxin-FOLFOX-IL-12
triple combination therapy engages memory response and
achieves long-term survival in colorectal cancer liver metastasis.
J Control Release. 2020:319(March 10):213–221. https://doi.org/10.
1016/j.jconrel.2019.12.053
76. Hu M, Wang Y, Xu L, An S, Tang Y, Zhou X, Li J, Liu R, Huang L.
Relaxin gene delivery mitigates liver metastasis and synergizes
Sheng et al. | 399
with check point therapy. Nat Commun. 2019:10(1):2993. https://
doi.org/10.1038/s41467-019-10893-8
77. Feng S, Agoulnik IU, Truong A, Li Z, Creighton CJ, Kaftanovskaya
EM, Pereira R, Han HD, Lopez-Berestein G, Klonisch T, et al.
Suppression of relaxin receptor RXFP1 decreases prostate cancer
growth and metastasis. Endocr Relat Cancer. 2010:17(4):1021–1033.
https://doi.org/10.1677/ERC-10-0073
Downloaded from https://academic.oup.com/jleukbio/article/113/4/383/6991105 by guest on 12 May 2023